Cystic Malformation of the PosteriorCerebellar Vermis in Transgenic Mice ThatEctopically Express Engrailed-1,a Homeodomain Transcription FactorDAVID H. ROWITCH,1,2* PAUL S. DANIELIAN,2 ANDREW P. MCMAHON,2 AND NATASA ZEC3

1Division of Newborn Medicine, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 021152Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 021383Department of Pathology, Children’s Hospital, Boston, Massachusetts 02115

ABSTRACT In WEXPZ-En-1 transgenic mice,Engrailed-1, a homeodomain-containing transcriptionfactor, is ectopically expressed in the developing brainunder control of the Wnt-1 enhancer. En-1 is a develop-mental regulatory control gene which has an essentialrole in the formation of the midbrain and cerebellum.Approximately 28% of WEXPZ-En-11 mice develop cys-tic malformations of the posterior lobe of the cerebellarvermis, fourth ventricular dilatation, and postnatal hydro-cephalus. These anatomic features are also foundamong the spectrum of posterior fossa malformationsin humans. Expression characteristics of the WEXPtransgene suggest that the neuropathology observed inWEXPZ-En-11 mice stems from overexpression of En-1during fetal and neonatal phases of cerebellar develop-ment. These observations raise the possibility thatabnormal regulation of Engrailed genes, or targets ofEngrailed, may be involved in the pathogenesis ofcystic central nervous system malformations of theposterior fossa in humans. Teratology 60:22–28,1999. r 1999 Wiley-Liss, Inc.

Cystic malformations of the posterior fossa in hu-mans are frequently associated with generalized hydro-cephalus and other central nervous system (CNS)anomalies. Such lesions have been anatomically andradiologically classified into: 1) Dandy-Walker malfor-mation, comprising cystic dilatation of the fourth ven-tricle, partial agenesis of the posterior (inferior) cerebel-lar vermis, and expansion of the posterior fossa withhigh position of the tentorium, frequently in associationwith generalized hydrocephalus; 2) Dandy-Walker vari-ant, in which the fourth ventricle cyst communicateswith the perimedullary subarachnoid spaces, in associa-tion with varying degree of upward rotation of thecerebellar vermis; and 3) retrocerebellar arachnoidpouch of the cisterna magna (or prominent cisternamagna), without cerebellar vermal dysgenesis or dilata-tion of the fourth ventricle (Raybaud, ’82; Barkovitch etal., ’89). The etiology and pathogenesis of posteriorfossa cystic malformations are poorly understood.Dandy-Walker malformation (DWM) has been associ-

ated with chromosomal abnormalities and other malfor-mation syndromes (reviewed in Murray et al., ’85;Bordarier and Aicardi, ’90; Ludwick and Norman, ’91;Norman et al., ’95), and familial cases have beendescribed (Hirsch et al., ’84; Russ et al., ’89). DWM hasbeen linked with exposure to certain teratogens such asisotretinoin and warfarin in humans (Ludwick andNorman, ’91; Norman et al., ’95) and 6-aminonicotin-amide in rats (Oi et al., ’96). Hereditary mouse hydro-cephalus, with cystic malformation of the posteriorcerebellar vermis similar to that seen in human Dandy-Walker syndrome, has been described by Bonnevie andBrodal (’46). Theories of the pathogenesis of cysticmalformations of the posterior fossa have been re-viewed by Raybaud (’82) and fall into roughly twogroups. Some authors have postulated that abnormalcerebrospinal fluid (CSF) drainage at the roof of thefourth ventricle during development (including absenceof normal anatomic sites of communication between thefourth ventricle and subarachnoid space) results incystic expansion of the fourth ventricle, with disruptedformation of the posterior vermis and generalized hydro-cephalus. Others have proposed that abnormal differen-tiation of dorsal rhombencephalic structures results inpartial agenesis of the posterior vermis, and formationof cystic fourth ventricle.

The cerebellum classically has been thought to derivefrom the rhombic lip. However, chick-quail neuroepithe-lial transplantation has demonstrated that midlinecerebellar structures, such as the anterior (superior)vermis, derive in part from the embryonic midbrain

Abbreviations used: CNS, central nervous system; CSF, cerebrospinalfluid; d.p.c., days post coitum.

Grant sponsor: NIH; Grant sponsor: Howard Hughes Medical Insti-tute; Grant sponsor: Wyeth Pediatric Research; Grant sponsor: Hu-man Frontiers Science Program; Grant sponsor: Joseph Boice EsserFellowship.

*Correspondence to: David H. Rowitch, M.D., Ph.D., now at theDepartment of Pediatric Oncology, Dana-Farber Cancer Institute, 44Binney St., Boston, MA02115. E-mail: david_rowitch@dfci.harvard.edu

Received 9 June 1998; Accepted 11 January 1999

TERATOLOGY 60:22–28 (1999)

r 1999 WILEY-LISS, INC.

(Martinez and Alvarado-Mallart, ’89; Hallonet et al.,’90). Orthotopic cells originating in the mesencephalicvesicle were found in a rostromedial region in all thecerebellar layers except the external granule layer,which derived exclusively from the metencephalon(Hallonet et al., ’90). Millet et al. (’96) used the molecu-lar marker Otx-2 to identify a population of cells,immediately rostral to the midbrain-hindbrain junc-tion, that was destined to contribute to anterior cerebel-lar structures. Thus, aberrant development in precur-sor cells of the caudal embryonic midbrain could inprinciple result in malformations of the anterior cerebel-lum and fourth ventricle.

Wnt-1 encodes a secreted signaling molecule and isexpressed in the neural plate from the time of condensa-tion of the first somite (8 days post coitum (d.p.c.) in themouse; Wilkinson et al., ’87). Mutation of Wnt-1 by genetargeting results in failure of development of the mid-brain and anterior hindbrain structures, including thecerebellum (McMahon and Bradley, ’90; Thomas andCapecchi, ’90; Mastick et al., ’96). Detailed analysis ofthe Wnt-1 null mutant has shown that failure ofmidbrain development leads to subsequent apoptosis incells of the anterior hindbrain and loss of rhombomere 1(McMahon et al., ’92; Serbedzija et al., ’96; Mastick etal., ’96). Since Wnt-1 is not expressed in the metencepha-lon per se (Wilkinson et al., ’87), one possibility is that asignal (e.g., growth factor) from the midbrain is re-quired to prevent cells of the anterior metencephalonfrom undergoing apoptosis (Rowitch et al., ’97). In anycase, it is clear that in addition to contributing precur-sor cells for metencephalic development, the mesen-cephalon must have an additional role in maintenanceof rhombomere 1, a process that either directly orindirectly involves Wnt-1.

The murine genes En-1 and En-2 are orthologues ofDrosophila engrailed and encode homeodomain-contain-ing transcription factors (Davis and Joyner, ’88; Daviset al., ’88). Mutation of En-1 by gene targeting results infailure of development of the cerebellum and colliculi(Wurst et al., ’94). The effects of En-2 gene targeting arerather more subtle, comprising abnormal cerebellarfoliation (Joyner et al., ’91). Functional redundancy ofEn-1 and En-2 in the embryonic midbrain-hindbrain isdemonstrated by the phenotype of the double mutant(En-1-/En-1-, En-2-/En-2-) embryo, in which there isfailure of development of the midbrain and rhombo-mere 1, similar to the Wnt-1 null phenotype (Wurst andJoyner, personal communication). The comparable phe-notypes of the Wnt-1 and Engrailed mutant micesuggested that these factors could participate in com-mon CNS developmental regulatory pathways (Joyner,’96). Indeed, transgenic expression of En-1 in thedeveloping mesencephalon of Wnt-1 null embryos issufficient to rescue early mes- and metencephalic devel-opment, suggesting that a key role of Wnt-1 signaling isto maintain Engrailed expression (Danielian and McMa-hon, ’96).

We examined a strain of transgenic mice in whichEn-1 is ectopically expressed in the embryonic mid-brain and hindbrain under the control of the Wnt-1enhancer (Fig. 1). Here we report that WEXPZ-En-11

mice develop postnatal hydrocephalus and a cysticmalformation of the cerebellar vermis in communica-tion with the fourth ventricle. These abnormalities arealso frequent components of human posterior fossamalformations. This mouse strain may therefore serveas a tool to study the pathogenesis of cystic malforma-tions of the posterior fossa in humans.

MATERIALS AND METHODS

Transgenic mice

The WEXPZ-En-1 and WEXP-lacZ lines of trans-genic mice were generated by pronuclear injection ofone-cell (C57BL6 3 CBA)F1 embryos with transgeneconstructs (Fig. 1), as previously described (Danielianand McMahon, ’96; Echelard et al., ’94). Offspringhemizygous for the WEXPZ-En-1 transgene were iden-tified by Southern blot analysis with a probe thatrecognized the WEXPZ-En-1 transgene array (Danie-lian and McMahon, ’96). Eight hemizygotes demonstrat-ing hydrocephalus at 3 weeks–2 months of age weresacrificed, and the CNS was dissected. The protocol foranimal experimentation had been previously reviewedand approved by the Standing Committee on the Use ofAnimals in Research and Teaching, Harvard Univer-sity.

Histological analysis

The brains of 8 WEXPZ-En-11 mice were treatedwith Bouin’s fixative and embedded in paraffin wax. Inone mouse, the brain was sectioned in the sagittalplane. In the remaining 7 mice, the forebrain wassectioned in the coronal plane and the brain-stem withattached cerebellum was sectioned in the horizontalplane. Serial sections at 6 µm were obtained for histo-logical analysis and were stained with hematoxylin-eosin.

Whole-mount b-galactosidase staining

Staining for b-galactosidase activity in the CNS ofWEXP-lacZ 19.5 d.p.c. transgenic fetuses was carriedout at 37°C for 12–24 hr, essentially as described(Whiting et al., ’91; Echelard et al., ’94). Samples werethen postfixed in 1% formalin and 0.2% gluteraldehyde,and frozen sections of 10 µm were counterstained withcresyl violet or neutral red prior to photography.

RESULTS

Three independent WEXPZ-En-11 transgenic founderlines have been observed to develop severe hydrocepha-lus, and one of these was chosen for extensive study atearly embryonic and fetal stages (Danielian and McMa-hon, ’96). Approximately 28% of hemizygous WEXPZ-En-11 animals developed hydrocephalus and died by1–2 months of age. Gross examination of the brain

CYSTIC CEREBELLAR MALFORMATION IN TRANSGENIC MICE 23

revealed hydrocephalus and a posterior fossa cyst (Fig.2A,B). To further characterize the cause of hydrocepha-lus in WEXPZ-En-11 mice, 8 clinically affected animalswere analyzed histologically (Table 1). In 6 of 8 of themice, the major histological abnormalities included 1)generalized dilatation of the ventricular system (Fig.2C,D), and 2) a cystically dilated posterior lobe of thecerebellar vermis, lined by markedly reduced cerebellarparenchyma, in broad communication with the fourthventricle (Fig. 2E–G).

The posterior vermis of the cerebellum in 6 of 8 of themice contained a large central cystic space (Fig. 2E–G),which in 3 of 8 also extended into both cerebellar lateralhemispheres. Folia of the cystically dilated posteriorlobe of the vermis, between the primary and secondaryfissure (including the declive, tuber vermis, and pyra-mid), were effaced in 6 of 8 of the mice. Folia posterior tothe secondary fissure (including the uvula and nodulus)were severely distorted by the cyst but not entirelyeffaced. Folia anterior to the primary fissure (i.e., all ofthe folia of the anterior vermis including the lingula,lobulus centralis, and culmen) were intact in all 8 of 8animals and were unaffected by the cyst (Fig. 2F,G).The cystic posterior lobe of the vermis was distendedrostrally such that it covered the intact anterior lobeand ended in the pineal region rostral to the anteriorlobe (Fig. 2G).

The cystic space in the cerebellum broadly communi-cated with the fourth ventricle (Fig. 2G). While thedilated fourth ventricle was lined by ependyma, thecystic space in the cerebellum did not have an ependy-mal lining, but was surrounded by markedly thinnedcerebellar white matter. There were scattered hemosid-erin-laden macrophages, deposits of hemosiderin, andkarryorhectic cells in the white matter adjacent to thecyst. Deep cerebellar nuclei were identifiable but werecompressed by the cyst. The cerebellar cortex overlyingthe cyst showed appropriate layering (including themolecular layer, internal granule cell layer, and Pur-kinje cell layer), but was moderately to markedly

reduced in thickness (Fig. 2E,E8). At the anterior pole ofthe cyst, the cerebellar tissue in the wall of the cystbecame patchy (Fig. 2F), such that the large areas ofthe cyst were covered only by the leptomeninges. Thecyst ended anteriorly in the pineal region as an out-pouching of leptomeninges.

All of the mice with cerebellar cysts also showedsevere generalized hydrocephalus (including dilatationof the fourth ventricle, aqueduct, third ventricle, andlateral ventricles), as well as tissue changes secondaryto hydrocephalus (including distortion of forebrain struc-tures and degenerative changes in periventricular tis-sues; Fig. 2C,D,G). Obstruction to CSF flow was notobvious at any level of the ventricular system.

In 2 of 8 of the mice, the brain-stem and the cerebel-lum were normal, without evidence of cerebellar cyst.In these mice, there was massive dilatation of thelateral ventricles without dilatation of the third ven-tricle, aqueduct, or fourth ventricle. No site of obstruc-tion to CSF flow was found.

WEXP transgene expression in the fetalcerebellum

In order to examine the pattern of WEXP-driventransgene expression in the fetal brain, we analyzedWEXP-lacZ mice, in which the reporter gene lacZ(encoding b-galactosidase) is expressed under control ofthe Wnt-1 5.5-kb regulatory element used in the WEXP-En-11 construct (Fig. 1). Early expression of lacZ fromthis transgenic line from 8.0–12.5 d.p.c. has beenpreviously described (Echelard et al., ’94) (Fig. 3).Interestingly, at 19.5 d.p.c., b-galactosidase stainingwas detected in tissues of the posterior cerebellum (Fig.2H). This finding was unexpected, since no endogenousWnt-1 expression was observed in the fetal cerebellumat 17.5 d.p.c. by in situ hybridization (Millen et al., ’94).Since b-galactosidase activity is generally a more sensi-tive indicator than RNA in situ hybridization, it is

Fig. 1. Structure of the WEXPZ-En-1 transgene. Schematic of the DNA construct used to generate thetransgenic line, WEXPZ-En-1, as described in Danielian and McMahon (’96). Engrailed-1 cDNA wascloned into the vector WEXPZ, placing it under control of Wnt-1 regulatory sequences located downstreamof the gene (Echelard et al, ’94). Note that the translation start (ATG) of Wnt-1 has been destroyed andthat a portion of sequence from the lacZ gene has been added to tag the mRNA. Figure modified fromDanielian and McMahon (’96).

24 D.H ROWITCH ET AL.

possible that low-level Wnt-1 expression was not de-tected by Millen et al. (’94). Alternatively, it is possiblethat the 5.5-kb Wnt-1 regulatory element lacks regula-tory sequences (present at the endogenous Wnt-1 locus)

that mediate repression of expression in the cerebel-lum.

DISCUSSION

Increased sophistication in the antenatal assessmentof malformations of the posterior fossa has resulted indetection of a spectrum of vermian lesions that canoccur in association with hydrocephalus or as part ofmultiple malformation syndromes. In such cases, it isimportant to establish a correct diagnosis for the pur-poses of determining prognosis, making appropriatetherapeutic decisions, and genetic counseling. A compre-

TABLE 1. Neuropathological findings in eighthydrocephalic WEXP-En-11 mice

Hydrocephalus 8/8Cyst in posterior vermis 6/8 (75%)Cyst in posterior vermis extending into cerebellar

lateral hemispheres3/8 (38%)

No cerebellar cyst; hydrocephalus limited to cere-bral lateral ventricles

2/8 (25%)

Fig. 2. Gross morphology and histological analysis of the brain in1–2-month-old WEXPZ-En-11 mice (A–G) and histochemical analysisof WEXP-lacZ1 mice at fetal stages (H). A, B: Gross appearance of thebrain in two cases. Note that the posterior cerebellum is partiallyreplaced by a cyst (arrows). C, D: Horizontal section of the cerebralhemispheres (C, C8) and brain-stem (D, D8) from wild-type (C, D) andWEXPZ-En-11 (C8, D8) mice. Note distended lateral ventricle (C8, LV)and fourth ventricle (D8, IV) due to generalized hydrocephalus. E–G:Horizontal (E, F) and parasagittal (G) sections of the cerebellum andbrain-stem of a WEXPZ-En-11 mouse, demonstrating a posteriorcerebellar cyst (C) in communication with the fourth ventricle. E,E8: The area between the cyst and fourth ventricle is indicated by a box(E), and is shown at 310 magnification (E8). Note that choroid plexus(cp) is directly overlaid by a cyst. F, G: The primary fissure, whichdivides the anterior and posterior vermis, is indicated by the dashedline. Note the cystically malformed posterior lobe of the vermis (dorsal

to the primary fissure in the horizontal section (F), and caudal to theprimary fissure in the parasagittal section (G)), as well as the intactanterior lobe of the vermis (ventral to the primary fissure in thehorizontal section (F), and rostral to the primary fissure in theparasagittal section (G)). Although both lateral hemispheres arepresent in WEXP-En-11 mice, only one lateral hemisphere is seen inthe horizontal section, due to distortion of the tissue by the cyst. G:Parasagittal section of the cerebellum (with attached forebrain andbrain-stem) of WEXPZ-En-11 mouse. The cystically distended poste-rior lobe of the vermis (C) extends anteriorly and covers the anteriorlobe. Broad communication between the cyst and fourth ventricle ispresent posteriorly. IV, fourth ventricle; III, third ventricle. H: b-galac-tosidase staining in the posterior cerebellum of a 19.5-d.p.c. WEXP-lacZ1 fetus. The section was counterstained with cresyl violet. Arrowindicates the posterior vermis. Cp, choroid plexus.

CYSTIC CEREBELLAR MALFORMATION IN TRANSGENIC MICE 25

hensive understanding of the pathogenesis of cysticmalformations of the posterior fossa would no doubthave implications for effective management of patientswith vermian dysgenesis.

Spectrum of neuropathologyin WEXPZ-En-11 mice

The major finding in 75% of the hydrocephalic trans-genic mice analyzed includes the cystic space within theposterior lobe of the vermis lined by the compressedtissue of the residual posterior vermis. The cystic space

was continuous with the dilated fourth ventricle, and in38% of the cases, the cyst extended into the lateralhemispheres. The anterior vermis was uninvolved bythe cyst. The entire ventricular system was markedlydilated, but the site of CSF obstruction was not appar-ent on the slides.

The cerebellar cyst (which preferentially involves theposterior vermis, and is contiguous with the fourthventricle), in association with generalized hydrocepha-lus, is a feature found among the spectrum of Dandy-Walker malformations in humans, and is also similar tofindings in the hereditary mouse hydrocephalus (Bro-dal et al., ’44; Bonnevie and Brodal, ’46). The hereditarymouse hydrocephalus (HMH) was considered to be theclosest animal anatomic equivalent of Dandy-Walkermalformation in humans, but is no longer in existence(Friede, ’89).

In the present hydrocephalic transgenic mice, therewas variable severity of cerebellar abnormalities, simi-lar to the variable severity of the cerebellar abnormali-ties in the HMH (Brodal et al., ’44; Bonnevie andBrodal, ’46). Brodal et al. (’44) speculated that thevariable severity of midline cystic cerebellar abnormali-ties in HMH could result from variable timing of theonset of hydrocephalus, with respect to the timing offormation of the vermis. An additional consideration inWEXPZ-En-11 mice is that slight variations in theexpression levels of En-1 from the transgene could playa role in the penetrance of the phenotype and develop-ment of hydrocephalus (Dobie et al., ’97). Given thatabout 2/3 of WEXPZ-En-11 mice survive, it is interest-ing to note that in humans there is a spectrum ofclinical severity of DWM, with Dandy-Walker variantpresenting as mild form. In future analysis, nonhydro-cephalic WEXPZ-En-11 mice will be analyzed to investi-gate possible subclinical malformations of the cerebel-lum.

Histological findings of hemosiderin deposits andsiderophages in the walls of the cyst in WEXPZ-En-11

mice are indicative of previous tissue destruction withhemorrhage. This is consistent with the theory ofBrodal et al. (’44) that the midline cerebellar cyst inhydrocephalic mice may be a result of the secondarytissue damage due to hydrocephalus. This finding alsoraises the possibility that dysgenesis of the posteriorcerebellar vermis (e.g., as seen in Dandy-Walker malfor-mation) may represent a secondary regressive or degen-erative feature rather than a primary failure of develop-ment, as previously speculated (Gibson, ’55).

Abnormal patterns of En-1 expression inWEXPZ-En-11 mice

Since hydrocephalus was observed in three separatefounder WEXPZ-En-1 transgenic animals (Danielianand McMahon, ’96), the phenotype can be linked toectopic En-1 expression in the Wnt-1 domain of expres-sion (Wilkinson et al., ’87; Echelard et al., ’94), asopposed to being an effect of a random integration eventin the genome. In order to interpret abnormal En-1

Fig. 3. Aberrant En-1 expression in the developing brain of WEXP-En-11 mice. Comparison of the pattern of b-galactosidase stainingdirected by the WEXP regulatory element in WEXP-lacZ transgenicembryos (A, C) to that of endogenous En-1 as revealed by whole-mount in situ RNA hybridization of wild-type mouse embryos (B, D).(A, B) Note that in WEXP-En-11 embryos at the one-somite stage (8.0d.p.c.), ectopic En-1 expression in the rostral midbrain would occur inthe approximate area indicated between arrows. (C, D) At the 25-somite stage (9.5 d.p.c.), an abnormal, increased dosage of En-1 wouldbe directed just rostral to the mid-hindbrain junction (arrow) by theWnt-1 regulatory sequences. E: Schematic of mes- and metencephaliccontributions to the vertebrate postnatal cerebellum, as shown byorthotopic grafting experiments (Hallonet et al., ’90). Cells from thecaudal mesencephalon contribute only to the anterior cerebellum(open area); structures in the posterior cerebellum derive exclusivelyfrom metencephalic anlage (hatched area). Figure modified fromEchelard et al. (’94), Rowitch and McMahon (’95), and Hallonet et al.(’90).

26 D.H ROWITCH ET AL.

expression in WEXPZ-En-11 mice, it is useful to reviewthe regulation of Wnt-1 and Engrailed genes in theembryonic brain. Wnt-1 and En-1 are contemporane-ously expressed in the murine neural plate at theone-somite stage (8.0 d.p.c.). While their respectiveexpression domains overlap at the presumptive mid-brain-hindbrain junction, Wnt-1 is expressed slightlymore rostrally than En-1 (Fig. 3A,B) (Davis and Joyner,’88; McMahon et al., ’92; Rowitch and McMahon, ’95;Rowitch et al., ’98). At the 25-somite stage (9.5 d.p.c.),En-1 and En-2 are expressed at the midbrain-hindbrainjunction and in rhombomere 1 (Davis and Joyner, 88;Davis et al., ’88) (Fig. 3D); Wnt-1 is expressed in thedorsal and ventral midbrain, as well as in a ring thatlies anterior to the midbrain-hindbrain junction (Wilkin-son et al., ’87) (Fig. 3C). Wnt-1 expression was notdetected in the murine cerebellum at 17.5 d.p.c. orpostnatally, while En-1 and En-2 showed changingspatial patterns of expression both at 17.5 d.p.c. andpostnatally (Millen et al., ’95).

In WEXPZ-En-11 mice there is misexpression of En-1at embryonic and fetal phases during CNS develop-ment (Danielian and McMahon, ’96). At the 0–1-somitestage (8.0 d.p.c.), ectopic En-1 expression occurs in arostral population of cells in the presumptive midbrain,while at the 25-somite stage (9.5 d.p.c.), after neuraltube closure, the WEXPZ-En-1 transgene results in anincreased dosage of En-1 at the midbrain-hindbrainjunction and dorsal midline. These points are illus-trated in Figure 3.

Engrailed-1 is a developmental regulatory controlgene essential in the normal formation of the embryonicmidbrain-hindbrain (Wurst et al., ’94). One possibilityis that misexpression of En-1 could cause a change incell fate leading to abnormal growth, differentiation,migration, or premature cell death of embryonic mesen-cephalic precursors destined to contribute to the fourthventricle and anterior cerebellum (Hallonet et al., ’90)(Fig. 3E). However, given that the neuropathology ofWEXPZ-En-11 mice is located in the posterior cerebel-lum, it is unlikely that neural plate expression of En-1in the midbrain at embryonic stages is to blame.

We detected WEXP-lacZ transgene expression in theposterior cerebellum, where cystic malformations arefound in WEXPZ-En-11 mice. We speculate, therefore,that it is the aberrant expression of En-1 at later fetaland neonatal stages (i.e., after the appearance of theprimary fissure) which is responsible for the cerebellarlesions. Interestingly, Baader et al. (’98) reported abnor-mal cerebellar development and Purkinje cell loss whenEn-2 was ectopically expressed at fetal stages, confirm-ing the detrimental nature of increased Engrailed genedosage during cerebellar development. It should benoted that our findings are at variance with the hypoth-esis of some authors that cystic malformations of theposterior fossa result from an arrest of hindbraindevelopment during embryogenesis (Friede, ’89). Insupport of our model, no morphologic or histologicabnormalities have been observed in the hindbrain of

WEXPZ-En-11 embryos at 10.5, 14.5, and hydrocepha-lus presented in the neonatal period (Danielian andMcMahon, ’96; Danielian and McMahon, unpublishedobservations). In future work, a detailed analysis of theprogression of cystic lesions will be undertaken. More-over, it will be important to establish the characteristicsof WEXPZ-En-1 transgene expression in the cerebel-lum and compare this with the dynamic expressionpatterns of endogenous En-1 and En-2 genes (Millen etal., ’95; Herrup and Kuemerle, ’97). Hart et al. (’72)described other associated CNS anomalies in 68% ofhuman cases of Dandy-Walker syndrome, includingnonspecific cerebral gyral anomalies, heterotopias, agen-esis of the corpus callosum, malformation of the poste-rior olivary nuclei, and hamartomas. WEXPZ-En-11

mice do not show associated abnormalities in all likeli-hood because the transgene does not express En-1outside the midbrain-hindbrain region (Echelard et al.,’94; Danielian and McMahon, ’96). In summary, boththe WEXP transgene expression characteristics anddevelopment of cerebellar cysts in the fetal-neonatalperiod suggest that neuropathology in WEXPZ-En-11

mice commences after embryogenesis is completed.

ENGRAILED gene regulation in human braindevelopment

Later stages of En-1 and En-2 expression have beendescribed in the mouse in detail (Millen et al., ’95).While Engrailed genes have essential roles duringbrain development, the target genes they regulate arecurrently the subject of investigation (Joyner, ’96; Lo-gan et al., ’96; Lee et al., ’97). It is of interest to notesimilarities in the distribution of Engrailed transcriptsin the mouse and human midgestational fetus, suggest-ing that these genes are under similar regulatorycontrol and may have similar developmental functionsin the two species (Zec et al., ’97). Moreover, theputative regulatory region of the human EN-2 geneshows a high degree of homology to that from the mouse(Song et al., ’96). Our findings demonstrate that ectopicexpression of En-1 in the brain of transgenic miceresults in cystic malformation of the posterior vermis,with variable generalized hydrocephalus. This raisesthe possibility that abnormal regulation of Engrailedgenes or their downstream targets may be involved insome cases of cystic posterior fossa malformations inhumans.

ACKNOWLEDGMENTS

The authors especially thank Hannah C. Kinney forextensive review of the neuropathological findings andhelpful discussions, and Pieter Diekkes for experttechnical assistance. This work was supported by agrant from the NIH to A.P.M. D.H.R. acknowledges aPhysician Postdoctoral Fellowship from the HowardHughes Medical Institute and a Wyeth Pediatric Re-search Grant. P.S.D. is the recipient of a postdoctoralfellowship from the Human Frontiers Science Program.N.Z. is the recipient of a Joseph Boice Esser Fellowship.

CYSTIC CEREBELLAR MALFORMATION IN TRANSGENIC MICE 27

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