function of the retinoic acid receptors ... - development · development and that rars are indeed...

INTRODUCTION

Fetuses from rat mothers reared on vitamin A-deficiient (VAD)diets exhibit severe congenital malformations known as thefetal VAD syndrome (Wilson and Warkany, 1948, 1949;Warkany et al., 1948; Wilson et al., 1953). These malforma-tions include abnormalities of the eyes, respiratory tract, heartand great vessels, urogenital system and diaphragm, while cleftpalate has been reported in the case of VAD pigs (Hale, 1933).Addition of retinol to the diet of VAD dams during pregnancyreversed nearly all of these malformations, a clear demonstra-tion that vitamin A is critical for normal development. Inter-estingly, the time of retinol administration during pregnancywas critical in order to prevent the appearance of a given mal-formation, suggesting that vitamin A could be required atseveral distinct stages of development (Wilson et al., 1953). Itis generally believed that retinoic acid (RA) is the active deriv-ative of vitamin A during development, as it is after birth

(except for vision, see Thompson et al., 1964; Wald, 1968);however, it has not been shown that RA can rescue VADembryos with the exception of cardiovascular development inquail embryos (Dersch and Zile, 1993). Furthermore, it iswidely accepted that the two families of retinoic acid nuclearreceptors (RARs and RXRs) act as transcriptional transducersof the retinoid signal during development. It is also thoughtthat the main isoforms (

α1 and α2, β1 to β4, γ1 and γ2) of thethree RAR types (RARα, β and γ), which have distinct patternsof expression during embryogenesis, may possess specifictransactivation characteristics enabling them to control theexpression of specific subset of genes during development(reviewed in Hofmann and Eichele, 1994; Chambon, 1994;Linney and LaMantia, 1994; Kastner et al., 1994; see Intro-duction of Lohnes et al., 1994 for additional references).

Thus, the highly diverse effects of the RA signal would beaccounted for by the multiplicity of functionally distinctreceptors (Leid et al., 1992; Chambon, 1994). This possibility

2749Development 120, 2749-2771 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

Compound null mutations of retinoic acid receptor (RAR)genes lead to lethality in utero or shortly after birth and tonumerous developmental abnormalities. In the accompa-nying paper (Lohnes, D., Mark, M., Mendelsohn, C., Dollé,P., Dierich, A., Gorry, Ph., Gansmuller, A. and Chambon,P. (1994).

Development 120, 2723-2748), we describe mal-formations of the head, vertebrae and limbs which, withthe notable exception of the eye defects, were not observedin the offspring of vitamin A-deficient (VAD) dams. Wereport here abnormalities in the neck, trunk andabdominal regions of RAR double mutant mice, whichinclude :(i) the entire respiratory tract, (ii) the heart, itsoutlow tract and the great vessels located near the heart,(iii) the thymus, thyroid and parathyroid glands, (iv) thediaphragm, (v) the genito-urinary system, and (vi) thelower digestive tract. A majority of these abnormalities

recapitulate those observed in the fetal VAD syndromedescribed by Joseph Warkany’s group more than fourtyyears ago [Wilson, J. G., Roth, C. B. and Warkany, J.(1953) Am. J. Anat., 92, 189-217; and refs therein]. Ourresults clearly demonstrate that RARs are essential for ver-tebrate ontogenesis and therefore that retinoic acid is theactive retinoid, which is required at several stages of thedevelopment of numerous tissues and organs. We discussseveral possibilities that may account for the apparentfunctional redundancy observed amongst retinoic acidreceptors during embryogenesis.

Key words: mouse, ontogenesis, retinoic receptor mutants, neuralcrest, heart, trachea, aortic arches, lung, kidney, genital tract,functional redundancy

SUMMARY

Function of the retinoic acid receptors (RARs) during development

(II) Multiple abnormalities at various stages of organogenesis in RAR double mutants

Cathy Mendelsohn*,†, David Lohnes*,‡, Didier Décimo, Thomas Lufkin§, Marianne LeMeur, Pierre Chambon

|

and Manuel Mark¶

Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique del’INSERM, Institut de Chimie Biologique, Faculté de Médecine, 11 rue Humann, 67085 Strasbourg Cedex, France

*Should be considered as equal first authors†Present address: Columbia University, Department of Physiology and Biophysics, 630 W 168th Street, New York, NY 10032, USA‡Present address: Institut de Recherches Cliniques de Montréal, Laboratoire de Biologie Cellulaire et Moléculaire, 110 Avenue des Pins-Ouest, Montréal, H2W 1R7,Canada§Present address: Brookdale Center for Molecular Biology, The Mount Sinai Medical Center, 1 Gustave L. Levy Place, New York, NY 10029-6574, USA|to whom reprint requests should be sent¶to whom correspondence should be addressed

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has been recently tested in vivo by generating null mousemutants for the various receptors (Li et al., 1993; Lohnes et al.,1993; Lufkin et al., 1993; Mendelsohn et al., 1994). Surpris-ingly, none of the abnormalities characteristic of the fetal VADsyndrome was observed in these mutants, thus suggesting ahigh degree of functional redundancy amongst the variousRARs. Compound RAR mutant mice were then generated toinvestigate this possibility, and as reported in the accompany-ing study (Lohnes et al., 1994), the double mutants died duringgestation or immediately after birth. In addition, these mutantsexhibited a larger number of malformations, including abnor-malities characteristic of the VAD syndrome, thus demon-strating that RA is the retinoid signalling molecule used duringdevelopment and that RARs are indeed transducers of thissignal in vivo. We have reported in the accompanying study(Lohnes et al., 1994), the craniofacial and skeletal abnormali-ties presented by RAR double mutant fetuses. We describehere the visceral abnormalities, and show that RA is requiredat many stages of development of a given tissue or organ. Theimplications of the present results and of those reported in thepreceeding study are discussed with respect to the multipleroles of RA in development and the apparent functional redun-dancy amongst the RARs.

MATERIALS AND METHODS

RAR double mutants were generated and analysed as described in theaccompanying study (Lohnes et al., 1994).

RESULTS

(A) Oesophagotracheal abnormalitiesIn 18.5 dpc wild-type (WT) fetuses, the right lung (rL, Fig.1a,h) is composed of four essentially separate lobes, a cranial(CL), a middle (ML) and a caudal lobe (DL) that are locatedon the right side of the thorax, and an accessory lobe that orig-inates on the right side, but is predominantly located on the leftside of the midline (Fig. 1a, and data not shown). In contrast,the smaller left lung (fL, Fig. 1a,h) is not subdivided. In amajority (6 out of 7) of 18.5 dpc αβ2 mutants analyzed onserial histological sections (Table 1), and in one fetus analyzedby whole mount, the left lung was absent (Fig. 1c), whereasthe right lung (rL, Fig. 1c,i; Table 1, and data not shown) wasmarkedly hypoplastic and consisted of 3 or 4 lobes. No portionof the right lung ever crossed the midline, due to hypoplasiaof the accessory lobe. In one αβ2 and in some αβ2+/− mutants,both the right and the left lungs were hypoplastic (compare rLand fL in Fig. 1a,b). In the case of agenesis of the left lung,the left stem bronchus was either missing or truncated. The sizeand lobulation of the lungs were apparently normal in all otherdouble mutants (see Table 1).

The oesophagotracheal septum (OTS in Fig. 1j) was absent(compare Fig. 1j and k) in all αβ2 and in 1 (out of 3) α1β2mutants, resulting in a complete lack of separation between theoesophagus (O) and the trachea (T). The epithelium of thisoesophagotracheal tube (OT, Fig. 1c,k) was of the columnarciliated type normally found in the trachea. This ciliatedepithelium also replaced the normally stratified squamousepithelium of the caudal oesophagus, and cardiac and proven-

tricular portions of the stomach (not shown). The oesophago-tracheal septum and the oesophageal and tracheal epitheliawere normal in all other mutant fetuses (Table 1).

Embryos were examined to investigate the origin of the mal-formations observed in 18.5 dpc mutants. In 11.5 dpc WTembryos, the division of the foregut by the oesophagotrachealseptum (OTS) is completed, yielding the trachea (T) on theventral side and the oesophagus (O) on the dorsal side (Fig.4a,c). In 11.5 dpc αβ2 mutants, no division had occurred; asingle gut tube (OT in Fig. 4b) was present and the lung budsarose directly from the ventral wall of this tube (not shown).Thus the absence of the oesophagotracheal septum in 18.5 dpcfetuses reflects an early morphogenetic arrest. In 11.5 dpc WTembryos, the right stem bronchus (rSB, Fig. 1d) had justbranched into the 4 lobar bronchi (e.g. LB, in Fig. 1d), whereasthe left stem bronchus (fSB, in Fig. 1d) remained unbranched.In contrast, the right stem bronchus had not branched in four11.5 dpc αβ2 embryos examined, while the left lung bud wascompletely absent in two of these embryos (e.g. Fig. 1e), i.e.in 11.5 dpc mutants the size and branching pattern of the rightlung bud resembled that of 10.5 dpc WT right lung. At 12.5dpc, the wild-type lung buds have acquired tertiary (segmental)bronchi (arrowheads in Fig. 1f). The size of the right lung andbranching pattern of right bronchi in the 12.5 dpc αβ2 embryoswere similar to the 11.5 dpc normal right lung (compare rSBand LB in Fig. 1d and g), again exhibiting a retardation indevelopment of approximately 24 hours; in this mutant, the leftlung (fSB) was present, but markedly hypoplastic. Thus, lunghypoplasia observed in 18.5 dpc mutants appears to be causedby a delay in bronchial branching, whereas left lung agenesismay be due to an early defect in lung bud formation.

(B) Abnormalities of the laryngeal and trachealcartilages and of the hyoid boneThe cartilaginous skeleton of the larynx is formed by thethyroid, cricoid and arytenoid cartilages, and that of the tracheaand extrapulmonary bronchi consists of segmentally arrangedC-shaped tracheal ‘rings’. Whereas the epithelia of the larynx,

C. Mendelsohn and others

Fig. 1. Comparison of the lungs, trachea and diaphragm between(a,d,f,h,j) wild-type (WT) and (b,c,e,g,i,k) RAR double mutantembryos and fetuses. (a-c) Views of the dorsal aspects of the lungsheart and aorta ; note that the oesophagus (O) was left in place in a,cbut was removed in b. (d,e) Transverse sections of 11.5 dpc embryosat the level of the cranial portion of the right lung. (f,g) Transversesections of 12.5 dpc embryos at the level of the cranial lobe of theright lung. (h,i) Frontal sections through the thoracic and abdominalregions of 18.5 dpc fetuses. (j,k) Sagittal median sections through theneck of 18.5 dpc fetuses. Abbreviations: AA, arch of the aorta; rAA,right arch of the aorta; CAA, cervical arch of the aorta; AC,arytenoid cartilage; CL, cranial lobe of the right lung; rC, rightcommon carotid; DA, descending aorta; DL, caudal lobe of the rightlung; DR, diaphragm; LE, liver; fL, left lung; LB, cranial lobarbronchus; rL, right lung; ML, median lobe of the right lung; MU,muscle of the caudal wall of the oesophagus or oesophagotrachea; O,oesophagus; rOS, retroesophageal subclavian artery; OTS,oesophagotracheal septum; OT, oesophagotrachea; RC, cricoidcartilage; fSB, left stem bronchus; rSB, right stem bronchus; SC,spinal cord; S, stomach; TC, thyroid cartilage; T, trachea; TR,tracheal rings; fV, left ventricle; VE, vertebral column; arrow heads,segmental bronchi. Magnifications: ×44 (d-g) ; ×9 (h,i) ×29 (j,k); thewhole-mounts in a-c are shown at the same magnification.

2751RARs in ontogenesis

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of the tracheobronchial tree and of the alveoli are derived fromthe endoderm of the foregut, the cartilaginous and smoothmuscular components of the trachea and bronchi are derivedfrom the surrounding lateral mesoderm.

All 18.5 dpc double mutants analysed by whole mountsshowed tracheal cartilage malformations. However, thesedefects varied greatly depending on the mutant genotype(Table 1; Fig. 2). As reported (Lohnes et al., 1993), the trachealrings of RAR γ null fetuses were variably fused in the ventralplane (TR, Fig. 2f). Strikingly, the tracheal rings disappearedin α1γ, α1α2+/−γ and αγ double mutants to be replaced by aventral cartilage and cartilaginous nodules (TR, Fig. 2g-i).Moreover, there was a marked reduction in the intensity ofalcian blue staining in αγ mutant tracheas, suggesting a defi-ciency in cartilage formation (Fig. 2i). In the β2γ mutants, thetracheal rings were severely malformed (TR, Fig. 2b). Thetracheal rings were only mildly malformed in α1β2 mutants,but completely disorganized in αβ2 mutants (TR, compare Fig.2a to c, d and e). RARβ2 transcripts are expressed both in theforegut endoderm and mesoderm, whereas RARγ expression isrestricted to the mesoderm (Dollé et al., 1990; Ruberte et al.,1990; our unpublished results). Consistent with these differ-ences in expression patterns, inactivation of either RARγ orRARβ2 in RARα1 or RARα (all isoforms) mutant back-grounds resulted in different anomalities of the tracheal carti-lages; for example, the ventral cartilaginous fusion was presentin α1γ, but not in α1β2 mutants (see Fig. 2c,g; see also Fig.2i,e,d).

The length of the trachea was markedly and specifically

reduced in the αγ and αβ2 mutants, with the bifurcation whichgives rise to the stem bronchi occurring prematurely (comparee.g. Fig. 2a with d, e and i; arrowheads). Interestingly, this wasnot seen in the α1γ and α1β2 mutants (compare Fig. 2c withe and g and h with i), thus the removal of the α2 isoform inα1γ and α1β2 mutant backgrounds was responsible for thesedefects. The left stem bronchus of the αβ2 mutant shown inFig. 2e was almost absent, reflecting the lack of the left lung(see above); however, the cartilaginous rings of the visibleportion of the right bronchus appeared regularly spaced.Further histological analysis revealed that the cartilages of themore caudal portions of the stem bronchi (not visible in wholemounts) were regularly spaced in αβ2, αβ2+/− and α1β2mutants, but fused in the α1γ, α1γα2+/−, β2γ and αγ mutants(data not shown). Since RARγ transcripts are expressed in thebronchial mesenchyme (Ruberte et al., 1990; Dollé et al., 1990and our unpublished results), it is likely that this receptor isinvolved in formation of the bronchial cartilages.

RAR double mutants which exhibited malformed thyroidcartilages, also had malformed and smaller arytenoid cartilages(Table 1). In RARγ single mutants, as in α1γ and β2γ mutants,these cartilages were normal in size and shape (TC and AC, inFig. 2f,g and b), but were smaller in the α1γα2+/− and unrec-ognizable in αγ mutants (TC and AC in Fig. 2h and star in Fig.2i). The thyroid and arytenoid cartilages were nearly normal inα1β2 mutants (Fig. 2c), while in αβ2 mutants their structurewas severely altered (TC and AC, Fig. 2d,e).

The cricoid cartilage was abnormally fused to the trachealrings in all double mutants and in RARγ single mutants (RC,


Table 1. Abnormalities of the respiratory tract, hyoid bone, cartilages, heart and outflow tract, aortic arch-derived greatvessels and diaphragm in 18.5 dpc RAR double mutant fetuses

RAR mutant genotype and number of 18.5 dpc fetuses examined

α1β2 αβ2+/− αβ2 α1γ α1γα2+/− αγ β2γ3g,10h 4g 7g,10h 5g,16h 5g,11h 5g,6h 3g,9h VAD

Respiratory tract abnormalitiesAgenesis of left lung 0/3 0/4 6/7 0/5 0/5 0/5 0/3 +Hypoplastic left lung 0/3 2/4 1/7 0/5 0/5 0/5 0/3 +Hypoplastic right lung 0/3 2/4 7/7 0/5 0/5 0/5 0/3 +Lack of oesophago-tracheal septum 1/3 0/4 7/7 0/5 0/5 0/5 0/3 +

Hyoid bone abnormalities 4/10e ND 10/10e 0/6 0/11 6/6f 0/9 NR

Abnormal cartilages(i) Laryngeal cartilages

Thyroid cartilage 0/10 ND 10/10 0/16 11/11 6/6 0/9 NRArytenoid cartilage 0/10 ND 10/10 0/16 11/11 6/6 0/9 NRCricoid cartilage

abnormal shape 0/10 ND 10/10 0/16 11/11 6/6 0/9 NRfused to tracheal rings 10/10 ND 10/10 16/16 11/11 6/6 9/9 NR

(ii) Tracheal cartilagesd 10/10 ND 10/10 16/16 11/11 6/6 9/9 NR

Ectopic cartilage nodules 2/3 0 1/7 0/5 0/5 2/5 0/3 NR

Heart outflow tract and aortic arch abnormalitiesPersistent truncus arteriosus (PTA) 1/3 2/4 7/7 0/5 0/5 5/5 0/3 +Dextroposed aorta 1/3 1/4 NA 0/5 2/5 NA 0/3 +High ventricular septal defect 2/3 3/4 7/7 0/5 3/5 5/5 0/3 +Other heart abnormalities 0/3 0/4 1/7a 0/5 1/5c 2/5a,b 0/3 +a,b

Abnormal aortic arch pattern 3/3 2/4 7/7 0/5 3/5 5/5 0/3 +

Diaphragmatic Hernia 0/3 1/4 1/7 0/5 0/5 0/5 0/3 +

With the exception of tracheal cartilages no abnormalities were ever observed in RARα, RARβ2 or RARγ single mutant fetuses.aPersistent atrioventricular canal; bthin myocardium; cdouble outlet right ventricule (DORV); dabnormalities varied with genotype; epartial fusion with thyroid

cartilage; fhighly abnormal shape; ganalyzed on serial histological sections; hanalyzed on whole-mount skeleton; see text.NR, not reported; ND, not determined; NA, not applicable.


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2754 C. Mendelsohn and others


Fig. 2b-i). As for the thyroid and arytenoid cartilages, the shapeof the cricoid cartilage in the γ, α1β2, α1γ and β2γ mutantswas nearly normal, whereas it was clearly abnormal in theα1γα2+/− fetuses, and unrecognizable in αβ2 and αγ mutants(compare RC in Fig. 2).

The body of the hyoid bone originates from neural crest cells(NCC) that populate the 2nd and 3rd branchial arches, thegreater horn being derived from the 3rd arch and the lesser hornfrom the 2nd arch. The size and shape of the hyoid bones of18.5 dpc γ, α1γ and α1γα2+/− mutants were normal (data notshown). In contrast, the hyoid bone in αγ mutants was highlyabnormal; the greater horns, body and lesser horns were unrec-ognizable (BO, LH and GH, compare Fig. 2j with k). In theβ2γ, α1β2 and αβ2 mutants, the shape of the hyoid bone wasnormal, but there was a fusion between the hyoid greater hornand the rostral horn of the thyroid cartilage in all αβ2 and someα1β2 fetuses (arrowheads in Fig. 2c and d).

Note that ectopic cartilages were also found in α1β2, αβ2and αγ mutants at either the base of the heart semilunar cusps,in the diaphragm or in the peritoneum (Table 1).

(C) Diaphragmatic herniaPosterolateral diaphragmatic defects, which result from afailure of closure of the pleuroperitoneal canals (which isnormally complete by 14.0 dpc; Kaufman, 1992), wereobserved in one of seven αβ2 and one out of four αβ2+/− 18.5dpc mutants but not in any other double mutants. In the αβ2mutant, there was an abnormal foramen in the right dorsalportion of the diaphragm (DR) permitting protrusion of a massof liver (LE) into the right pleural cavity (compare Fig. 1h andi). This herniated mass compressed the right lung (RL, Fig. 1i).In the affected αβ2+/− mutant, the diaphragmatic defect wasobserved on the left side. RARβ2 transcripts, but not RARγtranscripts are expressed in the anlage of the diaphragm at 12.5dpc (our unpublished results), suggesting that RARβ2 couldplay a role in its formation.

(D) Abnormalities of the heart and outflow tract The ventricular myocardium, which develops from cardiogenicplate mesoderm, was abnormally thin in two (out of five) 18.5dpc αγ mutant fetuses, (compare Fig. 3a with e and Fig. 3f withg), but not in the other mutants. The deficiency was particu-larly marked in the subepicardial or compact (outer) layer ofthe ventricular wall (CMY, Fig. 3f,g), while the trabecular(inner) layer (TMY, Fig. 3f,g) appeared less affected, thusimparting a spongy appearance to the myocardium reminiscentof that seen in 10.5-12.5 dpc normal embryos. In addition, oneof these two fetuses lacked the ventricular septum (VS,compare Fig. 3a with e). Myocardial deficiency, occurring asa consequence of a growth arrest, might account for theembryonic lethality in αγ mutants (see Lohnes et al., 1994),since neither renal agenesis (see below) or exencephaly (seeLohnes et al., 1994) are usually embryonic lethal in mouse orhuman (Kreidberg et al., 1993; Franz,1989 and refs therein).

Between 11.0 and 13.0 dpc, the aortic sac is divided longi-tudinally by the spiral-shaped aorticopulmonary septum (AP,Fig. 4c), (Fananapazir and Kaufman, 1988; Vuillemin andPexieder, 1989), a structure derived from rhombencephalicNCC (Kirby et al., 1983), thus giving rise to the ascendingaorta (AS, Fig. 4c) and to the pulmonary trunk (PT, Fig. 4c).Failure of this division to take place or to become completeresults in a persistent truncus arteriosus (PTA) receiving bloodfrom both ventricles (TA, Figs 3b,d, 4d). This defect wasobserved in all 18.5 dpc αβ2 (seven) and αγ (five) mutants(compare Fig. 3a and b). PTA, which appeared as a singleoutflow vessel on serial histological sections (TA, Fig. 3b), wasconnected with the heart through a valve with 4 semilunarcusps (TA:1-4, Fig. 3d), instead of the normal 6 cusps, namelythe 3 aortic cusps (AS: 1-3, Fig. 3c) and 3 pulmonary cusps(PT: 1-3, Fig. 3c) found in WT fetuses. The origin of this vesselfrom the right ventricle alone was evident in five αβ2 and inthree αγ mutants; in the remaining αγ and αβ2 mutants, PTAoverrode the ventricular septum (not shown). PTA was alsopresent in two (out of four) αβ2+/− fetuses, and in one (out ofthree) α1β2 18.5 dpc fetuses (Table 1). In one αγ fetus thepulmonary trunk and ascending aorta were septated distally,but undivided proximally; with this exception, however, therewas no evidence of partitioning in any part of PTA of αβ2,αβ2+/−, α1β2 and αγ mutants

. In all 18.5 dpc mutants with PTA (with the exception of the

one αγ mutant with PTA in which the ventricular septum wastotally absent, see above), the membranous portion of the ven-tricular septum (or pars membranacea) was incomplete,resulting in a high ventricular septal defects located (i) justbeneath the semilunar cusps of PTA and/or (ii) between theaortic vestibulum and the right ventricle (not shown). It is note-worthy that interventricular septal defects of the first typemight in fact correspond to the normally persisting interven-tricular foramen [i.e. FIV II in the terminology employed byVuillemin and Pexieder (1989)], which in the heart of WT miceforms the ostium of the aorta. In contrast, the communicationbetween the aortic vestibulum and right ventricle (i.e. FIV III;Vuillemin and Pexieder, 1989) is normally closed by 14.5-15.0dpc, thus its persistence in the 18.5 dpc double mutants ispathological. Other outflow abnormalities found in RARdouble mutants included four cases of dextroposition of theascending aorta combined with high ventricular septal defectand a case of double outlet right ventricle (Table 1). In dex-

Fig. 3. Comparison of the outflow tract of the heart, myocardium andaortic arches between 18.5 dpc (a,c,f,j) wild-type (WT) and (b,d,e,g-i,k-o;) RAR double mutant fetuses. (a,b,e,m,n) Frontal sectionsthrough the heart; (m,n) two aspects of the same heart: thehistological section in n is in a plane slightly ventral to that of m.(c,d) Transverse sections of the outflow tract at the level of thesemilunar cusps. (f,g) Aspects of the ventricular myocardium.(h,i) Frontal sections through the posterior mediastinum. (j-l) Frontalsections through the middle mediastinum. (o) Whole mount of theheart and arch of the aorta. Abbreviations: AA, arch of the aorta;rAA, right arch of the aorta; AP, aorticopulmonary septum; AS,ascending aorta; AS: 1-3, aortic semilunar cusps; fAT, left atrium;rAT, right atrium; AT, atrium; fC, left common carotid artery; rC,right common carotid artery; CAA, cervical arch of the aorta; CMY,compact layer of ventricular wall; TMY, trabecular layer ofventricular wall; D, ductus arteriosus; E, epicardium; fL, left lung; rL,right lung; fE, left external carotid; fi, left internal carotid artery; O,oesophagus; fOS, left retroesophageal subclavian artery; rOS,retroesophageal subclavian artery; PT, pulmonary trunk; fP, leftpulmonary artery; rP, right pulmonary artery; PT: 1-3, pulmonarysemilunar cusps; fS, left subclavian artery; rS, right subclavian artery;T, trachea; TA, truncus arteriosus; TA: 1-4, semilunar cusps of thetruncus arteriosus; TH, thymus; V, ventricle; fV, left ventricle; rV,right ventricle; fVC, left superior vena cava; rVC, right superior venacava; VE, vertebral column; VS, ventricular septum. Magnifications :×14 (a,b,e,h,i) ; ×34 (c,d) ; ×85 (f,g) ; ×68 (j-l) ; ×16 (m,n).

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troposed aorta (DA), this vessel arose partially from the rightventricle and thus straddled the ventricular septum (notshown). In double outlet right ventricle (DORV), the ascendingaorta (AS, Fig. 3m) and the pulmonary trunk (PT, Fig. 3m,n)arose separately from the right ventricle (rV, Fig. 3m,n). DA

and DORV were observed in α1β2, αβ2+/− and α1γα2+/−

fetuses. In addition, one αβ2 and one αγ mutant showed a per-sistent atrioventricular canal, possibly resulting from thefailure of the embryonic canal to divide into a right and a leftorifice (not shown). With these two exceptions, inflow abnor-


Fig. 4. Comparison of the aortic arches, and outflow tract of the heart between 11.5 dpc wild-type (a,c,e) and an α−/−/β2−/− embryos (b,d,f) andthymus (TH) and thyroid (TY) ectopias in 18.5 dpc mutant fetuses (g-i). (a,b; c,d; and e,f) Transverse sections at comparable levels of thedorsal aortic roots (fAR and rAR) and conotruncus (CT). (g-i) Frontal section through the pharynx (PH). Abbreviations: AP, aorticopulmonaryseptum; fAR, left aortic root; rAR, right aortic root; AS, ascending aorta; AT, atrium; BC, base of the skull; CR, conotruncal ridges; CT,conotruncus; HY, hyoid bone; O, oesophagus; OT, oesophagotrachea; OTS, oesophagotracheal septum; rP; right pulmonary artery; PH,pharynx; PT, pulmonary trunk; SC, spinal cord; T, trachea; TA, truncus arteriosus; TH, thymus; TH*, aberrant lymphoid tissue; TY, thyroidgland; fV, left ventricle; VE, vertebral column; f6, left 6th aortic arch; r6, right 6th aortic arch. Magnifications: ×74 (a-f,i) ; ×30 (g,h).


malities were not detected. PTA and heart abnormalities werenot observed in α1γ and β2γ fetuses (Table 1).

As expected from the analysis of 18.5 dpc mutants (seeabove), no sign of aorticopulmonary septation was observed ina majority (five out of six) of 11.5 dpc αβ2 and αγ embryos(compare AP, Fig. 4c with d). However, the 2 conotruncalridges always appeared to be normally developed in all of thesemutants (CR, compare Fig. 4e and f). These ridges are locatedin the ventriculo-arterial (conotruncal) region of the heart wherethey form upon fusion (by 14.5-15 dpc), the membranousportion of the ventricular septum, dividing the conotruncallumen into a pulmonary and an aortic vestibulum. Although theconotruncal ridges are continuous with the aorticopulmonaryseptum, they are not derived from NCC (Noden, 1991). Theseobservations support the conclusions that in mutants: (i)amongst the structures involved in septation of the outflow tractof the heart, only those that are derived from NCC are affected,and (ii) that the high ventricular septal defects seen at 18.5 dpcare most probably occurring secondarily to abnormal aorti-copulmonary septation in order to compensate for changes inblood flow. This view is supported by the constant associationof PTA, DA or DORV with ventricular septal defects in humanpatients (Gray and Skandalakis, 1972; Taussig, 1960).

(E) Abnormalities of the aortic arch-derived greatvesselsAortic arches are 5 paired arteries, which develop within themesectoderm of pharyngeal arches 1, 2, 3, 4 and 6 and transmitblood from the aortic sac to the dorsal aorta in the earlyembryo. The aortic arch pattern, which is symmetrical up to11.5 dpc (Kaufman, 1992; Fig. 5a), is subsequently modifieddue to the involution or persistence of specific aortic archesand segments of the dorsal aorta, thus becoming highly asym-metrical (Fig. 5b). In WT 18.5 dpc mice, only 2 of the aorticarches remain as connecting channels between the heart andthe descending aorta: the arch of the aorta (AA, Fig. 3j, seealso Fig. 5b) and the ductus arteriosus (D, Fig. 3j, see also Fig.

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Fig. 5. Diagrammatic representations of the normal embryonic (i.e.9.5-11.5 dpc) pattern of aortic arches (a), of the normal 18.5 dpcpattern of aortic arch (b) and of some abnormal patterns observed in18.5 dpc RAR double mutant fetuses (c-l). Note that duringdevelopment the 5 pairs of aortic arches are never presentsimultaneously, but they are represented in (a) in cumulativearrangement in order to emphasize their positional relationships. Theparts of the embryonic system which have regressed are representedby broken lines in b-l. Abbreviations: L. ao. root, left aortic root; R.ao. root, right aortic root; ao. sac, aortic sac; arch ao., arch of theaorta; L. arch ao, left arch of the aorta; R. arch ao, right arch of theaorta; asc. ao, ascending aorta; L. car. duct, left carotid duct; L. ceph.ao., left cephalic aorta; R. cerv. arch. ao, right cervical arch of theaorta; L. cerv. arch ao, left cervical arch of the aorta; L. com. car, leftcommon carotid artery; desc. ao., descending aorta; ductus art.,ductus arteriosus; ext. car., external carotid artery; L. ext. car, leftexternal artery; R. ext. car, right external carotid artery; innom,innominate artery; L. int. car, left internal carotid artery; R. int. car,right internal carotid artery; L. pulm. art, left pulmonary artery; R.pulm, right pulmonary artery; pulm. trunk, pulmonary trunk; L.retroes. subcl, left retroesophagal subclavian artery; R. retroes. subcl,right retroesophagal subclavian artery; L. subcl, left subclavianartery; R. subcl, right subclavian artery; truncus art, truncusarteriosus; L. 7th art., left 7th segmental artery; R. 7th art., right 7thsegmental artery.

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5b). The arch of the aorta, or systemic arch, which is formedfrom the left 4th aortic arch and from the left dorsal aortic root(the proximal part of the descending aorta; Barry 1951; Fig.5b), connects the left ventricle with the descending aorta. Theductus arteriosus which is formed from the distal portion of theleft 6th aortic arch, connects the right ventricle with the archof the aorta. (Barry, 1951; Fig. 5b). Aortic arch abnormalitiesare thought to result from the persistence of aortic archesand/or segments of the dorsal aorta that should normallyundergo involution and/or from disappearance of normally per-sisting channels (Wilson and Warkany, 1949; Gray and Skan-dalakis, 1972; Taussig, 1960).

(1) Aortic arch abnormalities in 18.5 dpc mutant fetusesAortic arch abnormalities were found in all 18.5 dpc doublemutants with the exception of α1γ and β2γ fetuses. Theyshowed great individual variations (Fig. 5; Table 2 in whichthe data are derived from observations made on serial histo-logical sections, and occasionally from whole mounts). For thesake of simplicity in the description, they were classifiedhereafter into 5 categories: (i) abnormalities characterized bythe persistence of the carotid duct, (ii) abnormalities charac-terized by persistence of the right aortic root, (iii) coarctation

of the aorta, (iv) aberrant origin of pulmonary arteries, (v) mal-formations of the aortic arches secondary to abnormal aorti-copulmonary septation.

(i) Abnormalities characterized by a persistence of thecarotid duct

A cervical arch of the aorta (CAA, Figs 1c and 3o; Gray andSkandalakis, 1972) was identified firstly by the proximity ofthe arch of the aorta to the larynx (see CAA, Fig. 1c) and,secondly, by the fact that the common carotid artery wasmissing on one side, thus resulting in the internal and externalcarotid arteries arising separately from the crest of the arch(f I and f E, Fig. 3o; Wilson and Warkany, 1949). This is likelycaused by a persistence of the carotid duct, i.e. the segment ofthe cephalic dorsal aorta located between the 3rd and 4th aorticarches (Fig. 5j,k) which should normally regress. Two cases ofcervical arch of the aorta (CAA) were observed, one on the leftside (Figs 5k, 1c, 3o), and the other on the right side (Fig. 5j).In both cases, the 4th aortic arches had failed to persist, bilat-erally. Both mutants with cervical arch of the aorta had addi-tional abnormalities, namely retroesophageal subclavianarteries (e.g. r OS in Figs 1c, 3h), PTA and agenesis of the leftpulmonary artery (Fig. 5j,k).


Table 2. Aortic arch and heart outflow tract abnormalities in 18.5 dpc RAR double mutantsRAR Aortic arch abnormalities (see Fig. 5) Associated double abnormalities of themutant 18.5 dpc Abnormal Abnormal outflow tract of thegenotype fetuses persistence involution Abnormality heart

α1β2 A (5i) R. ao. root, dist R6 L4, dist L6 R. 4th arch ao.; L. retroes. subcl. DAB (5e) R. ao. root L4 R. 4th arch ao.; Double asymmetrical arch ao. NDC (NI) dist L6 Isolated absence of ductus art. PTA

αβ2+/− A (5c) R. ao. root L. ao. root, dist. L6 R. 4th arch ao. PTAB (5b) None NormalC (5f) R. ao. root, dist R6 prox R6, prox L6 R. 4th arch ao.; Double symmetrical arch ao.; PTA

Aberrant origin of pulm. art.D (NI) None DA

αβ2 A (5d) R. ao. root L. ao. root, L6 R. 4th arch ao.; Absence of L. pulm. art. PTAB (5h) R. ao. root L6, R4

R. retroes. subcl.; Absence of L. pulm. art PTAC (5h) R. ao. root L6, R4D (5j) R. car. duct, R. ao. root L4, R4, L6 R. cerv. arch ao.; L. retroes. subcl.; Absence of PTA

L. pulm. artE (5h) R. ao. root L6, R4 R. retroes. subcl.; Absence of L. pulm. art. PTAF (NI) L6 Isolated absence of L. pulm. art. and ductus art. PTAG (5k) L. car. duct, R. ao. root L4, R4, L6 L. cerv. arch ao.; R. retroes. subcl.; Absence of PTA

L. pulm. art.α1γα2+/− A (5g*) R. ao. root R4 R. retroes. subcl. ND

B (NI) none L4 (partial) Coarctation of aorta DORVC (5g) R. ao. root R4 R. retroes. subcl.; Aberrant origin of R. pulm. art. DAD (5b) None NormalE (NI) None DA

αγ A (5l) R. ao. root L. ao. root, prox L6 R. 4th arch ao.; Aberrant origin of L. pulm. art. PTAB (5c) R. ao. root L. ao. root, dist L6 R. 4th arch ao. PTAC (NI) R. ao. root R4, dist L6 R. retroes. subcl. PTAD (5l) R. ao. root L. ao. root, prox L6 R. 4th arch ao.; Aberrant origin of L. pulm. art. PTAE (NI) R. ao. root L4 ; dist L6 R. 4th arch ao.; L. retroes. subcl. PTA

For a given genotype, A-G designate different fetuses; the relevant schematic representation in Fig. 5 is given in parentheses (5a-l panels)*; similar to Fig. 5g,but with the right pulmonary artery arising normally from the pulmonary trunk. NI, not illustrated; ND, not analysed (these mutants had no PTA, however adetailed analysis of the outflow tract was not possible due to an inappropriate plane of sectioning); R4 and L4, right and left 4th aortic arches, respectively; R. ao.root and L. ao. root, right and left aortic roots, respectively; R. car.duct. and L. car. duct, right and left carotic ducts, respectively; dist R6, distal portion of theright 6th aortic arch; L6, left 6th aortic arch which gives rise to the proximal part of left pulmonary artery and to the ductus arteriosus; prox L6, proximal portionof the left 6th arch (proximal part of the left pulmonary artery); dist L6, distal portion of the left 6th arch (ductus arteriosus); R. 4th arch ao., right 4th arch of theaorta; R. and L. retroes. subcl., right and left retroesophageal subclavian artery, respectively; R. and L. cerv. arch ao., right and left cervical arch of the aorta,respectively; DA, dextroposed aorta; PTA, persistent truncus arteriosus; DORV, double outlet right ventricle.


(ii) Abnormalities characterized by a persistence ofthe right aortic root

These abnormalities, by far the most frequent (Fig. 5; Table 2),can be subdivided into right 4th arch of the aorta (Fig. 5c-f,i,l)and right retroesophageal subclavian arteries (Fig. 5g,h). In thesimplest case of right 4th arch of the aorta, the left arch of theaorta was absent and replaced by the corresponding vessel onthe right side (rAA, Figs 1b, 3i; 5c,d). The arrangement ofmajor arteries derived from the right 4th arch of the aorta (i.e.subclavians, carotids) exhibited a mirror image of the normalpattern. In addition to the right sided arch of the aorta, the twomutants shown in Fig. 5c,d exhibited a PTA and one had anagenesis of the left pulmonary artery (Fig. 5d). A third variantis illustrated in Fig. 5e: as in the two preceding examples, theright dorsal aortic root failed to regress, and the abnormal right-sided arch of the aorta gave rise to the left and right commoncarotid arteries. Here, however, the left dorsal aortic root (i.e.the normal vessel) persisted, giving rise (as normally) to theleft subclavian artery, and connecting the heart with thedescending dorsal aorta via the pulmonary trunk and the leftductus arteriosus (i.e. the normal distal portion of the 6th arch).This is one of the many possible forms of double arch of theaorta (Wilson and Warkany, 1949; Gray and Skandalakis,1972; Taussig, 1960). In the last variant of this group (Fig. 5f),both the left and the right 4th aortic arches and aortic roots hadpersisted and formed a symmetrical double arch of the aorta;each component of the double arch gave rise to a commoncarotid and to a subclavian artery (Fig. 5f); additional defectsincluded a PTA, and the left and right pulmonary arteries aroseaberrantly from this double arch of the aorta (see below).

In normal embryos, the left subclavian artery arises entirelyfrom the 7th left segmental artery which is connected to theleft dorsal aortic root. In contrast, the proximal portion of theright subclavian artery is formed in part from the right 4thaortic arch, whereas its distal portion is formed from right 7thsegmental artery (Barry, 1951; Fig. 5a,b). The right subclavianartery normally arises from the innominate (brachiocephalic)artery (Fig. 5b). In the simplest case of right retroesophagealsubclavian artery, this vessel arose as the most distal ratherthan as the most proximal branch from an otherwise normalleft arch of the aorta (Fig. 5g,h). The subclavian artery (r OS,

Fig. 3h) passed to the right behind the oesophagus (O, Fig. 3h)and in front of the vertebral column (VE, Fig. 3h) to reach itsnormal destination, namely the right forelimb. This abnormal-ity is probably caused by the abnormal regression of the right4th aortic arch and the persistence of the right aortic root whichnormally disappears (Table 2). In the variant shown in Fig. 5g,the aortic arch pattern is otherwise normal, whereas that shownin Fig. 5h has additional malformations, namely PTA andagenesis of the left pulmonary artery. Note that a mirror-imageabnormality (i.e. retroesophageal left subclavian) existed insome cases of persistent right 4th arch of the aorta (f OS, Fig.3i; see also Fig. 5i). Right and left retroesophageal subclavianarteries were also observed in mutants with cervical arch of theaorta (see above and Fig. 5j,k).

(iii) Coarctation of the aortaIn the one mutant with coarctation of the aorta (Table 2), thearch of the aorta between the ductus arteriosus and the leftcommon carotid artery was markedly narrowed, possibly as aresult of an abnormal incomplete regression of the left 4thaortic arch (compare AA, Fig. 3j and k; Barry, 1951).

(iv) Agenesis and aberrant origin of pulmonaryarteries

A case of aberrant origin of left and right pulmonary arteriesfrom their respective ipsilateral arches of the aorta between theorigins of the subclavian and carotid arteries is illustrated inFig. 5f. This is likely due to the abnormal regression of theproximal segments of the left and right 6th aortic arches andabnormal persistence of the distal segment of the right 6thaortic arch. An aberrant origin of the left pulmonary arteryfrom the subclavian artery (Fig. 5l) represents a variant of thepreceeding condition.

Agenesis of the left pulmonary artery was always observedin αβ2 and αβ2+/− fetuses with agenic left lungs (Fig. 5d,h,j,k).In this case, the absence of the left pulmonary artery is likelyto be secondary to lung agenesis.

(v) Some malformations of the aortic arch patternlikely to be secondary to abnormal or aberrantaorticopulmonary septation

An aberrant origin of the right pulmonary artery from the

Table 3. Thymus, thyroid and parathyroid glands abnormalities RAR mutant genotype and number of 18.5 dpc fetuses examined

α1β2 αβ2+/− αβ2 α1γ α1γα2+/− αγ β2γ3 4 7 5 4 5 3 VAD

Thymus abnormalities- Small, single lobed, median thymus 0 0 1/7 0 0 0 0 NR- Persistent cervical thymusa 0 0 0 0 0 5/5 0 NR- Accessory (cervical) thymus bodiesb 1/3 0 5/7 0 1/4 NA 0 NR- Aberrant pharyngeal lymphoid tissuec 1/3 1/4 2/7 0 0 1/5 1/3 NR

Thyroid abnormalities- Small thyroid 1/3 0 2/7 0 1/4 1/5 0 NR- Ectopic thyroidd 0 0 1/7 0 0 3/5 0 NR

Ectopic parathyroidse + 0 + 0 + + 0 NR

aThymus found bilaterally in the neck; no trace of thymic tissue in the anterior mediastinum; bthe two lobes of the thymus are in their normal location, butaccessory thymus bodies are also present in the neck region; cthis condition may represent a form of ectopic accessory thymus (Gray and Skandalaky, 1972);dectopic thyroid tissue was found either in contact with the hyoid bone (i.e. rostral to its normal location) or (in part) within the anterior mediastinum (i.e. caudalto its normal location); eno numbers are given, since ectopic parathyroid glands are sometimes difficult to identify; NA, not applicable; NR, not reported.

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ascending aorta was observed in one mutant (rP, Fig. 3l; Fig.5g), whereas the left pulmonary artery arose from thepulmonary trunk, as usual. The developmental fault underly-ing this condition must have been improper alignment of theaorticopulmonary septum with respect to the 6th archprimordia (Wilson and Warkany, 1949; Gray and Skandalakis,1972).

The ductus arteriosus was absent in all of the mutants witha PTA (see examples in Fig. 5c,d,j,k). Absence of the ductusarteriosus is almost invariably associated with PTA in mice andhumans (Franz, 1989; Gray and Skandalakis, 1972). It iscurrently assumed that absence of significant blood flowthrough the ductus arteriosus due to the presence of a muchlarger aorticopulmonary communication permits the distalportion of the left 6th aortic arch (which normally regressesonly in the immediate postpartum) to undergo involutionearlier in fetal life (Gray and Skandalakis, 1972; Taussig,1960). However, the possibility that alteration of the hemody-namic pattern caused by an outflow tract abnormality such asPTA might explain all of the other aortic arch abnormalitiesappears unlikely, since it is clear from the present data thatthese two types of malformations can occur independently.Both types of malformations are also known to exist indepen-dently in rats (Wilson and Warkany, 1949) and humans (Grayand Skandalakis, 1972).

(2) Early abnormalities of the aortic arch patternThe most frequent aortic arch abnormalities found in 18.5 dpcmutants were associated with abnormal persistence of the rightdorsal aortic root and abnormal regression of the left 6th arch(Table 2). In WT embryos at 11.5 dpc, i.e. when the aortic archsystem is still bilaterally symmetrical and prior to completeaorticopulmonary septation, the left and right aortic rootsreceive blood from the heart via the ipsilateral 3rd, 4th and 6tharches (Kaufman, 1992). In mutant embryos, the 3rd and 4thaortic arches were bilaterally present in all 11.5 dpc αβ2 (four),11.5 dpc αγ (two) and 10.5 dpc α1β2 (two) mutants examined.However, in three of these embryos, the calibres of both theleft 4th arch and of the ipsilateral aortic root (which togetherrepresent the normal anlage of the arch of the aorta) weremarkedly reduced compared to the same structures on the rightside (compare fAR and rAR, Fig. 4a with b). The left and right6th aortic arches were barely visible in 11.5 dpc mutantsexamined (r6 and f6, compare Fig. 4c with d), and their distalportions (on the left side, the future ductus arteriosus) werenever identified in αγ and αβ2 mutants at later developmentalstages (i.e. from 12.5 to 18.5 dpc, data not shown). Lastly, inone 10.5 dpc α1β2 mutant, the calibres of both the right 4tharch and ipsilateral aortic root were reduced compared to theirleft homologues (not shown).

Taken together, these data clearly indicate that at least someof the aortic arch abnormalities observed in 18.5 dpc fetusesare determined before the stage at which specific archesnormally start to regress. Since the 6th aortic arches becomefirst visible at a stage when the septation of the aortic sac isalready underway, it is not possible to assess whether theirsmall size in the 11.5 dpc mutants corresponds to a primaryeffect of the mutations or is secondary to PTA. In any event,our findings indicate that, in mutant embryos with PTA, theductus arteriosus might disappear early in development, before12.5 dpc.

(F) Abnormalities of the thyroid, parathyroid andthymus glandsRhombencephalic NCC together with the endoderm of theprimitive pharynx give rise to the thyroid, parathyroid andthymus glands (Le Douarin, 1981). These gland anlagen sub-sequently ‘migrate’ caudally to their definitive locations. In18.5 dpc WT fetuses, the thyroid gland is seen in close contactwith the lower part of the larynx and the first three cartilagi-nous tracheal ‘rings’. The parathyroid glands are embedded inthe posterolateral part of each lobe of the thyroid gland. Thetwo lobes of the thymus are located beneath the sternum,within the superior part of the anterior mediastinum. Mostmutants exhibited at least one form of glandular ectopia (Table3), including cervical thymus (TH, Fig. 4g), thyroid beneaththe hyoid bone (TY, Fig. 4g and h), and rostrally displacedectopic parathyroids not associated with the thyroid gland (notshown). In addition, aberrantly located lymphoid tissue wasfound in the larynx of some double mutant fetuses (Table 3;TH*, Fig. 4i), resembling in this respect the lymphoid nodulesthat have been proposed to correspond to ectopic accessorythymus glands in humans (Gray and Skandalakis, 1972).

(G) Abnormalities of the urinary systemAbnormalities of the kidneys and ureters were found in α1β2,αβ2+/−, αβ2, α1γα2+/− and αγ mutants at 18.5 dpc (Table 4).Renal abnormalities fell into 3 broad categories: renal agenesis,aplasia and hypoplasia (Gray and Skandalakis, 1972). Renalagenesis and renal aplasia were observed specifically and con-sistently in αγ mutants, and these two abnormalities eventuallycoexisted in the same 18.5 dpc fetus: from a total of fivefetuses, two fetuses had bilateral agenesis, two exhibitedbilateral aplasia, and the last one showed agenesis on one sideand contralateral aplasia (Table 4, and data not shown). Inagenesis, no trace of renal tissue existed. This abnormality wasalways associated with agenesis of the ipsilateral ureter. Inrenal aplasia, small retroperitoneal masses of tissue containingrandomly dispersed kidney tubules and glomeruli were foundcaudal to the normal location of the kidney. The renal pelviswas always absent. The ureters were absent or atretic, i.e. hada markedly reduced caliber and lacked the caudal portion. Inaddition, the urinary bladder was absent in one 18.5 dpc αγmutant fetus.

Embryos were examined to investigate the possible originof renal agenesis in 18.5 dpc αγ mutants. In 11.5 dpc WTembryos, the epithelial ureteric bud (U, Fig. 6c), which arisesfrom the caudal part of mesonephric duct (Wolffian duct), hadcome into contact with the condensed metanephric mes-enchyme (N, Fig. 6c). In the two 11.5 dpc αγ mutants, theureteric bud was absent on one or both sides, and when presentfailed to reach the metanephric blastema (U, compare Fig. 6cand d). At this time, the nephric mesenchyme was bilaterallypresent (N, Fig. 6c,d) and showed no evidence of increasedapoptosis (see Kreidberg et al., 1993). The parenchyma of 12.5dpc WT kidneys contain numerous tubules (arrowheads in Fig.6e). Analysis of a 12.5 dpc αγ mutants revealed that the nephricmesenchyme was bilaterally present (N, Fig. 6f), but showedno signs of histological differentiation. In addition, a largenumber of pycnotic nuclei were present (not visible at the mag-nification of Fig. 6f). Interestingly, this mutant had no ureters(compare U, Fig. 6e and f). In one 14.5 dpc αγ fetus (not



shown), a rudimentary kidney was present on one side, whileon the other side there was no trace of nephric tissue and noureter.

In renal hypoplasia, the renal parenchyma at 18.5 dpc,although conspicuously smaller than normal (compare K, Fig.6a and b, and Fig. 6g and h), was often subdivided into corticaland medullary regions (CO and ME, compare Fig. 7e and f).This abnormality was found bilaterally in all αβ2 mutants andin a majority of cases (seven out of ten), it was associated withdilation of the renal pelvis (hydronephrosis; compare PL inFig. 6g and h) and of the ureter (hydroureter; e.g., compare fUin Fig. 6i and j), indicating that some renal excretory functionmust have occurred. The hypoplastic kidneys were oftenectopic, i.e. located in the lower lumbar or sacral regionsinstead of the normal upper lumbar site (compare K, in Fig. 6aand b). At the histological level, the cortical region of thehypoplastic kidneys contained abnormally large glomeruli(GL) and convoluted tubules (TU) (compare Fig. 7e and f);moreover, this cortical region always lacked the peripheralnephrogenic zone (Z, compare Fig. 7e and f), where nephronscontinue to be produced after birth. Bilateral renal hypoplasiawas also conspicuous in three (out of four) αβ2+/−, two (out ofthree) α1β2, and two (out of five) α1γα2+/− mutants, at 18.5dpc but was always less severe than in the αβ2 mutants (Table4).

Agenesis of caudal ureters and ectopic ureteral openingsconstitute anatomical and functional obstructions respectively,resulting in the accumulation of urine in the urinary tract,leading to hydronephrosis and hydroureter. All αβ2 mutants(seven out of ten, see Table 4) exhibiting hydronephrosis andhydroureter displayed abnormalities of caudal ureter. Ectopi-cally terminating ureters (e.g. f U, Fig. 7b) opened into the

urethra instead of into the base of the urinary bladder, theirnormal termination site. Agenesis of caudal ureter was char-acterized by a failure of one or both ureters to join any part ofthe lower genito-urinary tract (compare rU in Fig. 6i and j).Hydronephrosis and hydroureter with an otherwise normalgenito-urinary tract was observed in one β2γ mutant.

(H) Abnormalities of the genital tract and lowerdigestive tractMale genital ducts develop mainly from mesodermal epithe-lium of the Wolffian (or mesonephric) duct (epidydimis, vasdeferens and seminal vesicles) and from endoderm of the uro-genital sinus (e.g. prostate). Abnormalities of the genital tractwere observed only in the two αγ, and in one (out of two)α1γα2+/− 18.5 dpc mutant males examined (see Table 4).Bilateral agenesis of epidydimis, vas deferens and seminalvesicles was found in one αγ mutant male and was associatedwith bilateral renal agenesis. Bilateral dysplasia of epidydimisand vas deferens was observed in one αγ and in one α1γα2+/−

male: these two abnormal structures displayed a succession ofdilated and stenotic portions. Moreover, in both mutants, thevas deferens stopped at a short distance from its normalopening in the urethra, whereas the seminal vesicles appearednormal (α1γα2+/− mutant) or were missing (αγ mutant) (Table4, and data not shown). However, prostate buds were presentin these males. Interestingly, the caudal-most Wolffian ductwas always lacking in αγ mutants examined at 11.5 dpc (two),12.5 dpc (one) and 14.5 dpc (one) (W, compare Fig. 6e and f).In αβ2+/−, αβ2, α1γ and β2γ mutant males, the genital tractwas apparently normal. The testes appeared histologicallynormal in all mutants (data not shown).

The female genital ducts develop from mesodermal epithe-

Table 4. Uro-genital tract abnormalities in 18.5 dpc RAR double mutantsRAR mutant genotype and number of 18.5 dpc fetuses examined

(males : females)

α1β2 αβ2+/− αβ2 α1γ α1γα2+/− αγ β2γ1 : 2 4 : 0 4 : 6 2 : 3 2 : 3 2 : 3 1 : 2 VAD

Kidney abnormalitiesRenal agenesis (uni- or bilateral) 0 0 0 0 0 3/5 0 NRRenal aplasia (uni- or bilateral) 0 0 0 0 0 3/5 0 NRRenal hypoplasia (bilateral) 2/3 3/4 10/10 0 2/5 0 0 NRHydronephrosis 2/3 0 7/10 0 0 0 1/3 NR

Ureter abnormalitiesAgenesis (partial or total, uni- or bilateral) 2/3 0 6/10 0 0 5/5 0 +Ectopia (uni- or bilateral) 2/3 0 5/10 0 0 0 0 +

Absence of anal canal 0 0 5/7 0 0 0 0 NR

Abnormalities of the genital tractFemale

Agenesis of the uterustotal 0 NA 6/6 0 0 0 0 +partial (body)* 2/2 NA 0 0 0 3/3 0 +

Agenesis of the cranial vagina 2/2 NA 6/6 0 0 3/3 0 +Male

Bilateral agenesis or dysplasia of the vas deferens 0 0 0 0 1/2 2/2 0 +Bilateral agenesis of seminal vesicles 0 0 0 0 0 2/2 0 +

Pseudohermaphroditism 0 0 0 0 0 0 0 +

Agenesis of urinary bladder 0 0 0 0 0 1/5 0 NR

*The body of the uterus was lacking, but the uterine horns were uni- or bilaterally present.NA, not applicable; NR, not reported.

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lium of the Müllerian (or paramesonephric) duct and fromendodermal epithelium of a dorsal outgrowth of the urogenitalsinus, the vaginal plate (for a review see Thiedemann, 1987).In 18.5 dpc WT females, the separate cranial Müllerian ductsrepresent the anlage of the oviduct (Fallopian tube) and uterinehorns; the fused caudal Müllerian ducts (M, Fig. 6i) representthe anlage of the body of the uterus (see Fig. 6i) and of thecranial part of the vagina (V, Fig. 7a; Thiedemann, 1987 andrefs therein). In 18.5 dpc αβ2 female mutants, all the deriva-tives of the Müllerian duct were always lacking (compare Min Fig. 6i and j, and V in Fig. 7a and b; Table 4). The two α1β2and the three αγ female mutants exhibited a related genital tractphenotype: they always lacked the cranial vagina and body ofthe uterus. However, the oviduct/uterine horn portion of theMüllerian ducts was always present at least on one side (Table4). In the remaining female mutants, the genital tract wasapparently normal. The vaginal plate, which represents theanlage of the caudal vagina (Thiedemann, 1987 and refstherein), was present in all double mutants. The histologicalstructure of ovaries in double mutants was always normal.

The genital tract of αβ2 mutant embryos was analyzed inorder to investigate the possible origin of these female genitalduct abnormalities. Müllerian ducts were absent in the two 12.5dpc and in the two 13.5 dpc αβ2 mutants examined (M,compare Fig. 7c and d). In contrast, only the caudal Müllerianducts only, appeared to be lacking in α1β2 and αγ mutants(data not shown). Note that male and female Müllerian ductsare identical between 12.5 and 13.5 dpc in WT embryos(Dyche, 1979).

Based on the findings that experimental interruption ofWollffian ducts arrests the progress of Müllerian ducts(Gruenwald, 1941; Didier, 1971 and refs therein), it has beensuggested that the formation of Müllerian ducts is dependenton Wolffian ducts. In five αβ2 female mutants examined at18.5 dpc, a tract of mesenchymal tissue was observed alongthe entire length of the putative genital ducts (compare GT,Fig. 6i with 6j), and normal vestiges of Wolffian ducts (i.e.epoophoron cranially, and Gartner’s cyst caudally) were foundat both extremities of this tract of mesenchymal tissue (e.g.compare GC in Fig. 7a and b). Taken together these observa-tions indicate that the Wolffian ducts (i) were initially formed(in order to account for the presence of Gartner’s cyst and theepoophoron), and (ii) underwent normal regression. In thesame context, it is noteworthy that the male genital tractappeared normal in all four 18.5 dpc αβ2 mutants, and the

Wolffian ducts were complete in all αβ2 mutants examined at12.5 dpc (two) and 13.5 dpc (two).

Agenesis of the anal canal was observed only in (five out ofseven) 18.5 dpc αβ2 mutants; the terminal end of the rectumwas separated from the skin by a thick layer of mesenchyme(not shown).

DISCUSSION

(A) RA is required for several steps inmorphogenesis of foregut derivativesThe endoderm of the primitive thoracic foregut and the asso-ciated lateral mesoderm represent the putative anlagen of thelarynx, pharynx, oesophagus, tracheobronchial tree and lungparenchyma. Some abnormalities of thoracic foregut deriva-tives found in RAR double mutants can be ascribed to devel-opmental arrests (i.e. agenesis of the left lung and/or of theoesophagotracheal septum), growth retardation (hypoplasia ofthe left and right lung) or impaired cellular differentiation(ciliated metaplasia of the stratified squamous oesophagealepithelium). However, the malformations of the laryngeal car-tilages and tracheal rings have no counterparts during normalembryogenesis.

Left lung agenesis, hypoplasia of the left and/or right lungsand agenesis of the oesophagotracheal septum, which wereconstantly associated in αβ2 double mutant fetuses, have beenreported in VAD rat fetuses (Warkany et al., 1948; Wilson et


Table 5. Aortic arch abnormalities in RAR double null mutants and vitamin A-deficient fetusesAortic arch abnormalities RAR mutants (Fig. 5) VAD

Right cervical arch of the aorta 5j +Left cervical arch of the aorta 5k +Right 4th arch of the aorta 5c,d,e, f, i, l +Right retroesophageal subclavian artery 5,g,h,k +Left retroesophageal subclavian artery 5i,j NRDouble aortic arch symmetrical and asymmetrical 5e,f +Absence of left pulmonary artery 5d,h,j,k +Aberrant origin of pulmonary arteries from ipsilateral arch of the aorta 5f NRAberrant origin of left pulmonary artery from the left subclavian artery 5l NRAberrant origin of right pulmonary artery from the ascending aorta 5g +Coarctation of the aorta NI NR

NI, not illustrated; NR, not reported.

Fig. 6. Comparison of the kidney and genito-urinary tract between(a,c,e,g,i) wild-type (WT) and (b,d,f,h,j) mutant embryos and fetuses;(a,b) Bouin-fixed whole mounts of 18.5 dpc males ; note that thesmall kidney in the mutant (K in b) is also ectopic, i.e. it liesabnormally close to the testis (T) and away from the adrenal gland(AD). (c,d) Transverse sections through the 11.5 dpc nephricblastema (N). (e,f) Transverse sections through the 12.5 dpc nephricblastema (N). (g,h) Transverse sections through the 18.5 dpc kidneys(K). (i-j) Transverse sections through the pelvic cavity of 18.5 dpcfemales. Abbreviations: AD, adrenal gland; DA, dorsal aorta; G, gut;GT, genital tract; IN, intestines; K, kidney; M, Müllerian duct; N,nephric mesenchyme; OB, umbilical artery; PL, renal pelvis; PR,peritoneal cavity; R, rectum; T, testis; U, ureteric bud; UB, urinarybladder; UG, urogenital sinus; fU, left ureter; rU, right ureter; VE,vertebral column; W, Wolffian duct. Arrowheads denote renal tubuli.Magnifications : ×75 (c,d,i,j) ; ×45 (e,f); ×19 (g,h); a and b are shownat the same magnification.

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al., 1953). Left and right lung defects are determined at twostages, through alterations of distinct RA-dependent processes.Analysis of 11.5 and 12.5 dpc αβ2 mutants indicates that leftlung agenesis and hypoplasia of both lungs result from theabsence of left lung budding and altered bronchial branching,respectively. Both processes are dependent on interactionsbetween the foregut endoderm and its surrounding mesoderm(Spooner and Wessels, 1970). However, they are initiated at

two stages (i.e. 10.0 dpc for lung budding and 11.0 dpc forbronchial branching) and exhibit different mesodermalinductive requirements: in vitro, lung budding can be triggeredby a variety of embryonic mesenchymes, whereas bronchialbranching is strictly dependent on bronchial mesenchyme(Spooner and Wessels, 1970; Schuger et al., 1993). Further-more, vitamin A must be administrated to VAD rat embryosbefore the equivalent of 9.5 dpc in the mouse to prevent lung


Fig. 7. Comparison of the kidney and of the genito-urinary tract between (a,c,e) wild-type (WT) and (b,d,f) αβ2 fetuses at 18.5 dpc (a,b,e,f) andat 13.5 dpc (c,d). (a,b) Sections at similar levels of the urethra (UT) and rectum (R). Note that the mutant left ureter (fU) was found to terminatein the urethra at a short distance caudal to this plane of section. (c,d) Sections at similar levels of the gonads (GO). Abbreviations: CO, corticalregion of the kidney; GC, Gartner’s cyst; GL, glomeruli; GO, gonad; M, Müllerian duct; ME, medullary region of the kidney; PL, renal pelvis;R, rectum; TU, convoluted tubules; UT, urethra; fU, left ureter; V, cranial vagina; W, Wolffian duct; Z, nephrogenic zone. The large arrow in epoints towards the renal pelvis. Magnifications: ×79 (a,b); ×158 (c-f).


agenesis, whereas lung hypoplasia can be prevented by vitaminA administration at 10.5 dpc, i.e. after the onset of lungbudding (Wilson et al., 1953).

In WT embryos, lung budding and formation of theoesophagotracheal septum occur almost concomitantly. BothRARα and RARβ2, but apparently not RARγ, play a role atearly stages of respiratory tract development. Agenesis of theoesophagotracheal septum was associated with normal lungsin one RARα1β2 mutant, supporting the conclusion that lungbudding and the formation of the oesophagotracheal septumalso represent distinct RA-dependent events. However, dis-ruption of the second RARβ2 isoform allele in a RARαβ2+/−

background is sufficient to generate agenesis of the left lungand lack of formation of the oesophagotracheal septum(compare RARαβ2+/− and αβ2 double mutants in Table 1).Consistent with a distinct role of RARβ2, in situ hybridizationresults show that RARβ2, but not RARγ, is expressed in theforegut endoderm (Ruberte et al., 1991; Dollé et al., 1990 ourunpublished results). Interestingly, RARβ2 has also beenshown to be expressed at early stages in the foregut endodermof chick embryos (Smith, 1994). In the mouse, RARβ2expression, which begins at 9.5 dpc, i.e. prior to lung budformation, continues in tracheal and lung bud epithelia.However, by 11-12 dpc, RARβ2 is not detected in the epithe-lium of the most distal branching bronchi, nor in the closelyassociated mesenchyme, but its expression remains in theepithelium of more proximal bronchi and in peripheral lungmesenchyme (our unpublished results). Thus, during lungbranching RARβ2 may act to generate a mesenchymal signalacting at a distance on bronchial branching. In any event, it isclear that RARγ, which is weakly expressed in lung mes-enchyme (Dollé et al., 1990; our unpublished data), cannotreplace the function of RARβ2 during branching morphogen-esis.

During late fetal life and early postpartum (15.0 dpc to day4), the oesophageal lumen is lined with non-ciliated andciliated cells, the former being much more abundant. At 16.0dpc and later, most of the non-ciliated cells are of the squamous(i.e. adult) type (Calvert et al., 1991). In all 18.5 dpc αβ2 andin one α1β2 fetuses, the differentiation into squamous cellswas impaired, since the entire oesophageal lumen and thecardiac and proventricular portions of the stomach were linedexclusively with ciliated cells, which was never observed inRARγ double mutants. Since both RARβ2 and RARγ tran-scripts are expressed in the oesophageal epithelium (Dollé etal., 1990 and our unpublished results), RARβ2 may be specif-ically required for oesophageal epithelium differentiation.However, since this abnormality was always associated with acomplete lack of oesophagotracheal septation, it cannot beexcluded that it is related to an abnormal contact with theamniotic fluid known to contain soluble factors involved inepithelial cell maturation in the digestive tract (Calvert et al.,1991 and refs therein). Note that, in contrast, vitamin A defi-ciency in adult animals induces a squamous metaplasia of thenormally ciliated tracheal epithelium (Wolbach and Howe,1925; Boren et al., 1974). Interestingly, the RARαβ2 mutantsundivided oesophagotracheal tube lined with ciliated epithelialcells resembled the gut tube of more primitive vertebrates (e.g.fishes), suggesting that RA has been instrumental for theappearance of new foregut derivatives during evolution.

(B) Abnormalities of the heart and aortic arch-derived great vesselsAbnormalities of heart and great vessels found in RAR doublemutants consisted of myocardial deficiency, interventricularseptal defects, outflow tract abnormalities, persistent atrioven-tricular canal and abnormal aortic arch pattern. All of theseabnormalities have been described in the offspring from VADrats (Wilson and Warkany, 1949; Wilson et al., 1953; Tables1 and 5). Thus, the present data demonstrate that RARs aretransducing the retinoid signal necessary for proper myocar-dial growth, aorticopulmonary and ventricular septation, andpatterning of aortic arches.

The heart outflow tract abnormalities found in 18.5 dpc RARdouble mutants included PTA, dextroposed aorta (DA) anddouble outlet right ventricle (DORV). PTA, which was themost frequent defect found in all αβ2 and αγ and in some α1β2and αβ2+/− fetuses, but not in α1γ, α1γα2+/− and β2γ fetuses,was due to the lack of formation of the aorticopulmonaryseptum at 11-13 dpc. In agreement with this, PTA could beprevented in VAD rat fetuses by administration of vitamin Aat the equivalent of mouse 10.5 dpc (Wilson et al., 1953).

(1) An initially asymmetrical aortic arch system may beresponsible for the abnormal and highly variabledefinitive aortic arch pattern of RAR double mutants In 18.5 dpc WT fetuses, the arrangement of the great arterieslocated near the heart is remarkably constant (see Fig. 5). In18.5 dpc, α1β2, αβ2+/−, αβ2, α1γα2+/− and αγ fetuses, but notin α1γ and β2γ fetuses, this arrangement was remarkablyvariable and with a few exceptions abnormal, including (i)hypoplasia (i.e. coarctation of the aorta) (ii) agenesis (i.e.absence of the ductus arteriosus, of the innominate artery, ofthe left or right common carotid arteries or of the normalsystemic arch) (iii) aberrant origins (e.g. retroesophageal sub-clavian artery, aberrant origin of the pulmonary artery), and(iv) malposition of the systemic arch, (i.e. cervical arch of theaorta, right arch of the aorta). In a majority of cases, multiplearterial abnormalities were present in the same fetus (see Fig.5 and Table 5).

In WT embryos, the aortic arch system is bilaterally sym-metrical up to 11.5 dpc. Between 12.0 and 13.5 dpc, specificsegments of this system (i.e. the left and right carotid ducts,the right dorsal aortic root and the distal segment of the right6th aortic arch) regress and disappear yielding an asymmetri-cal pattern (Kaufman, 1992). From the first time of theirappearance, the aortic arches are functional since they connectthe heart with the dorsal aorta and issue major arterial stems.As blood is pumped through the symmetrical aortic archsystem a complex hemodynamic pattern of blood pressures andvelocities evolves by putting different stresses on the varioussegments of the arterial vessels in such a manner as todetermine their subsequent persistence or involution (Moffat,1959; Rychter, 1962; Pexieder, 1969). In this respect, it is note-worthy that abnormalities of the aortic arch pattern can bedetected in αβ2 and α1β2 mutant embryos as early as 10.5 and11.5 dpc, i.e. at a time when the WT pattern is still bilaterallysymmetrical. These early abnormalities consist of hypoplasiaof aortic arches or segments of the dorsal aorta: (i) reductionof the calibre of the left 4th aortic arch and aortic root (ascompared with their right homologues) (ii) reduction of the

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calibres of both the right 4th aortic arch and aortic root (ascompared with their left homologues) and (iii) concomitantreduction of the calibre of left and right 6th aortic arches (ascompared with WT counterparts). Smaller calibres of both theleft 4th aortic arch and left dorsal aortic root may deviate theblood flow towards the right side, thus preventing the normalinvolution of the right arch of the aorta. The left systemic archmay subsequently involute (resulting in a mirror-image con-figuration of the normal definitive pattern) or persists (resultingin a definitive double arch of the aorta), while the formation ofaberrant left or right cervical arch of the aorta may be due toan initial reduction of the calibre of both right and left 4thaortic arches. A decrease of the calibre of a 4th aortic arch butnot of the ispilateral aortic root may lead to aberrant retro-oesophageal subclavian arteries. The initial bilateral narrowingof the 6th aortic arches observed in 11.5 dpc mutants was sys-tematically associated with, and is likely to be secondary to,the absence of septation of the aortic sac (PTA). Since thedistal portion of the right 6th arch normally disappears before13.5 dpc and both the proximal portions of the left and the right6th aortic arches normally make only minor contributions tothe pulmonary arteries (Moffat, 1959), this early abnormality,per se, is likely to result solely in the absence of the ductusarteriosus.

(2) Abnormalities of aorticopulmonary septation andaortic arches are consistent with a cardiac neural crestdeficiency It has been shown in quail-chick chimeras that rhomben-cephalic neural crest cells (NCC), which originate from theneural folds at an axial level corresponding to the mid-oticplacode (the future rhombomere 6) to the caudal, unsegmented,portion of the rhombencephalon (3rd somite level), form thetunica media of the 3rd, 4th and 6th aortic arches (LeLièvreand Le Douarin, 1975; Noden, 1991; Kirby and Waldo, 1990and refs therein). After reaching the pharyngeal arches 3, 4 and6, some NCC continue their migration towards the aortic sacwhere they participate in its septation. These cranial NCC,which contribute to the walls of the aortic arches arteries 3 to6 and to the aorticopulmonary septum, are referred to ascardiac NCC. Excision of premigratory cardiac NCC in thechick results in a high incidence of (i) heart outflow tractdefects, including PTA, DA and DORV, (ii) aortic arch abnor-malities, including left arch of the aorta (in birds, the defini-tive systemic arch is the right one), double aortic arch, coarc-tation of the aorta and abnormal origin of the subclavian andpulmonary arteries and (iii) high interventricular defects(Kirby et al., 1983; Nishibatake et al., 1987; Kirby et al., 1985;Bockman et al., 1987; Kirby and Waldo, 1990 and refstherein). Inflow abnormalities, such as persistent atrioventric-ular canal were also detected. From these cardiac NCC ablationstudies, it was concluded that a mesectodermal cell deficiencywas directly responsible for PTA and most of the aortic archabnormalities. The other outflow tract defects (i.e. DA andDORV) and all the inflow abnormalities as well as high inter-ventricular septal defects were ascribed to alterations in theblood flow caused by a PTA or an abnormal aortic arch pattern(Kirby and Waldo, 1990 and refs therein). That the heartoutflow tract and aortic arch abnormalities observed in chickfetuses following ablation of cardiac NCC are strikinglysimilar to those found in our RAR double mutant fetuses

strongly suggests that the cardiac NCC may be quantitativelyand/or qualitatively deficient in RAR double mutants.

It is unlikely that these cardiac NCC deficiencies are alreadydetermined at the premigratory stage. Vitamin A can indeedprevent the appearance of aortic arch abnormalities and PTAin VAD rat offspring provided that it is administered beforethe equivalent of mouse 9.5 dpc and 10.5 dpc, respectively(Wilson et al., 1953), whereas all cardiac NCC have alreadymigrated away from the mouse neural tube at 9.0 dpc (Serbedz-ija et al., 1992). Thus the RA signal may be important forcardiac NCC late in their migration or at the time of formationof the aortic arch tunica media and/or of the aorticopulmonaryseptum. In RAR double mutants, RA-dependent processes willbe impaired, and more or less pronounced cardiac NCC defi-ciencies may occur in the various arches on a stochastic basis,leading subsequently to aberrant patterns. Preventing the RAsignal to be transduced might therefore be equivalent toablation of the cardiac NCC. The spatial expression patterns ofRARs are also consistent with a role in controlling the fate ofmigratory or postmigratory rather than premigratory NCC (seeLohnes et al., 1994).

It is noteworthy that ablation of cardiac NCC also results inthyroid, parathyroid and thymic ectopia and hypoplasia(Bockman and Kirby, 1984; reviewed in Kirby and Waldo,1990), similar to those seen in RAR mutants. In contrast, theectopic cartilage nodules that were observed in the heart valvesof RAR mutants may correspond to a defect in the specificationof some cardiac NCC, since similar ectopic cartilage noduleshave been found in the heart valves following grafting of mes-encephalic NCC at the level of cardiac NCC (Kirby, 1989).

(C) Abnormalities of the kidney and ureter aregenerated at distinct developmental stagesThe definitive kidney (metanephros) and the ureter developfrom both the mesenchymal metanephric blastema (i.e. thecaudal portion of the intermediate mesoderm) and the epithe-lial ureteric bud, which arises from caudal mesonephric duct(Wolffian duct). The metanephric mesenchyme gives rise toexcretory metanephric units (nephrons) comprising the kidneytubules (i.e. the convoluted tubules and the loop of Henle) andthe glomeruli. In contrast, all the components of the drainagesystem including ureters, renal pelvis and collecting tubulesoriginate from the ureteric buds (reviewed in Torrey, 1985).The essential features of metanephric development are (i)ingrowth and dichotomous branching of epithelial uretericbuds into metanephric mesenchyme and (ii) conversion of themesenchyme into epithelial kidney tubules (reviewed bySaxén, 1987). It is well established that kidney histomorpho-genesis requires reciprocal epithelial-mesenchymal interac-tions; branching of the ureteric bud is dependent upon somespecific inductive signal generated by the metanephricblastema, whereas kidney tubule differentiation within themetanephric blastema is triggered by an inductive stimulusfrom the ureteric bud (Saxén, 1987).

Abnormalities of the kidney found in RAR double mutantfetuses consisted of developmental arrests (i.e. renal agenesis,aplasia and hypoplasia) and ectopia. Renal agenesis and aplasiawere found exclusively in αγ mutants, and renal agenesis wasalways associated with agenesis of the ipsilateral ureter. His-tological analysis of 11.5, 12.5 and 14.5 dpc αγ embryosindicated that renal agenesis is most probably caused by a



failure of the ureter anlage to bud from the caudal Wolffianduct or, if budded, to reach the metanephric blastema, thusresulting in the disappearance of the latter, as suggested by thepresence of a large number of pycnotic nuclei. These earlydefects of the ureteric bud were always associated with, and insome case might be secondary to, the agenesis of the caudal-most Wolffian duct (see results section and below). Thus, renalagenesis might be determined at 11.0 dpc, which correspondsto the onset of ureteric bud formation, or even earlier at 10.5dpc when the caudal Wolffian duct normally reaches thecloaca. From the current knowledge on kidney organogenesis(see above), we also conclude that complete ureteric regressionmost probably occurred in the 18.5 dpc αγ mutant fetus, whichexhibited kidney aplasia and no ureter.

Hypoplastic kidneys, often ectopically located caudal to theirnormal position, were observed in all αβ2 and in some αβ2+/−,α1β2 and α1γα2+/− mutants. The size of the αβ2 metanephricblastema and its relationship with the ureteric bud appearednormal at 11.5 dpc (data not shown). That the mutant kidneysstopped growing at some stage of development is indicated bythe absence of the nephrogenic zone at 18.5 dpc. Thus, in αβ2mutants, the ureteric bud reaches the metanephric mesenchymeto generate nephrons, but branching morphogenesis is arrestedat some later stage. These data indicate that RA and specific pairsof RARs are required for two steps of kidney morphogenesis(formation of the ureteric bud and growth of the metanephricblastema), as well as for the ascent of the kidneys from theiroriginal sacral location to a lumbar site. RARγ appears to playa distinct role at early times, whereas RARβ2 appears to be moreimportant at later stages. In this respect, we note that RARβ2transcripts are selectively located in metanephric mesenchymeat the time of branching morphogenesis (D. Décimo and P.Dollé, unpublished results). Interestingly, agenic or hypoplastickidneys have not been reported in the fetal VAD syndrome,whereas ectopias were frequently observed.

Ectopic ureteric openings in the urethra or agenesis of thecaudal ureter, which were observed in all αβ2 and some α1β2fetuses, often coexisted in the same fetus. Ectopic uretericopenings most probably result from a failure of the caudal endof the ureters to migrate cranially toward the vesical portion ofthe urogenital sinus (Monie, 1975), whereas partial agenesismust be attributed to regression rather than incompleteformation of caudal ureters, since both kidneys and cranialureters were present. These two abnormalities also appear tobe determined at different stages, since administration ofvitamin A to VAD embryos reduced the incidence of uretericinterruptions up to the equivalent of mouse day 12.0 dpc,whereas ureteric ectopia was less frequent following vitaminA administration up to 14.0 dpc (Wilson et al., 1953). Inter-estingly, RARβ2 but not RARγ transcripts were detected inboth the mesenchyme and the epithelium of the urogenitalsinus adjacent to the ureter, but not in the ureter itself (ourunpublished observations). Thus, the defect in uretermigration/attachment reflects the lack of a RA-induced signalemanating from the urogenital sinus or associated mes-enchyme, whose generation would involve RARβ2 (orRARα), but not RARγ.

(D) Several steps in morphogenesis of the male andfemale genital tracts are affected in RAR mutants The Wolffian duct first appears at 9.0 dpc in the upper thoracic

region, then grows caudalwards until it reaches the cloaca at10.5 dpc (Kaufman, 1992). The agenesis of the seminalvesicles and caudal vas deferens seen in day 18.5 dpc αγmutants is likely to reflect the absence of the caudal Wolffianducts seen in 11.5 dpc αγ embryos. Interestingly, although therostral Wolffian ducts were always present in the analysed 11.5dpc αγ embryos, their derivatives (the epidydimis and rostralvas deferens) were absent (αγ fetuses) or severely malformed(αγ and α1γα2+/− fetuses) at 18.5 dpc. In VAD rats, Wolffianducts were always complete, but seminal vesicles were oftenagenic and this defect could be prevented by vitamin A admin-istration up to the equivalent of mouse 12.0 dpc (Wilson et al.,1953). Thus, RA is most probably required at two stages ofmale genital duct development, i.e. before 11.5 dpc for theformation of at least the caudal Wolffian duct, and after 11.5dpc for the morphogenesis of the epidydimis and rostral vasdeferens.

The female genital ducts develop from the Müllerian ductsand from the vaginal plate. The cranial portion of the Müllerianducts becomes identifiable at 12.5 dpc, and its caudal growthis completed at 14.0 dpc. The midline union of the Müllerianducts which is complete at 17.0 dpc (Thiedemann, 1987) formsthe body of the uterus and the cranial vagina. All abnormali-ties of the genital duct observed in 18.5 dpc αβ2 female fetusesmight have resulted from either a defect in the formation or afailure in the persistence of the Müllerian ducts. SinceMüllerian ducts were not identifiable in 12.5 dpc αβ2 mutantembryos, abnormalities of the genital ducts observed in 18.5dpc αβ2 female fetuses result from a defect in the formationof the Müllerian duct, most probably occurring before thepossible expression of the Müllerian inhibiting substance bythe embryonic gonad (Münsterberg and Lovell-Badge, 1991).Furthermore, our results indicate that this defect is not due toa primary defect in the formation of Wolffian ducts which areknown to be required for the induction and growth of Müllerianducts (Gruenwald, 1941; Didier, 1971 and refs therein). Theless severe phenotype observed in α1β2 and αγ female fetuses,namely agenesis of the body of the uterus and of the cranialvagina, reflects the absence of caudal Müllerian ducts only.This absence may possibly occur through different mecha-nisms in these two instances, since caudal Wolffian ducts werelacking in αγ mutants, but not in α1β2 mutants.

Absence of the uterus and cranial vagina were also observedin VAD fetuses, in which they were related to the agenesis orincomplete development of the Müllerian ducts or to the failureof these ducts to unite caudally (Wilson and Warkany, 1948).That vitamin A administration to VAD embryos prevented theagenesis or incomplete formation of Müllerian ducts only upto the equivalent of mouse 9.5 dpc (Wilson and Warkany,1953) strengthens the view that there is an early requirementfor RA for their formation. However, vitamin A administrationat the equivalent of mouse 12.0 dpc could still prevent thefailure of Müllerian ducts to unite. These observations indicatethat RA is required for multiple steps in the morphogenesis ofthe female reproductive tract, i.e. formation of the rostral andcaudal portions of the Müllerian duct at early stages, andmidline union of the caudal Müllerian duct at a later step.

Abnormal persistence of Wolffian ducts in females andMüllerian ducts in males resulting in pseudohermaphroditictendency was observed in offspring of VAD rats (Wilson andWarkany, 1948), but not in RAR mutants. Together with a

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shorter ventral retina (see Lohnes et al., 1994), this is the onlyaspect of the fetal VAD syndrome which was not recapitulatedin the RAR double mutants analyzed in this and in the accom-panying study (Lohnes et al., 1994).

CONCLUSIONS

RAR mutant abnormalities, RA target genes andpattern of expression of RARsOne fundamental question regarding the retinoid signallingpathway is how a simple compound such as RA can induce somany diverse and complex responses. Our study (see alsoLohnes et al., 1994) clearly shows that these responses areelaborated through the RARs. Our mouse RAR mutants shouldprove invaluable models to elucidate further this signallingcascade, both regarding the nature of the involved develop-mental events and the RA-target genes controlling theseevents.

The congenital abnormalities described here and in theaccompanying study (Lohnes et al., 1994) could result fromalterations in several cell autonomous or non-autonomousdevelopmental processes, including: axial patterning, as illus-trated by homeotic transformations of cervical vertebrae; spec-ification of cell fate and/or migration, as exemplified by theappearance of ectopic cartilaginous nodules likely derivedfrom NCC, and by malformations of most cranial and cardiacmesectodermal derivatives; cell proliferation and/or differen-tiation, which is likely to be the case for the myocardial celldeficiency and limb defects; epithelial-epithelial inductiveinteractions involved in the formation of ectodermal placode-derived structures, as evidenced by defects in derivatives of theotic and lens placodes; and morphogenetic processes whichrely on epithelial-mesenchymal inductive interactions, such aslung budding and bronchial branching, and kidney and salivarygland branching morphogenesis. There are a number of RA-responsive genes that are likely involved in the aboveprocesses (see Gudas et al., 1994, for review). These includetranscription factors (e.g.

Hox genes, N-myc), cell adhesionmolecules (e.g. laminin B1), growth factors (e.g. PDGF,TGFβs, BMPs) and growth factor receptors (e.g. EGF receptor,PDGF receptor).

The vertebral homeotic transformations, which are strik-ingly restricted to the cervical region, may provide oneexample of a cell autonomous effect of retinoids. As discussed(see Lohnes et al., 1994), the similarity of these malformationsto those observed in some Hox-deficient animals suggests thatRA may control axial patterning through direct control ofexpression of certain Hox genes. The chondrification of themeninges and other structures such as the persistent retrolen-ticular mesenchyme in RAR mutants offers an example of RAinvolvement in cell fate specification, and suggests that factorsinvolved in the initiation of chondrogenesis may be aberrantlyexpressed in RAR mutants. One possible candidate could bethe chondrogenic factor BMP4, which is known to be down-regulated by RA treatment in embryocarcinoma cells (Rogerset al., 1992).

Cellular migration depends on the expression of celladhesion and extracellular matrix proteins (Erickson andPerris, 1993). Retinoids have been shown to regulate the

expression of a number of these proteins, including N-CAM(Husmann et al., 1989) and laminin B1 (Vasios et al., 1989,1991). Perturbation of the expression of such proteins couldlead to abnormal NCC migration (Bronner-Fraser et al., 1991)and contribute to the malformations of NCC-derived elementsseen in RAR double mutants. Alternatively, these NCC defectscould be attributed to postmigratory events, possibly involvinggrowth factor or growth factor receptors. In this respect, thereare striking similarities between the phenotypes of Patch(which harbor a deletion of the PDGF receptor α locus) andRAR mutant mice. In both types of mutants, the defects areobserved primarily in cardiac and cranial NCC derivatives (e.g.skull bones, thymus, cornea, aorticopulmonary septum), butnot in gangliogenic NCC (Morrison-Graham et al., 1992).

A number of structures in RAR mutants appear to have beengrowth-arrested, among which is the myocardium. Severalretinoid target genes have been implicated in normal cardio-genesis, including TGFβs and N-myc (reviewed in Gudas etal., 1994). Evidence supporting a role for these target genesinclude the finding that fetal VAD leads to down regulation ofTGFβ1 in the developing heart (Mahmood et al., 1992). Fur-thermore, mice deficient for N-myc exhibit a myocardial defi-ciency similar to that observed in RARαγ double mutants(Moens et al., 1993; Charron et al., 1992). That RA plays arole in branching morphogenesis is indicated by the defects inthe lungs and kidneys of RAR mutants. Retinoid-responsivegenes that are likely involved in lung branching morphogene-sis include N-myc (Moens et al., 1993; Stanton et al., 1992),laminin B1 (Schuger et al., 1991) and the EGF receptor(Schuger et al., 1993). Interestingly, the mitogenic effect of RAappears to originate from the mesenchymal population ofdeveloping lungs (Schuger et al., 1993; see below).

From the above examples, it is clear that RA can play a rolein many developmental processes through the regulation of abroad number of genes with diverse functions. Although thetarget genes and target cells/tissues directly affected in theRAR mutants remain to be determined, many of the candidategene products (e.g. growth factors, extracellular matrixmolecules) can act at a distance and in a number of distinctmorphogenetic events. It is therefore not surprising that the siteof expression of RARs and the localization of the affectedstructures do not always coincide in the mutants. The defectsin branching morphogenesis of the lung may correspond tosuch a situation (see above). In all of these instances tissuerecombination experiments will be required to determinewhich tissue is primarily responding to RA.

Many structures whose development is affected by admin-istration of RA excess and is believed to require retinoidsduring ontogenesis (e.g. some regions of the CNS, neurogenicNCC; for reviews see Morriss-Kay, 1991, 1993; Maden, 1994;Linney and LaMantia 1994) appear unaffected in the mutantsexamined to date. Since RARβ1 and β3 isoforms (in additionto RARβ2) are expressed in some of these structures (ourunpublished results), RARβ (all isoforms) and RARβcompound mutants are required to assess the possible role ofRA in their development. Alternatively, retinoids may not berequired for some of these RAR-positive tissues to developproperly (Linney and LaMantia, 1994). In this respect, it isworth remembering that even though RARγ is expressed in allprecartilage condensations, irrespective of their embryologicalorigin (Ruberte et al., 1990), only a subset of cartilage-derived



structures are affected in RARαγ mutants, clearly indicatingthat, in some cases, the expression of RARγ is gratuitous withregard to cartilage histomorphogenesis.

Redundancy and variations in penetrance andexpressivityTaken together, the high degree of interspecies conservation ofeach individual RAR isoform throughout vertebrate evolution,the differential distribution of their transcripts during embryo-genesis and in the adult (particularly for RARβ and γ tran-scripts which are often mutually exclusive), and the apparentspecificity of their transactivation functions in transfected cells,have suggested that each RAR isoform may control theexpression of a specific subset of RA target genes, thus con-tributing to the highly diverse effects of RA (see Chambon,1994 for a review). Unexpectedly, homozygous null fetuses forthe RARα1, RARβ2 or RARγ2 isoforms or all RARα isoforms(Mendelsohn, 1994; Lohnes et al., 1993; Li et al., 1993; Lufkinet al., 1993) did not display any obvious abnormalities, and thecongenital abnormalities exhibited by RARγ null fetuses weremuch more discrete than anticipated from its pattern ofexpression (Ruberte et al., 1990). The results reported here andin the accompanying study (Lohnes et al., 1994) support ourearlier suggestion that there could be considerable functionalredundancies between members of the RAR family (Lohnes etal., 1993; Lufkin et al., 1993).

It is particularly striking that in a number of cases the samemalformations were apparently generated in different doublemutants (e.g. RARαγ and RARαβ2, see Tables in this and inthe accompanying study of Lohnes et al., 1994). These abnor-malities cannot be attributed simply to a lack of normalexpression of the RARβ isoforms (see Lohnes et al., 1994).Thus, at one extreme, our present observations may be inter-preted as indicative of complete redundancy between all RARsfor transcriptional control of all RA target gene expression. Inthis scenario, the conservation of RAR isoform A/B regionswould not correspond to a requirement for isoform specificAF-1 transactivation functions (for refs see Leid et al., 1992;Kastner et al., 1994; Chambon, 1994). The only requirementwould be to reach a certain threshold level of RAR in a givencell at a given time, which could be achieved through any com-bination of RAR isoforms. Multiple RAR genes with multiplepromoters and specific patterns of expression would haveevolved only to fulfil these purely ‘quantitative’ spatiotempo-ral requirements. Thus, in order to elicit a developmental defectin a cell expressing both RARα, RARβ2 and RARγ, it mightbe sufficient to inactivate either α and β2, α and γ, or β2 andγ to be below a critical threshold, whereas in a cell expressingRARα at a low level and RARγ at a high level, it might be suf-ficient to inactivate RARγ to be below this threshold. Interest-ingly, in several instances, an abnormality that is not presentedby RARα1γ mutants appears in RARα1γ α2+/− mutants and,furthermore, an abnormality not present in RARα1γα2+/−

mutants may be generated in RARα (all isoforms) RARγdouble mutants (see Tables). Generating RARα2γ doublemutants is necessary to determine whether these RARα2dosage effects correspond to a quantitative requirement forRARα1 and RARα2 isoforms or rather reflect a specificfunction of RARα2.

In view of the importance of inductive interactions duringvertebrate ontogenesis, it seems more likely that the generation

of the same malformation in two different double mutantsreflects an ‘interaction’ between two RA-dependent eventsinvolved in a given developmental pathway. For instance, thegeneration of the same malformation in RARαγ and RARαβ2double mutants may correspond to the control of a growthfactor receptor by RARαγ in a given cell type, and of the cor-responding growth factor in another cell type, with RARα andγ, and RARα and β2 being functionally redundant. Alterna-tively, the appearance of the same malformation in twodifferent double mutants may correspond to two sequentialdefects in a given cell type, the results of which cannot be phe-notypically distinguished.

At the present time, functional redundancies appear toprovide the simplest explanation to account for the appearanceof the VAD fetal abnormalities only in RAR double mutants.However, our results do not exclude more complex develop-mental scenarios in which multiple molecular defectsgenerated by non-redundant receptors, each acting on specificsubsets of RA target genes, would have to be combined togenerate visible (phenotypic) abnormalities (see Thomas,1993). In this respect, we note that the RARα1 and RARβ2mutations, which on their own have no detrimental effectsduring development and in the adult, results in a number offetal abnormalities and death shortly after delivery, whencombined. It should also be kept in mind that the RARα andRARγ mutations are ultimately lethal at various stages afterbirth (Lohnes et al., 1993; Lufkin et al., 1993), and that thestrong conservation of each RAR isoform across vertebratessuggests that each likely possesses at least one specificfunction. Examining the expression of a battery of putativeRA-responsive genes in the various RAR mutants is necessaryto determine whether the apparent phenotypic redundancytruly reflects a functional redundancy at the molecular level.Ultimately, the substitution in the mouse genome of one RARisoform by another is required to determine whether, and towhich extent, these receptors are functionally redundant.

For a given mutation, variability in the penetrance of a givenabnormality between different animals, and variability of itsexpressivity within an animal, were very frequently encoun-tered in the present and accompanying study of Lohnes et al.(1994) (see Tables). These variabilities may be related, at leastin part, to variations in the levels of expression of redundantRARs. Variability in penetrance may be due to variations inthe genetic background of the present null mutants, which isnot homogenous. Increase in the penetrance of some abnor-malities observed in RAR mutants upon sequential removal ofRARγ, RARα1 and RARα2 (i.e. RARγ, RARα1γ,RARα1γα2+/− and RARαγ mutants; see Tables) might reflectvariations in the expression of functionally redundant RARγ,RARα1 and RARα2 in different animals depending on theirgenetic background. Alternatively, or concomitantly, varia-tions in the levels of factors (e.g. transcription factors) whichmay act synergistically with RARs, may also account for theobserved variations in penetrance. Variations in expressivityare particularly striking. Some of them probably result fromstochastic variations in the amount of redundant receptors incontralateral cells of symmetrical structures. For instance,agenesis of the Harderian glands is either unilateral or bilateralin RARγ mutants (Lohnes et al., 1993), whereas it is alwaysbilateral in RARα1γ and β2γ mutants (see Table 4 of Lohneset al., 1994); RARγ null cells with RARα1 or RARβ2 below

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certain threshold levels may not respond to the RA signal, thusleading to the agenic phenotype, whereas cells with sufficientlevels of RARα or β2 would respond. In other cases, stochas-tic variations of the levels of other synergistic factors mayaccount for variations in expressivity. This may be the case forthe dramatic variations in expressivity that are observed in theforelimbs of RARαγ mutants (see Lohnes et al., 1994). It islikely that these frequent variations in penetrance and expres-sivity of the phenotypes exhibited by the various RAR mutantsis a reflection of the complexity of the molecular mechanismsthat underlie the developmental events controlled by retinoicacid.

We would like to thank Dr T. Pexieder for a critical reading of themanuscript, Dr A Dierich and P. Dollé for their collaboration and allmembers of the retinoid group for useful discussions; B. Weber, C.Fischer and V. Giroult and the technical staff of the animal facilityfor excellent help; B. Boulay, J.M. Lafontaine and C. Werlé for theillustrations and the secretarial staff for assembling the manuscript.C. M. was supported by a fellowship from the NIH (5F32 GM13597-03) and from the ARC, D. L. was a recipient of a fellowship from theMRC Canada and T. L. was a recipient of a fellowship from theAmerican Cancer Society (PF-2919) and from the Fondation pour laRecherche Médicale. This work was supported by funds from theInstitut National de la Santé et de la Recherche Médicale, the CentreNational de la Recherche Scientifique, the Centre Hospitalier Uni-versitaire Régional, the Association pour la Recherche sur le Cancer,the Human Frontier Science Program and the Fondation pour laRecherche Médicale.

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(Accepted15 July 1994)

function of the retinoic acid receptors ... - development · development and that rars are indeed...

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