THE ANATOMICAL RECORD 222:170-176 (1988)

Development of the Pulmonary Vein in the Arne r i can Al I i g at0 r (Alligator mississippiensis)

LYNN M. KUTSCHE AND L.H.S. VAN MIEROP Department of Pediatrics, Division of Research, University of Florida College of Medicine,

Gainesville, FL 32610

ABSTRACT The origin of the embryonic common pulmonary vein in terrestrial vertebrates is still uncertain. Most earlier studies in nonhuman embryos describe the vein as entering the sinus venosus. The currently prevailing view, however, based largely on the study of human material, is that the embryonic common pulmonary vein is associated with the left atrium from its inception.

We recently observed the pulmonary vein entering the sinus venous part of the right atrium in several normal dog embryos of a stage comparable to horizon XIV in man (Streeter: Contrib. Embryol. Carnegie Inst. Wash., 31:53,1945). In slightly older specimens the vein entered the left atrium just to the left of septum primum. This observation, and the fact that some atrial septal and pulmonary venous anomalies in man still await a plausible pathogenetic explanation, stimulated a restudy of the origin of the vein. The alligator was used because we already had prepared a large number of closely graded serially sectioned embryos for other purposes. Wax plate reconstructions clearly showed that the common pulmonary vein entered the left side of the sinus venosus. With the formation of the atrial septum, this part of the sinus venosus is “pinched off” and becomes incorporated into the left atrium, thus trans- ferring the pulmonary venous ostium to that atrium.

Several aspects of mammalian cardiovascular devel- opment remain controversial or unclear. This may be partially due to the fact that development of the car- diovascular system begins very early and progresses rapidly to near completion while other organ systems are still in an early stage of development. Also the still- very-incomplete histologic differentiation of the cardi- ovascular tissues in these young embryos makes dis- tinction between structures difficult. One still puzzling facet of cardiovascular embryology is the genesis of the pulmonary vein, even though much has been written on the subject during the past century. There is no agreement on whether the early embryonic single pul- monary vein initially enters the (common or left) atrium (Tandler, 1913; Davies and MacConaill, 1937; Streeter, 1945; Patten, 1953; Neill, 1956; Los, 1958, 1968; Van Praagh and Corsini, 1969; Van Mierop, 1979; Rammos et al., 1987) or the sinus venosus (Schmidt, 1870; Goette, 1875; Boas, 1883; His, 1885; Hochstetter, 1903; Flint, 1906-7; Fedorow, 1910; Brown, 1913; Buell, 1922). Nor is there agreement on whether it develops as a sprout or evagination from the posterior (dorsal) wall of the sinuatrial region before connecting with the pharyngeal venous plexus, or whether it develops in situ as part of the plexus, gaining a secondary connection with the heart.

Chang’s paper (1931) is unclear concerning initial entry and Auer (1948) and Butler (1952) have the pul- monary vein entering the sinuatrial region. Nearly all proponents of a sinus venosus entry of the common pulmonary vein have studied nonhuman vertebrates and published their observations prior to 1923. Those

favoring a left atrial entry generally based their con- clusions on the study of human embryos and, except for Tandler (19131, published their findings after 1936. All of these later studies found the pulmonary vein to be present for the first time in embryos of 4.5-6 mm crown- rump (C-R) length (Streeter’s horizon XIII-XIV), clearly already entering the left atrium. This led to the as- sumption that the embryonic pulmonary vein enters the left atrium from its inception.

In six well-prepared, serially sectioned normal dog embryos (comparable to horizon XIV in man) from two different litters the common pulmonary vein entered the sinus venosus part of the right, rather than the left atrium. This and the fact that certain types of atrial septal and pulmonary venous anomalies in man still await a plausible pathogenetic explanation stimulated us to restudy the origin of the embryonic common pul- monary vein. We had obtained a large number of em- bryos of the Florida alligator to study the developing arterial pole of the heart. Serially sectioned embryos of appropriate age were used for this study.

MATERIALS AND METHODS Freshly laid eggs of the American alligator (Alligator

mississippiensis) obtained from a commercial breeder were incubated at 30.5”C. A total of 85 embryos were obtained, of which 34 were of the appropriate devel- opmental stages.

Received November 10, 1987; accepted April 5, 1988.

0 1988 ALAN R. LISS, INC.

DEVELOPMENT OF THE PULMONARY VEIN 171

L A A

L P A + / -

R A A

R A

RPCV \ .

PCV

Fig. 1. Dorsal view of adult alligator heart showing the junction of the right and left pulmonary veins just cranial to the lee precaval vein. The pulmonary vein then enters the left atrium by means of a short common stem. CV: coronary vein, GC: gubernaculum cordis, UR)A lefi(right) atrium, URIAA: 1efHright)aortic arch, L(R)PA: left(right)pulmonary ai- tery, UR)PCV 1eftfright)precaval vein, UR)V left(righth.entricle, PCV pstcaval vein, PV: pulmonary veins.

Embryos were fixed in Carnoy’s fluid, which in our experience is preferable to Bouin’s or other aqueous fixatives commonly used for embryological material. Carnoy’s produces little shrinkage and tends not to col- lapse thin-walled structures such as the cardiac atria. After two changes each of absolute alcohol, methyl ben- zoate and benzene, the embryos were embedded in Par- aplast. The specimens were serially sectioned at 10 pm since we were primarily interested in organ morphology rather than cellular detail. We also wanted to minimize compression of the sections as much as possible for use in reconstruction. Sections were stained with hematox- ylin-phloxin-saffron. Wax plate reconstructions were made of six embryo hearts (Sack‘s [19661 modification of Born’s method).

Adult alligator hearts were fixed in 10% alcoholic formalin in the distended state. For this purpose all arteries and veins leaving and entering the heart were ligated except one precaval and one pulmonary vein, which were cannulated. Fixative was run into the heart through the cannulae at a pressure of about 20 cm of water and the hearts were submerged in the same fix-

ative. After fixation the cardiac chambers and great arteries were opened either by the creation of appro- priate windows or by sectioning in a frontal plane.

OBSERVATIONS Adult Heart

The relations of the great veins in the adult alligator heart are shown in Figure 1. Right and left precaval veins and the postcaval vein enter the sinus venosus as does the major coronary vein. The sinuatrial ostium is guarded by two large, equal-size sinuatrial valves and is oriented obliquely relative to the long axis of the heart. A single right and single left pulmonary vein join to form a very short, common stem which enters the cranial part of the left atrium immediately to the left of the atrial septum. This septum is completely closed and, as in birds and unlike mammals, there is no fossa ovalis. The right wall of the short, wide common pul- monary vein forms part of the atrial septum and is, in fact, thinner and more translucent than the remainder of the septum. As seen from the right atrium, the atrial septum is smooth.

Embryological Material

The noncardiovascular features used to determine stage of development are listed in Table 1. Sections of the developing eye used for staging purposes are shown in Figure 2.

AM 110-113 The mesenchyme around the pharynx behind the heart

is scant and no blood vessels are visible. There are three distinct branchial clefts and the first three branchial arch arteries (“aortic arches”) are present. Of these, the second arch artery is small; the fourth arch artery is incomplete. The cardiac loop is well developed and there is abundant cardiac jelly. The mesenchyme surround- ing the pharynx forms the dorsal wall of the sinus ven- osus and a true dorsal mesocardium therefore is not present. There are two shallow evaginations from the dorsal wall which extend into the retrocardiac mesen- chyme.

AM 117

The first three branchial arch arteries are present but the fourth is still small and incomplete. The sixth arch artery has barely made its appearance. The con- dition of the sinoatrial region is as described above ex- cept that the right evagination is less prominent (Fig. 3A). AM 36, 1 19-1 21

The first branchial arch artery is small; the second is minute. The third is large and the fourth is now complete. A rudimentary fifth arch artery is present, closely associated with the fourth and the as-yet-incom- plete sixth arch artery. The right evagination of the sinus venosus is no longer present (Fig. 3B). AM 122

The first two aortic arch arteries are tiny. The third and fourth arch arteries are large. The sixth is present but small. In the heart the atrioventricular canal region contains early atrioventricular cushions. The left-sided evagination of the sinus venosus has acquired a lumen

172 L.M. KUTSCHE AND L.H.S. VAN MIEROP

Fig. 2. Sections of the eye of alligator embryos. A 5-mm-C-R-length embryo, AM-113. B: 6-mm embryo, AM-117. C: 9-mm embryo, AM-120.

D. 10-mm embryo, AM-122. E: 10.5-mm embryo, AM-123. F. 10.5-mm embryo, AM-126.

and has connected to vessels in the retrocardiac mes- pulmonary venous ostium consists of mesenchyme which enchyme, thus establishing the pulmonary vein which is bulging into the sinus venosus (Fig. 3D,E). still enters the sinus venosus (Fig. 3C). The left horn of the sinus venoms is demarcated from the atrium by AM 24-279 37-40 the formation of a sinoatrial fold which is particularly In these embryos the atrial septum has reached the prominent on the left (Fig. 4). The pulmonary venous left sinoatrial fold (which forms the roof of the left sinus ostium is flanked on its right side by endothelium cov- horn and part of the floor of the left atrium) but has ered mesenchyme of the now-much-narrower sinomes- not fused with it. However, the part of the sinus venosus enchymal junction and on the left by a shallow fold of which contains the pulmonary venous ostium now clearly the sinus wall. The roof (cranial part) of the common has become part of the left atrium. atrium is separated into right and left components by a thick myocardial ridge covered by a thin layer of en- AM I 2t 31-339 41-44 docardial cushion tissue. This ridge continues in a dor- The atrioventricular canal cushions have not fused socaudal direction, blending with the mesenchyme to yet but the dorsocaudal part of the atrial septum has the right of the pulmonary venous ostium. fused with the roof of the left sinus horn, completing

the pinching off process which transfers the pulmonary vein to the left atrium (Fig. 3F). The pulmonary vein AM 123, 124, 126

The atrioventricular canal cushions have enlarged is a sizable vessel. The left sinus valve has made its and the truncoconal cushions have made their appear- appearance to the right of the atrial septum. ance. The atrial septal ridge has become more promi- nent, extends to the right of the pulmonary venous AM 45-48 ostium, and is approaching the left side of the sinoatrial The left sinus valve is well developed and the lateral fold (the “Vorkammerboden” of Fedorow, 19101, threat- cushions of the atrioventricular canal have made their ening to pinch off that part of the sinus venosus into appearance. The primary cushions, however, have not which the pulmonary vein enters. The part of the atrial yet fused. Ostium primum is closed and ostium secun- septal ridge furthest caudad and to the right of the durn is present. The portion of the sinus venosus which

DEVELOPMENT OF THE PULMONARY VEIN 173

Fig. 3. Sections at the level of the sinus venosus. A-C, E, F are cut transversely; D is cut midway between transverse and frontal. The em- bryonic pulmonary vein initially enters the left side of the sinus venosus (A-E). Because D is cut frontotransversely, the pulmonary vein appears to enter the left atrium. A Embryo AM-117. B: Embryo AM-120. C: Embryo AM-122. D: Embryo AM-123. E: Embryo AM-126. F: Embryo

AM-41. A: atrium, €3: bronchus, DA dorsal aorta, E: esophagus, L(R)A left(right)atrium, UR)CCV left(right)common cardinal vein, LSH: lea sinus horn, SAF: sinuatrial fold, SP: atrial septum (“septum primurn”), SV: sinus venosus, V ventricle. Arrow indicates pulmonary venous os- tium. Double-headed arrow indicates sinuatrial junction. Bar = 0.2 m.

contains the pulmonary venous ostium has become an integral part of the left atrium.

DISCUSSION According to Schmidt (1870) the pulmonary vein en-

ters the left portion of the sinus venosus in a pig embryo of 7 mm CR length and a similar description is given by Brown (1913) for a 5-6-mm cat embryo. His (18851, in a study of early human embryos, also found the pul- monary vein opening into the left part of the sinus ven- osus as did Goette (1875). The part of the sinus venosus which contains the pulmonary venous ostium then sec- ondarily becomes part of the left atrium. According to Boas (18831, the early embryonic pulmonary vein in lung fish, amphibians, and reptiles also enters the left, smaller, portion of the sinus venosus, but he does not clearly describe how it comes to enter the left atrium. The most detailed description of the transfer of the pul- monary venous ostium from the sinus venosus to the left atrium is provided by Fedorow (1910) in amphibi- ans, reptiles, birds, and mammals. Fedorow considers the vein an outgrowth of the dorsal wall of the sinus venosus based on his observation of an endothelial pro-

liferation extending into the mesenchyme around the foregut, dorsal to the sinus venosus. The part of the sinus venosus receiving the common pulmonary vein becomes part of the atrium as a result of differential growth. The originally wide sinoatrial ostium narrows with the formation of a fold of the cardiac wall a t the junction between the sinus venosus and the left atrium. This fold, referred to by Fedorow as the ‘Vorkammer- boden” (atrial floor), extends from left to right, with the pulmonary venous ostium remaining cranial to it. The developing, increasingly prominent atrial septum ex- tends caudad along the dorsal sinuatrial wall, to the right of the pulmonary venous ostium, and then fuses with the atrial floor, thus transferring the pulmonary venous ostium to the left atrium.

Our study in the alligator embryo essentially agrees with Fedorow’s account. We have also found that the common pulmonary vein originally enters the left side of the sinus venosus. Its ostium is derived from the left of a pair of evaginations of the dorsal, mesenchymal wall of the transverse part of the sinus venosus. The right evagination normally disappears. Infolding of the sinuatrial wall separates the sinus venosus from the atrium producing on the right, the right sinus valve

Figure 3 C-F

DEVELOPMENT OF THE PULMONARY VEIN

TABLE 1. Noncardiovascular features used to determine stage of development

175

Embryo no. AM- 110-113

117

36,119- 121

122

123,124 126

24-27, 37,40

1,2,31- 33,41-

44 45-48

Devel. stage C-R Incub. length age Tracheobronchial (mm) (days) Limbs Nose Eye tree Reese’ H-H2

5.0

6.0

9.0

10.0

10.5

11.5

12.5

15.0

8-9

9-10

9-10

10-11

10-12

12-13

13-14

16-17

No limb buds present

Early appearance of limb buds

Distinct limb buds

Distinct limb buds

Limb buds as long as wide

Limb buds longer than wide

Manus and pes paddle shaped

Well defined manus and

Slight depression of nasal placode

Shallow olfactory pits

Clearly visible olfactory pits

Deep olfactory pits

Olfactory pits short tunnels -

Lens vesicle slightly flattened. No retinal pigment

flattening of lens. No retinal pigment

Elongated posterior lens fibers

Lumen of lens obliterated

Increased

Fktinal pigment visible at high magn.

Retinal pigment visible at medium magnification

Retinal pigment dense

Indication of lung buds

Early lung buds

Primary bronchi present

Primary bronchi elongated

Secondary bronchi present

Trachea long, well developed lung buds

Third order bronchi present

12 17

12 18

13 19

13 20

14 20

15 24

16 25

17 26

‘Reese: Reese (1915). ‘H-H: Hamburger and Hamilton (1951)

and, on the left, the wall between the left precaval vein and the left atrium (the equivalent of the coronary sinus- left atrial wall in mammals). This left-sided component of the sinuatrial fold represents the “Vorkammerboden” of Fedorow. The atrial septum (the homologue of the mammalian septum primum) extends caudad along the dorsal wall of the transverse part of the sinus venosus, just to the right of the pulmonary venous ostium. It eventually reaches and fuses with the “Vorkammer- boden” just above the entrance of the left precaval vein into the sinus venosus. Thus, the small, left-sided part of the sinus venosus, into which enters the common pulmonary vein, becomes part of the left atrium. Al- though most of the atrial septum in the adult alligator contains myocardium, the part to the right of the pul- monary venous ostium does not. It is derived from the retrocardiac mesenchyme with part of it eventually forming the right wall of the common pulmonary vein.

In summary, our observations show that the early embryonic pulmonary vein initially enters the left cen- ter part of the sinus venosus, rather than the left atrium.

Fig. 4. Wax plate reconstruction of the heart of embryo AM-122. The common pulmonary vein clearly enters the sinus venosus at this stage of development, but w d be transferred to the left atrium when the atrial septum fuses with the sinuatrial fold (the “Vorkammerboden” = atrial floor). AVRC: atrioventricular canal region, CA: common atrium, ESOPH esophagus, URIACV: left(right)anterior cardinal vein, UR)PB: left(right) pnmary bronchus, PV pulmonary veins, PVO: pulmonary venous os- tium, SP atrial septum, SV sinus venosus, TC = truncoconus. *Indicates the ‘Vorkammerboden,” actually the left side of the sinuatrial fold. Insert indicates plane of section. Bar= 0.2 nun.

176 L.M. KUTSCHE AND L.H.S. VAN MIEROP

With the development of the sinuatrial fold and fusion of the atrial septum with the left center part of this fold just to the right of the common pulmonary venous os- tium, the small part of the sinus venosus which receives the pulmonary vein becomes part of the left atrium.

ACKNOWLEDGMENTS Supported by Clinical Investigator Award 5K08

HL00885 from the National Heart, Lung, and Blood Institute. We thank Debra J. Abraham for assistance in the preparation of this manuscript.

LITERATURE CITED Auer, J . 1948 The development of the human pulmonary vein and its

Boas, J.E.V. 1883 Beitrage zur &ologie der Amphibien. Morphol. Jahrb.,

Brown, A.J. 1913 The development ofthe pulmonary vein in the domestic

Buell, C.E. 1922 Origin of the pulmonary vessels in the chick. Contrib.

Butler, H. 1952 Some derivatives of the foregut venous plexus of the

Chang, C. 1931 On the origin of the pulmonary vein. Anat. Rec., 50.1-

Davies, F., and M.A. MacConailll937 Cor biloculare, with a note on the

Fedorow, V. 1910 Uber die Entwickelung der Lungenvene. Anat. He&,

Flint, J.M. 1906-7 The development of the lungs. Am. J. Anat., 6:l-

Goette, A. 1875 Die Entwickelungsgeschichte der Unke. (Cited by Brown,

Goette, A. 1905 Uber den Ursprung der Lungen. Zoo1 Jahrb., 21.141-

major variations. Anat. Rec., 101.581-594.

8:169-187.

cat. Anat. Rec., 7:299-329.

Embryol. Carnegie Inst. Wash., 14.11-26.

albino rat, with reference to man. J. Anat., 86t95-109.

8.

development of the pulmonary veins. J. Anat., 71:437-446.

40.529-607.

137.

1913).

160.

Hamburger, V., and H.L. Hamilton 1951 A series of normal stages in the development of the chick embryo. J. Morphol., 88t49-92.

His, W. 1885 Anatomie Menschlicher Embryonen. Vogel, Leipzig, Vol. 3, pp. 129-184.

Hochstetter, F. 1903 Die Entwicklung des Blutgefaszsystems. In: Hert- wig's Handbuch der Entwicklungslehre der Wirbeltiere, Vol. 3 (cited by Fedorow, 1910).

Los. J.A. 1958 De Embrvonale Ontwikkeling. van de Venae Pulmonales en de Sinus Coronahus Bij de Mens. Tgesis. Leyden, The Nether- lands.

Los, J.A. 1968 Embryology. In: Pediatric Cardiology. H. Watson, ed. CV. Mosby, St. Louis, pp. 301-319.

Neill, C.A. 1956 Development of the pulmonary veins. With reference to the embryology of anomalies of pulmonary venous return. Pedi- atrics, 18t880-887.

Patten, B.M. 1953 Human Embryology. 2nd edition. The Blakiston Co.. -_ Inc., New York, pp. 644-646.-

Rammos, S., A.C. Gittenberger-de Groot, A. Oppenheimer-Dekker, and H.H. Kramer 1987 A development background of abnormal pulmo- nary venous return. Embryol. Hefte, 1.32 (Abstract).

Reese, A.M. 1915 The Alligator and its Allies. G.P. Putnam & Sons, New York.

Sack, W.O. 1966 Rapid wax plate modeling. Anat. Rec., 154.233-242, Schmidt, F.T. 1870 Bidtrag ti1 kundskaben om hjerteb undviklungshis-

tone. Nord. Med. Arkiv, 2(Nr. 23) (Cited by Brown, 1913). Streeter, G.L. 1945 Development horizons in human embryos. Descrip-

tion of age group XIII, embryos about 4-5 mm long, and age group XIV, period of indention of the lensvesicle. Contrib. Embryol. Car- negie Inst. Wash., 31.53.

Tandler, J . 1913 Anatomie des Herzens. In: Handbuch der Anatomie des Menschen. K. von Bardeleben, ed. Gustav Fischer, Jena, p. 20.

Van Mierop, L.H.S. 1979 Morphological development of the heart. In: Handbook of Physiology-The Cardiovascular System. 1. R.M. Berne, ed. Am. Physiol. Soc., Bethesda, pp. 1-28.

Van Praagh, R., and I. Corsini 1969 Cor triatriatum: Pathologic anatomy and a consideration of morphogenesis based on 13 postmortem cases and a study of normal development of the pulmonary vein and atrial septum in 83 human embryos. Am. Heart J., 78t379-405.