Cerebro-Craniofacial and Craniofacial Malformations: An Embryological Analysis

CHRISTL VERMEIJ-KEERS, M.D. R. F. MAZZOLA, M.D.

J. C. VAN DER MEULEN, M.D. M. STRICKLER, M.D. Leiden, The Netherlands

A macro- and/or microscopical study on the normal and abnormal development of the forebrain with the eyes, nose, and cranium, was performed in 139 mouse embryos, 120 normal and 19 abnormal human embryos and fetuses, and in about 2,300 human skulls. The results suggest that from the embryological point of view, a distinction should be made between facial defects involving the brain and/or the neural elements of the eyes, i.e., the cerebro-craniofacial dysplasias, and mal- formations of the face and cranium only, called the craniofacial dyspla- sias. Both groups can be subdivided into early or primary defects (in embryos <17 mm C-RL) and late or secondary defects (in embryos >17 mm C-RL). Almost all of the primary defects can be considered to originate from disorders occurring during the transformation of the brain and face. The secondary defects concern defective differentiation of neurectoderm and of the mesenchyme into bone centers, cartilage, and muscles. All of the defects in question can be explained by insufficient cell proliferation, degeneration, and/or differentiation. New terminology is proposed.

Introduction the literature some of the malformations have been given an embryological explanation in Knowledge of the normal embryonic and terms of the fusion (His, 1892) and merging fetal growth processes in the human brain, (Patten, 1961) theories of the facial processes, face, and cranium, is of practical value to whereas others-the rare craniofacial clefts- those who wish to understand the deviations do not fit into these schemes (Kawamoto, that occur during pathologic development. In - 1976). The neural crest cell migration theory, how- Dr. Chr. Vermey-Keers is in the Department of Anat- - ever, supported by Weston (1963) and John- omy and Embryology, University of Leiden, Wassenaar- ston (1966), leads to a possible explanation of seweg 62, 2333 AL Leiden The Netherlands, Tel.nr.: 071 - 148333 tst. 2993. Dr. R. F. Mazzola is affiliated with the some of these rare abnormalities seen in man Cattedra di Chirurgia Plastica Recostruttiva (Johnston, 1975). According to this hypothe- nell: Universita di Milano, I - 20122 Milan, Italy. Prof.Dr. sis, the neural crest cells, which are responsible J. C. van der Meulen is with the Department Plastic and for the origin of the skeletal and connective Reconstructive Surgery, University Hospital, Rotterdam, The Netherlands, and Prof.Dr. M. Stricker is affiliated tissues of the face, migrate subepidermally with the Service de Chirurgie Plastique de la face et from the ectoderm of the neural fold to their Stomatologie, Centre Hospitalier de Nancy, F - Nancy, destination, and form most of the mesen- France. Address correspondence to Dr. Chr. Vermeij- chyme of the facial processes. If they do not Keers migrate completely, are deficient in number Presented at the 4th International Symposium on Orbital Disorders on August 31, 1981 in Amsterdam, The due to defective formation or proliferation, or Netherlands. undergo abnormal differentiation, a malfor- 128

Vermeij-Keers ef al., CRANIOFACIAL MALFORMATIONS 129

mation will ensue, the severity depending on normal outgrowth of, for example, the facial the stage in which the deviation occurs. On swellings in human and mouse embryos (Ver- this basis it seemed possible to explain the mei-Keers, 1972 b, 1975 a; Gaare, 1976; Poel- pathogenesis of anomalies such as some cases mann and Verme-Keers, 1976; Gaare and - of cyclopia, severe hypotelorism, midline fa- Langman, 1980) and of the neural tube cial clefts, and the Treacher Collins syndrome. (Schluter, 1973; Geelen and Langman, 1977). However, recent microscopical studies Verme-Keers and Poelmann (1980) sug- (Vermeij-Keers and Poelmann, 1980) on the gested that a relatively high frequency of neural crest in mouse embryos showed that it physiological cell degeneration locally in the is improbable that crest cells migrate. In that neural crest is related to disorganization of study only one aspect of cell migration is the epithelium and breakdown of the basal considered, i.e., the active movements of cells lamina, as a result of which the neural crest or sheet of cells relative to the surrounding cells lost their epithelial arrangement. tissues. Movements of cells within an epithe- Johnston (1966, 1975), Noden (1975), Le lium or displacement caused by proliferation Lievre and Le Douarin (1975), and Le are not taken into account. The following can Douarin (1975) describe migration of neural be stated in this respect. The neural crest loses crest cells in the head-neck area on the basis its epithelial arrangement locally before the of transplantation experiments. This type of stage of neural fold elevation, even before the experiment has, however, several drawbacks. stage of neural plate formation, i.e., in pre- In the first place, the basal lamina of the somite stages. Thus, this event starts much neural crest becomes disrupted to a far greater earlier than is generally accepted. extent than occurs under physiological con- Cells that have been separated from the ditions. Secondly, cell-free zones into which neural crest join the mesoderm and do not cells might migrate can be introduced artifi- migrate to large cell-free zones between the cally. Thirdly, during the operations a num- mesoderm and the surface ectoderm, as stated ber of cells are undoubtedly damaged, and by some authors (Johnston, 1975). It should this artificial cell degeneration could play an be kept in mind here that cell-free spaces additional role in the disruption of the epithe- occurring in sections of intact mouse embryos lium. Thus, the transplantation procedures should be considered fixation artifacts, as in- might even induce the process of cell migra- dicated by van Oostrom, (1972) and tion. Finally, none of these experiments has Keers and Poelmann (1980). shown conclusively that an individual crest Vermeij-Keers and Poelmann (1980) have cell migrates from the neural fold to, for shown that due to outgrowth of the neurec- example, one of the facial processes, without toderm to the neural plate, of the plate to the proliferation. neural groove, and of the groove to the neural Identification of the contribution of the tube, the neural crest is shifted first laterally neural crest cells to the mesenchyme requires and then dorsally and medially relative to the a marker system developed specifically for the notochordal plate (Figure 1). During this crest cells. Use can be made of, for instance, transformation the neural crest "drops" cells immunological techniques. From present which retain their position and divide imme- knowledge it may be concluded, without in- diately. High local proliferative activity of voking the theory of neural crest cell migra- these cells can explain the development and tion, that the basic developmental processes outgrowth of the facial processes (Vermey- occurring in the cephalic region (cell prolif- Keers and Poelmann, 1980). A similar process eration, degeneration, and differentiation) is described by Gasser (1979) for the growth- can lead to normal craniofacial morphogene- associated movements of the somites and by sis (Vermeij-Keers and Poelmann, 1980). As Poelmann (1981) concerning the formation of we shall see, interference with these processes the embryonic mesoderm. may give rise to the various malformations Apart from the migration theory, Johnston observed in man. (1975) suggested that in man massive cell death could be involved in the pathogenesis Materials and Methods of brain-eye-face malformations. However, The embryonic development of the face the local occurrence of massive cell degener- and brain in man is not only extremely com- ation is a phenomenon associated with the plex but also difficult to study. When avail- 130 _ Cleft Palate Journal, April 1983, Vol. 20 No. 2 able, very young human embryos are usually fusion of the prosencephalon was studied in a in a bad cellular condition. The components 9.5 mm C-RL human embryo cut transversely of the craniofacial region are initially visible into 10 um sections. Abnormal development on the surface of the embryo but submerge as of the nose with a bilateral persistent bucco- the outgrowth of the neural folds and facial nasal membrane and a bilaterally cleft lip was processes proceeds, and become almost invis- investigated in 10 um thick transverse sections ible. Therefore, one must section the embryo, of a 20 mm C-RL human embryo. but in doing so one loses the three-dimen- Macroscoricar OmsERvATIONS In Human sional picture. Recourse must be had to vol- SKULLs. The variations found in the localiza- ume reconstructions, le. transparent (Ver- tion and direction of different facial clefts led meij-Keers, 1967; 1972 a) and graphic recon- us to examine normal human skulls to obtain structions (Tinkelenberg, 1979). The above- a basis for the explanation of certain abnor- mentioned limitations and the availability of malities. About 2,300 human skulls, repre- scanning electron microscopy explain the re- senting an unselected collection with respect searcher's unrelenting desire to study other to craniofacial defects, were used to assess the species in the hope of filling the gaps in the occurrence of agenesis of bones, the absence information on man. Animal models have of sutures, and the presence of persistent and indeed partially solved the problem, particu- extra sutures. larly because in the early stages of embryonic development the morphological differences Results between the human and non-human facial Earcty DEVELOPMENT OF THE FOREBRAIN and brain structures are small, but our present Anp Exes in ReratIONs To THE NaAsaL FIELDS knowledge still does not cover all of the stages (Pracopes). Scanning-electron- and light-mi- of cerbrofacial morphogenesis. croscopical observations in mouse embryos: Normar DEvEropmENT: Human anp Mu- day 6.6 First indication of the prospective rInE MaTERIAL. In the attempt to arrive at an neurectoderm (Poelmann, 1980; Vermeij-Keers over-all morphological description of the nor- and Poelmann, 1980) (Figure 1). mal early development of the brain and face, day 7.3 Development of the neural crest (Ver- meij-Keers and Poelmann, 1980). we studied 75 human embryos and 120 mouse day 7.4 Neural plate stage (Figure 1). embryos (CPB-S strain) microscopically. The day 7.5 Development of the neural folds. human embryos ranged from 3 to 28 mm day 7.6 First appearance of the optic primor- crown-rump length (C-RL) (3-8.5 weeks of dium. gestation) and the mouse embryos were aged day 8.1 Forming of the optic sulci (Figure 2). 6.0 to 11.0 days post coitum (p.c.). The scan- day 8.4 Forming of the optic vesicles by evag- ning electron microscope was used to study 19 ination (Figure 3). mouse embryos aged between 7.6 and 10.5 day 8.5 Fusion of the prosencephalon (Figure days p.c. ' 4). Appearance of the nasal fields as bilateral The normal differentiation of the mesen- ectodermal thickenings composed of cuboidal to chyme into the cranial bone centers was in- cylindrical epithelium and separated by the an- terior neuropore (Figure 5). vestigated microscopically in 60 human em- day 8.8 Closure of the anterior neuropore bryos and fetuses ranging from 17 to 155 mm (Figure 5). Occurrence of cell degeneration be- in C-RL (7-20 weeks). fore, during, and after closure of the anterior Amnormar Human MatERIAL. The follow- neuropore (Figure 6), followed by breakdown of ing studies were done in abnormal human the basal lamina. Presence of cuboidal to flat embryos and fetuses: A macroscopical inves- epithelium at the site of the closed anterior tigation concerning the missing elements neuropore, in the area between the nasal fields. (eye(s), nose and/or bony structures) in cyclo- The nasal fields are in continuity with the pro- pia and hypotelorism was performed in 8 spective lens placodes. human fetuses aged between 14 and 28 weeks day 9.8 The nasal placodes develop within the nasal fields. The neurectoderm is now com- of gestation and in 2 skulls of human fetuses pletely separate from the surface ectoderm, and aged about 24 and 28 weeks of gestation. The the cerebral vesicles are distinct bulges. localization and direction of facial clefts were day 9.8 to 10.7 Transformation of the nasal determined in 7 human fetuses and 2 embryos placodes via nasal grooves (Figure 7) into nasal (including one 22 mm C-RL embryo sec- tubes with interposition of e.g., the epithelial tioned frontally) and 4 human skulls Non- plate of Hochstetter. Cell degeneration in the Vermeij-Keers ef al., CRANIOFACIAL MALFORMATIONS 131

FIGURE 1. Schematic representation of the cephalic region of mouse embryos in four stages (7.3, 7.4, 7.6, and 8.3 days p.c.) based on transversely oriented microscopical sections at the same magnification. The direction of the section of the 8.3-day-old embryo lies between the transverse and frontal planes. n = notochordal plate; m = mesoderm; ne = neurectoderm; le = lateral ectoderm. In: Vermeij-Keers, Chr. and Poelmann, R. E.; The neural crest: a study on cell degeneration and the improbability of cell migration in mouse embryos. Neth. J. Zool. 30: 78, 1980.

epithelial plate of Hochstetter is observable be- eration (12 mm) with breakdown of its basal fore, during, and after fusion of the facial swell- laminas, resulting in fusion of the three processes; ings (Vermeij-Keers, 1972 b; Poelmann and Ver- 2. two plates between the lateral nasal and meij-Keers, 1976) coupled with breakdown of maxillary processes: one in the face, the other in the basal lamina. the nasal lumen: (a) the naso-lacrimal duct is day 11.0 Disappearance of the bucco-nasal isolated by cell degeneration from the medial membrane, due to a degenerative phenomenon. border of the epithelial plate on the facial side (12-15 mm), (b) the meatus nasi inferior evolves TransrormatIOnNs or tHE BRAIN aND Fack. from the plate; during fetal development it opens Light-microscopical observations in normal again, on the side of the nasal lumen (no cell human embryos: degeneration present in this plate). 3.0-4.0 mm C-RL (3-4 weeks)-The neuro- Outgrowth of the two medial nasal processes porus anterior is closed. The nasal fields emerge. _ in the interplacodal area, which are separated 6.0-6.5 mm C-RL (4.5 weeks)-Development by a wide groove, the internasal groove (7 mm of the nasal placodes within the nasal fields. We C-RL). The region between the interorbital and call the area between the nasal placodes the the internasal grooves is called the triangular interplacodal area (Figure 8). First indication of area (Figure 9). Cerebral vesicles are distinct (8.0 the cerebral vesicles, i.e., "doming" of the lateral mm C-RL). walls of the forebrain. 12.0-17.0 mm C-RL (6-7 weeks)-Transfor- 7.0-12.0 mm C-RL (5-6 weeks) mation of the primitive oral cavity by outgrowth mation of each nasal placode via nasal groove of swellings into this cavity. Development, on into nasal tube by the outgrowth, in a fronto- both sides in its ectoderm, of a rather narrow caudal direction, of three facial swellings (the epithelial plate between the maxillary and man- maxillary process and the medial and lateral dibular processes, this plate extending forward nasal processes) separated from each other by from the vicinity of the ear anlage to the corner grooves. This outgrowth is coupled with inter- of the primary mouth opening (Figure 9). As a positioning of epithelial plates, not only in the result of cell degeneration combined with break- superficial facial region but also deep in the down of the basal lamina, an epithelial cord nasal lumen: remains, the organ of Chievitz (17 mm). Between 1. the epithelial plate of Hochstetter at the the same boundaries another epithelial plate site of contact between the three swellings (7-12 develops in front of this organ, i.e., the first mm); disappearance of this plate by cell degen- anlage of the parotid gland (17 mm). 132 Cleft Palate Journal, April 1983, Vol. 20 No. 2

FIGURE 2. Scanning electron micrograph of a mouse embryo aged about 8.0 days p.c., showing the optic sulci (}). (Cranio-frontal view).

The outgrowth of the mandibular processes which is accompanied by breakdown of the basal or nandibular arch takes place without formation laminas. of an epithelial plate in the median line (Figure 9). Closure of the optic fissure (17 mm C-RL) DeverormENT or somE or tHE MEemBRranE occurs, without cell degeneration. Bones or tue Skuut in Normar Human Em- Presence of the primitive choana, ie. the BRYOS AND FETUSES. opening between the primitive nasal and primi- 17-155 mm C-RL (7-20 weeks)-The left and tive oral cavities, after the breakthrough of the right parts of the upper jaw are formed from bucco-nasal membrane (degeneration process, separate elements, the premaxilla and the max- 17 mm). illa. The premaxilla, which bears two incisor 17.0-27.0 mm C-RL (7-8.5 weeks)-The out- teeth, differentiates on both sides from the mes- growth and differentiation of the nasal septum enchyme of the medial nasal process and has (the medial nasal processes and triangular area) two bone centers (Arey, 1965). The maxilla, the in the frontocaudal direction leads to the disap- palate, and the zygomatic, nasal, and lacrimal pearance of the internasal and interorbital bones each develop mainly from a single center, grooves. The distance between the eyes shows a whereas the vomer, the frontal and the mandible relative decrease (Figure 9). are formed by paired centers. The first mem- The development of the secondary palate is brane bone centers arise in 17 mm embryos, e.g. combined with, for example, the formation of an in the mandible and maxilla. epithelial plate between the two palatine pro- The formation of sutures of the viscerocran- cesses (+ 27 mm C-RL). This plate too loses it ium starts in embryos of about 25 mm C-RL, continuity by degeneration of its epithelial cells, ie., with the symphysis of the mandible, whereas Vermeij-Keers ef al., CRaANIOFACIAL MALFORMATIONS 133

FIGURE 3. Scanning electron micrograph of a mouse embryo aged 8.4 days p.c. Prosencephalon with the optic vesicles preparatory to fusion. (Frontal view).

in the neurocranium it occurs in fetuses of about on bone (Figure 10). The narrow palate is 5.5 cm C-RL and almost all sutures are present closed. The deformed vomer, consisting of two in specimens of 7.5 mm C-RL (14 weeks of triangular bone centers, is joined to the flat development). palate occipitally and connects the two pter- ApBnormaLt Human EmsBrvos, FEtusEs, anp ygoid processes which, as it were, block the secondary choana. Macroscopical observations on cyclopia and hypo- Macroscopical observations of the facial clefts: telorism: One human embryo, 6 of the fetuses, and 4 Cyclopia-a single eye or closely approxi- human skulls show a cleft in the primary mated eyes with all integrades, synophthal- palate uni- or bilaterally, ie., cheilognathos- mus, and synorbitism, in a single orbit-is chisis with or without palatoschisis. The clefts represented in our material by five fetuses; in the primary and secondary palates are three fetuses showed hypotelorism (see also located at sites of epithelial plates. The facial Table 1). swellings are not fused with each other, and In both cyclopic fetal skulls-one with a the same holds for the palatine processes. bony proboscis constructed of three bone cen- Some of the fetuses show incomplete clefts. ters and the other showing a single frontal Fetus Eb 148 has a cleft across the maxilla, bone developed from one midline bone center i.e., an oro-ocular cleft, on the left side of the without proboscis-the ethmoid, the nasal face, adjacent to the normal nose. and lacrimal bones, the nasal septum, and the Light-microscopical observations: 9.5 mm C-RL two premaxillae show agenesis. The maxillae embryo with the prosencephalon not closed. on both sides have fused and form one block Neither the optic sulci nor the nasal fields are 134 Cleft Palate Journal, April 1983, Vol. 20 No. 2

FIGURE 4. Scanning electron micrograph of a mouse embryo aged 8.5 days p.c. Prosencephalon fused except for the anterior neuropore (1), which separates the two nasal fields (nf). (Frontal view, heart removed). present. The development of the branchial Macroscopical observations in human skulls: arch system corresponds with that of a normal Agenesis of bone was observed only for 11.0 mm C-RL human embryo. nasal bones, in 6 of the 2,300 skulls. 22.0 mm C-RL embryo with a cleft on the Absence of sutures is understood not only left side of the upper lip. The facial swellings the normal situation (in which fusion of su- on the right side are normally fused and tures occurs during aging) and premature Hochstetter's epithelial plate has disappeared synostosis, but also their absence as an effect completely due to cell degeneration. The ep- of "cleft" formation. The last was observed in ithelial plate on the left side, which shows no 2 cases, in one of which the zygomatic arch cell degeneration and has uninterrupted basal showed a break at the site of the temporozy- laminas, is still present (Figure 11). gomatic suture (the temporal process of the 20.0 mm C-RL embryo with underdevel- zygomatic and the zygomatic process of the oped flat nose. Bilateral atresia is combined temporal bone were both shortened and with persistence of the bucco-nasal membrane rounded) and in the other the frontal suture and partial persistence of the epithelial plates was missing (Figure 12). of Hochstetter, resulting in a lip with a cleft Persistent sutures (e.g., the frontal suture, on both sides. No signs of development of the the incisive suture) and remnants of sutures future inferior concha or the Jacobson organ. (e.g., in the symphysis of the mandible), were The naso-lacrimal ducts are absent. The pal- seen frequently. atine processes are in a vertical position, Le. Extra sutures also occur. An extra suture the normal situation for this developmental represents a suture within a membrane bone stage. developing normally from a single bone cen- Vermeij-Keers ef al., CRANIOFACIAL MALFORMATIONS 135

FIGURE 6. Detail of Figure 5, showing the anterior neuropore with degenerating cells and macrophages (1).

ter. This phenomenon was observed 4 times localization of the extra sutures differed (e.g., unilaterally in the maxilla, not always on the lateral or medial to the infra-orbital foramen). same side. Two of these extra sutures were The bipartite zygomatic was present uni- or present in the frontal process of the maxilla bilaterally in 12 skulls and the bipartite or and two in the body of the maxilla. The tripartite nasal bone in 6 skulls. 136 Cleft Palate Journal, April 1983, Vol. 20 No. 2

FIGURE 7. Scanning electron micrograph of a mouse embryo aged 10.0 days p.c. (Heart removed.) Nasal placodes have been transformed into nasal grooves by the medial nasal (mnp), lateral nasal (Inp), and maxillary processes (mp). ing = internasal groove.

Discussion dysplasias (for this term, see van der Meulen et al., in press) and defects of the face and Introduction of a new classification of mal- cranium only, called the craniofacial dysplasias. formations in the cephalic region after the Both groups can be subdivided into early or publication of so many topographically orien- primary and late or secondary developmental tated examples (Pfeifer, 1967, 1974; Mazzola, defects (dysplasias). 1976; Tessier, 1976), is only useful if a new CEereEBRO-Crantoracitat DysPLAsIAS approach has been taken, i.e., if the underly- Early or primary defects <17 mm C-RL. The ing embryological mechanisms have been onset of early cerebro-craniofacial defects oc- studied and taken into account. The result is curs before the embryo has reached the 17 a simpler classification of the known malfor- mm C-RL stage. In all 8 cases representing mations, with groups of comparable defects cyclopia (synophthalmus and synorbitism) (for the clinical aspects and nomenclature, and hypotelorism, which correspond roughly reference is made to van der Meulen et al., in with types I-IV of DeMyer et al. (1964), and press). in 2 cyclopic skulls, the interplacodal area In the first place, a distinction should be (the future medial nasal processes) is missing. made between facial malformations with in- This situation forms the embryological basis volvement of the brain and/or the eyes, in for the experimental induction of cyclopia by other words the cerebro-crantofacial defects or draining of the interplacodal area after clo- k ~

Vermey-Keers ef al., CRANIOFACIAL MALFORMATIONS 137

......

++ 45......

epithelium flat to cuboidal

cuboidal to cylindrical 3

high cylindrical otis FIGURE 8. Graphic reconstruction of a 6.5 mm C-RL human embryo showing the forebrain with the developing eyes and both nasal placodes; frontal view. High cylindrical epithelium = nasal and lens placodes. ipa = interplacodal area, ov = optic vesicle. sure of the anterior neuropore (Venneman, same period as the differentiation of the nasal personal communication). The missing inter- placodes (6.0-6.5 mm C-RL)-does not occur placodal area can be explained by insufficient in cases of holoprosencephalia. The absence outgrowth of the surroundings of the optic of the interplacodal area in types I-IV leads sulci, probably due to the absence of sufficient to fusion of the two nasal fields. In cyclopia proliferation of the mesoderm or to insuffi- the nasal placode(s) do not develop at all, cient cell degeneration in the neural crest, the giving cyclopia without proboscis (2 of our 5 latter resulting in the dropping of too few cases), or differentiate within the nasal fields mesectodermal cells. Lieuw Kie Song and above the eye vesicle(s), giving cyclopia with Been (1980) induced this insufficient drop- proboscis (3 examples) and extreme hypote- ping by using laser rays to destroy the entire lorism with -a septated proboscis (one exam- neural crest in the prosencephalic part of the ple). Hypothetically, the latter group of ex- neural tube of the chick embryo. amples (with proboscis) might be explained The optic vesicles can fuse totally (one eye, as follows. Normally (mouse embryos: this one orbit), partially (two eyes, one orbit), or study; human embryos: O'Rahilly (1966, almost (hypotelorism), depending on the de- 1967); O'Rahilly and Gardner (1971); see also gree of growth retardation. the abnormal embryo of 9.5 mm C-RL), the Furthermore, according to DeMyer et al., nasal fields develop after the anlage of the eye (1964), these types are holoprosencephalic. vesicles, and, furthermore, the nasal fields are Development of the cerebral vesicles of the larger than the nasal placodes and in conti- prosencephalon-which normally starts after nuity with the ectoderm of the future lens the formation of the eye vesicles but in the placodes. Dislocation of the nasal placode(s) 138 _ Cleft Palate Journal, April 1983, Vol. 20 No. 2

\_"))>) & : m <- -*) \////\ })

my 15 mm CRL adult FIGURE 9. The face of a 15 mm C-RL human embryo (drawing of a glass-plate reconstruction) with the facial swellings indicated in relation to an adult face. Localization of epithelial plates is indicated by dotted lines. jog = interorbital groove; ta = triangular area; ing = internasal groove; Inp = lateral nasal process; mp = maxillary process; mnp = medial nasal process; m = mandible.

TABLE 1. Cyclopia and Hypotelorism

Fetus No. Facial Features Other Craniofacial Defects

1 cyclopia: microcephaly, agnathus with astomus single eye in single orbit, arhinia without proboscis 2 synophthalmus: microcephaly, i.e., right head in a case of fused eyes in single orbit, arhinia without dicephalus; left head is normal proboscis 3 _ synophthalmus: microcephaly eyes not completely fused in single orbit, arhinia with proboscis 4 synorbitism: anencephaly two eyes in single orbit, arhinia with pro- boscis 5 synorbitism: microcephaly, agnathus with astomus two eyes in single orbit, arhinia with pro- boscis 6 extreme hypotelorism but separate orbits, microcephaly, agnathus with astomus arhinia with septated proboscis 7 hypotelorism; flat nose; agenesis premax- acrania with encephalocele illae, median part upper lip and nasal septum; cleft palate 8 hypotelorism; flat nose; agenesis premax- cranio-rachischisis illae, median part upper lip and nasal septum; cleft palate

in the nasal field(s) outside the range of the in DeMyer's types III and IV and in two of maxillary pocess(es) would lead to the mal- _ our cases of hypotelorism (Table 1, nos. 7 and formation. 8) are transformed only by the lateral nasal The fused nasal placodes of the flat noses and maxillary processes. In type V of DeMyer Vermeij-Keers ef al., CRANIOFACIAL MALFORMATIONS 139

FIGURE 10. Frontal view of the skull of a neonate with cyclopia (neurocranium removed via a horizontal section}. iof = infra-orbital foramen; oc = optic canal; p = proboscis.

FIGURE 11. Frontal section of a 22 mm C-RL human embryo with cleft lip (Heidelberg collection). The persistent epithelial plate on the affected side shows no signs of cell degeneration (1).

140 Cleft Palate Journal, April 1983, Vol. 20 No. 2 __

FIGURE 12. Neonatal skull with defective formation of the frontal suture. Both frontal bone centers show arrested growth. et al. (1964) (see also Mazzola, 1976), showing rubella syndrome (Vermeij-Keers, 1975 b), hypotelorism with premaxillae anlage, the in- causes anophthalmia or microphthalmia and terplacodal area is present but too narrow. underdevelopment of the viscerocranium on From the embryological point of view the the affected side. eyeballs-except for the lens-form part of Cell degeneration plays a part in the nor- the brain, and therefore malformations of the mal development of the mammalian eyeball eyeballs (neural elements) are grouped with (Silver and Hughes, 1973) and lens (Schook, the cerebro-craniofacial defects. However, the 1980). Silver and Hughes (1974) postulated influence of the lens on the size of the eyeball that lack of cell degeneration in the early is very important. For instance, primary con- developmental stages of the eye inhibits the genital aphakia, occurring e.g. as part of the invagination of the optic vesicle and thus Vermeij-Keers ef al., CRANIOFACIAL MALFORMATIONS 141 leads to anophthalmia or microphthalmia. separate organs, holds the key position in the Moreover, the optic fissure will not be formed. developing face. Abnormal topic fissure development (Silver In view of the changes-not only in the and Hughes, 1973) and improper closure of shape of the face but also in that of the nasal the fissure (Arey, 1965) cause congenital co- luman-associated with the formation of the lobomata of the iris, ciliary body, retina, and/ organ of Jacobson and the inferior nasal mea- or choroid tunic. Normally, the fissure closes tus it is unlikely that ingrowth of the epithe- without cell degeneration (Silver and Hughes, lium of the nasal placodes, as described by 1974) and with fusion of its basal lamina, 1.e., Peter (1913), Andersen and Matthiessen intra-epithelial fusion. (1967) and many others, takes place. Further- Late or secondary defects =17 mm C-RL. Age- more, the outgrowth of the. maxillary nesis of the corpus callosum, a structure which processes is not coupled with changes in the normally originates in the 54 mm C-RL stage shape of the first branchial arch. For this last (Hochstetter, 1919), is often combined with reason, the maxillary process must represent extreme hypertelorism. Other examples con- a separate swelling and does not form part of cern arhinencephalia with arhinia and pro- the mandibular arch. boscis, and arhinencephalia with trigonoce- Two fusing mandibular swellings described phalia, in which premature synostosis of the by, for instance, Patten (1953) were not ob- metopic suture has taken place or the frontal served in our material. bone has originated from a single bone center. The localization of the naso-lacrimal In this last case the external nose can be groove is very important with respect to the normal (Warkany, 1975). For suggestions con- eye anlage. At the site of the groove an epi- cerning the development of arhinia, see below thelial plate is laid down by outgrowth of the under Craniofacial malformations (early defects). maxillary and lateral nasal processes, from CrantoracIaAt DysPLASIAS which the naso-lacrimal duct develops by cell Early or primary defects <17 mm C-RL. The degeneration. The naso-lacrimal groove does transformations occurring in the human face not end in the medial corner of the eye, as normally take place after the closure of the many authors state, but more lateral to it in neuroporus anterior lying between the two the swelling representing the developing lower nasal fields. The location of the anterior neu- eyelid (Fig. 9). It is the interorbital groove ropore, i.e., between the two nasal fields, is that ends at the medial corner of the eyes (12 described in mouse embryos in this study, and mm stage; Vermeij-Keers, 1972 a). O'Rahilly (1967) and O'Rahilly and Gardner Besides outgrowth (cell proliferation), cell (1971) have described the same in human degeneration is mandatory for normal early embryos (for further details, reference is made embryonic development (Vermey-Keers, to Vermeij-Keers et al., in prep.). 1972b; Vermeij-Keers and Poelmann, 1980; Disturbances of the closing mechanism, for Silver and Hughes, 1974; Menkes, 1968). In instance absence of cell degeneration with general, the transformation of the face can be breakdown of the basal lamina (ie., of inter- described as follows. Swellings grow out to- epithelial fusion of the anterior neuropore) wards each other; at places of contact, epithe- can explain dermoid (syn.: midline) cysts and lial plates develop, with or without cell degen- midline fistulas, which are generally described eration, but always with changes in the shape as separate entities (e.g. Mazzola, 1976). The of the face, of the nasal lumina, and/or of the nasal placodes develop within the nasal fields oral cavity. (6.0-6.5 mm C-RL) separated by the inter- The following primary defects are based on placodal area. Around both nasal placodes disorders associated with development and three swellings develop, grow out with respect transformations of the facial swellings. to the prosencephalon, and transform the Nasal aplasia. To underline the fact that the placodes, via the nasal groove, into the nasal nose develops from two nasal elements, we tube (Vermeij-Keers, 1967,1972 a). All three propose that the term nasal aplasia be substi- swellings participate in the formation of the tuted for half-nose (i.e., one nasal element) floor of the tube and the primary palate (Pe- and arhinia for agenesis of the nose (i.e., both ter, 1913; Vermeij-Keers, 1972 a). nasal elements). Thus the nose, initially composed of two The observations made in the 20 mm C- 142 _ Cleft Palate Journal, April 1983, Vol. 20 No. 2

RL human embryo may be of some interest early embryonic development of the face up for the explanation of this anomaly. This to and including the 17 mm C-RL stage, and embryo shows bilateral persistence of the that ofthe secondary palate from the 27 mm bucco-nasal membrane, undeveloped naso- C-RL stage on. lacrimal ducts, and underdeveloped nasal lu- Primary clefts can be caused by several factors: mina, which means that Jacobson's organ and I. Absence of cell proliferation, that is, out- the inferior concha failed to form. There are growth of the facial swellings, as a result of no primitive choanae and the mesenchyme of which the processes do not come into con- the primary palate is in continuity with that tact with each other and the epithelial plates cannot develop. of the future base of the skull (sphenoid bone), II. Absence of cell degeneration in the ecto- thus forming a block of mesenchyme. Nor- derm of the epithelial plates. Here, sufficient mally, the bucco-nasal membrane breaks proliferation has occurred for the swellings through by cell degeneration in the 17 mm to make contact and to lay down an epithe- stage, thus giving rise to the development of lial plate. the primitive choana. In cases of nasal aplasia A. Discontinuity of the basal lamina does and arhinia, degenerative phenomena have not occur (see description of the left cleft of probably failed to occur. the 22 mm C-RL embryo); the plate opens Another possible explanation can be found again and forms a groove (compare the very early in development when the nasal development of the inferior nasal meatus). Both I and II A lead to the formation of fields/nasal placodes should but do not differ- complete clefts. entiate (see the abnormal human embryo of B. Discontinuity of the basal lamina by cell 9.5 mm C-RL). Normally, the future lateral degeneration takes place, i.e., interepithelial and medial nasal and the maxillary processes fusion, but subsequent cell degeneration is are situated round the nasal placode at 6.0- insufficient and the epithelial plate does not 6.5 mm C-RL (Vermeij-Keers, 1972 a). In the disappear completely. The remnants of the absence of the placode(s) the affected side(s) epithelial plate open again to form a groove. of the nose, with, e.g., the naso-lacrimal A tissue bridge remains between the medial duct(s), will not develop and bone tissue can and lateral walls. The result is an incomplete subsequently differentiate within the non- cleft. __ III. An incomplete cleft can also develop as a transformed mesenchymal block. result of a combination of I and II. Nasal aplasia with proboscis. An abnormal The true or primary clefts all coincide localization of the nasal placode within the with a site of an epithelial plate: nasal field probably explains the occurrence a. between the lateral nasal and maxillary of arhinia or nasal aplasia with proboscis (see processes on the one hand and the medial under Cerebro-crantofacial malformations). How- nasal process on the other, ever, Kirchmayr (1906) and McLaren (1955) b. between the lateral nasal and maxillary described cases with an almost normally de- processes, c. between the maxillary and mandibular veloped nose in association with proboscis and processes, other primary defects, i.e., cleft lip and palate. d. between the palatine processes. Theoretically, this combination of a normal All of the primary clefts show an ectoder- nose with proboscis could be the result of mal defect along the extension of the affected differentiation of a double nasal placode epithelial plate. In other words, the ectoderm within one nasal field. above the affected epithelial plats has not Nasal duplication. The explanation proposed closed. The localization indicated under a, for the existence of an almost normal nose coinciding with the epithelial plate of Hochs- with proboscis could also hold for nasal or tetter, corresponds with the common cleft lip rhinal duplication. Theoretically, this dysplasia (cheiloschisis or cheilognatoschisis). The na- could be the result of differentiation of a solacrimal groove marks the cleft mentioned double nasal placode within one nasal field. under b, and is often combined with a com- In the more severe cases with an extra orbit mon cleft lip, resulting in a defect running (diprosopia) (see Mazzola, 1976) the possibil- from the mouth through the nose to the swell- ity of conjoined twins should be considered. ing of the lower eyelid lateral to its inner Primary clefts. The true clefts or primary corner (comp. Morian I, 1887, oro-naso-ocular clefts can be explained on the basis of the cleft). Vermeij-Keers ef al., CRANIOFACIAL. MALFORMATIONS 143

The ectodermal part of Tessier's "no.: 7 tween two membrane bones at sites of sutures cleft" (1976), which represents maxillo-man- (see van der Meulen et al., in press, e.g. max- dibular dysplasia or macrostomia, corre- illo-zygomatic, zygo-frontal, and intermandi- sponds with c. Type d represents the cleft bular dysplasia and, in this report the discus- palate. sion of the skulls without the temporo-zygo- The defects of the bones and musculature matic suture and the frontal suture). In these (e.g., the facial muscles) are the result of these cases one or both bones can grow unequally. abnormalities of development. Point 3 concerns bony defects with a highly Late or secondary defects z=17 mm C-RL. In variable localization within a membrane bone principle, closure of the ectoderm of the face (see Tessier's, 1976, nos. 10 and 11; 4 and 5, is completed in the 17 mm stage. All three i.e., Morian II and III, 1887; see also van der "facial" epithelial plates have developed, and Meulen et al., in press, e.g. maxillary dyspla- the mesenchymal cores of the swellings have sia, and in this report fetus Eb 148). fused. Now, differentiation of the mesen- Point 4 relates to, e.g., the frontal process chyme, i.e., into bone centers, cartilage, and of the maxilla. facial muscles, can take place. The most im- Points 1-4 can be classified under the term portant factors during the late development dysostosis. of the face are the number and degree of Point 5 refers to synostosis between bones outgrowth of the bone centers. Defective dif- of the viscerocranium and/or neurocranium. ferentiation causes what are called the second- The suture formation in the viscerocranium ary defects or dysplasias. starts with that of the mandible in embryos In general, the bone centers grow out and of about 25 mm C-RL, 8th week of develop- make contact with each other. At the places ment. Suture formation in the neurocranium of contact, sutures develop. Their localization begins in the 55 mm stage and has finished in and direction can differ very widely between fetuses of about 7.5 cm C-RL (14 weeks of the left and right sides of one skull and be- gestation). : tween skulls. After birth, some sutures ossify, Defective differentiation into cartilage of, e.g., the frontal suture in the second year. e.g., the nasal septum and alar cartilages, This particular suture can also persist as the results in the bifid nose or internasal dysplasia metopic suture. (see this classification and also van der Meu- Differences in the number of bone centers len et al., in press) and "clefting" of the nasal in, for instance, the nasal and zygomatic ala or nasoschisis, respectively. bones and the maxilla, occur in "normal" Internasal dysplasia. During formation of the skulls, as indicated by extra sutures. These facial swellings situated around the two nasal extra sutures can differ in localization and placodes, the internasal groove develops in direction; for example, an extra suture in the the midline between the two medial nasal maxilla can lie laterally or medially from the processes (7 mm stage). Because we consider infra-orbital foramen. the early development of the nose as that of - Secondary defects can arise in this devel- two separate organs, (see above), we have opmental period, due to the absence of: introduced the term internasal groove to re- 1. anlage of bone centers, place the terms area infranasalis (e.g., Peter, 2. outgrowth of bone centers between two 1913) and infranasal groove (Sedano et al., adjacent membrane bones, 1970). Moreover, our terminology expresses 3. outgrowth of one or more bone centers the concept of outgrowth of the facial swell- in membrane bones having multiple ings. The medial nasal processes develop in bone centers, and the area between the two nasal placodes, 4. hypoplasia of bone(s) or of parts of which we call the interplacodal area. We bone(s) after normal development of the found it necessary tG introduce this term be- bone centers, and cause we consider the embryological term 5. premature synostosis. fronto-nasal process unacceptable on the Point 1 concerns, e.g., absence of the malar grounds that, unlike the nasal and maxillary bone in the Treacher Collins syndrome and, processes, this entity is not a swelling. I in the present study, agenesis of the nasal Due to insufficient outgrowth of the nasal bones. septum in the fronto-caudal direction (17-27 Point 2 refers to bony defects situated be- mm C-RL), the internasal groove will not 144 Cieft Palate Journal, April 1983, Vol. 20 No. 2

disappear and the midfacial part will remain degeneration, and/or differentiation. Combi- wide. The result is internasal dysplasia (see nations of primary and secondary defects be- van der Meulen et al., in press). The most longing to the cerebro-craniofacial, craniofa- severe cases show, in combination with the cial, and cerebro-cranial groups are possible. persistence of the internasal groove, other sec- Acknowledgment: The authors thank Dr. ondary defects such as hypertelorism and a G.J.R. Maat for advice concerning the study body defect situated between the two premax- of the skulls, J. Koppenberg for the collection illae and/or a median cleft lip. of the data, and Dr. R.E. Poelmann and Mrs. Hypertelorism. Physiologically, embryos =17 M.M.T. Mentink for the performance of the mm C-RL show hypertelorism (Fig. 9). Dur- scanning-electronmicroscopical techniques of ing normal outgrowth of the mid-facial part this study. in the fronto-caudal direction, the distance between the eyes shows a relative decrease. If References this development does not take place, the AnperseEn, H. and MattHiessen, M. E., Histochemistry hypertelorism will persist. of the early development of the human central face Nasoschisis. In this defect the early devel- and nasal cavity with special reference to the move- opment of the nose has been completely nor- ments of fusion of the palatine processes. Acta Anat., 68: mal and both external nostrils are present, 473-508, 1967. but the latter show a notch or even two Arry, L. B., Developmental Anatomy. Philadelphia and London, W. B. Saunders Company, 7th Ed., 1965. notches, the result of defective differentiation DeMyrEr, W., Zeman, W. and PaimEr, C. G., The face of the mesenchyme into cartilages of the ala. predicts the brain: diagnostic significance of median Besides aplasia or hypoplasia of bone or facial anomalies for holoprosencephaly (arhinen- cartilage, the secondary defects include im- . cephaly). Pediatrics, 34: 256-263, 1964. perfect facial muscles, with or without asso- GaAARE, J. D., Cell degeneration during the fusion of the nasal processes in mice. Anat. Rec., 184: 407, 1976. ciated anomalies of ectodermal origin. Defects GaARE, J. D. and Lanoman, J., Fusion of nasal swellings characterized by interrupted ectodermal lin- in the mouse embryo. DNA synthesis and histological ing can be classified as secondary clefts. features. Anat. Embryol. 159, 1: 85-99, 1980. The explanation of secondary defects holds Gasser, R. F., Evidence that sclerotomal cells do not migrate medially during normal embryonic develop- for both viscerocranium and neurocranium. ment of the rat. Am. J. Anat., 154: 509-524, 1979. In general, it may be concluded that the GEELEN, J. A. G. and Lanoman, J., Closure of the neural distinction between primary and secondary tube in the cephalic region of the mouse embryo. Anat. defects, which applies to the cerebro-cranio- Rec., 189: 625-640, 1977. facial and craniofacial dysplasias, also holds His, W., Die Entwickelung der menschlichen und thier- ischer Physiognomien. Arch. Anat. Physiol., Anat. Abt.: for the group of cerebro-cranial malforma- 384-424, 1892. tions (see Table 1, primary defects: anenceph- HocHmstETTER, F., Beitrage zur Entwicklungsgeschichte aly and craniorachischisis, and secondary de- des menschlichen Gehirns. I. Teil, Wien und Leipzig, fects: acrania with encephalocele). Franz Deuticke, 1919. The cases of agnathus with astomus (Table Jounston, M. C., A radioautographic study of migration and fate of the cranial neural crest cells in chick 1) are based on persistence of the bucco-pha- embryo. Anat. Rec., 156: 143-155, 1966. ryngeal membrane and absence of outgrowth Jounston, M. C., The neural crest in abnormalities of of the mandible. the face and brain. Ed. Daniel Bergsma. Morphogen- Many malformations in the zygo-temporo- esis and Malformations of Face and Brain. The Na- tional Foundation - March of Dimes; Birth Defects: mandibular corner are accompanied by de- Original article series Vol. XI, No. 7. New York, Alan fects of the external and middle ear, both R. Liss, Inc., 1975. being derivatives of the branchial arches, and Kawamoto, H. K., The kaleidoscopic world of rare cra- rarely of the internal ear, which develops out niofacial clefts: Order out of chaos (Tessier classifica- of the auditory placode. This combination of tion). Clin. Plast. Surg., 3: 529-572, 1976. KircHmaAyR, L., Ein Beitrag zu den Gesichtsmissbildun- defects is easy to explain by the fact that the gen. D, Ztschr. f. Chir., 81: 71-81, 1906. basic processes involved in the transforma- LEDouvARIN, N., The neural crest in the neck and other tions of the external, middle, and internal ear parts of the body. Ed. Daniel Bergsma. Morphogenesis are identical to the transformations of the face and Malformations of Face and Brain. The National Foundation-March of Dimes; Birth Defects: Original and, furthermore, the two occur synchro- article series Vol. XI, No. 7, New York, Alan R. Liss, nously. Inc., 1975. All the groups of defects mentioned here Le Lievrr, C. S. and LE DouarRin, N., Mesenchymal can be explained by insufficient proliferation, derivatives of the neural crest: analysis of chimaeric Vermeij-Keers ef al., CRANIOFACIAL MALFORMATIONS 145

quail and chick embryos. /. Embryol. exp. Morph., 34: Publishing Company, 1976. 125-154, 1975. ' . ' SeHruUTER, G., Ultrastructure observations on cell necrosis LiEuw Kis Sonc, S. H. and Bren, W., Median facio- during formation of the neural tube in mouse embryos. cerebral anomalies in chick embryos resulting from Z. Anat. Entwickl. Gesch., 141: 251-264, 1973. local destruction of the anterior most parts of the early ScHook, P., A spatial analysis of the localization of cell neural plate and neural crest. Acta. Morph. Neer!.-Scand., division and cell death in relationship with morpho- 18: 231-252, 1980. genesis of the chick optic cup. Acta Morph. Neerl.-Scand., Mazzora, R. F., Congenital malformations in the fron- 18: 213-229, 1980. tonasal area: Their pathogenesis and classification. SeEpano, H. O., Comrn, M. M. Jr., Jirasex, J. and Clin. Plast. Surg., 3: 573-609, 1976. Gorin, R. J., Frontonasal dysplasia. J. Pediat., 76: McLarEn, L. R., A case of cleft lip and palate with 906-913, 1970. polypoid nasal tubercle. Br. J. Plast. Surg., 8: 57-59, SIcver, J. and HucnHuEs, A. F. W., The role of cell death 1955. during morphogenesis of the mammalian eye. /. MrEnxrEs, B., Localized necrosis and necrobiosis in terato- Morph., 140: 159-170, 1973. genesis. Rev. Roum. d'Embryol. Cytol.: ser. d'Embryol., 5: Siver, J. and HucHuEs, A. F. W., The relationship be- 139-149, 1968. tween morphogenetic cell death and the development MortAN, R., Ueber die schrage Gesichtsspalte. Arch. K/iin. of congenital anophthalmia. /. Comp. Neur., 157: 281- Chir. 35: 245-288, 1887. 302, 1974. Nopen, D. M., An analysis of the migratory behavior of T'eEssiEr®, P., Anatomical classification of facial, cranio- avian cephalic neural crest cells. Dev. Biol., 42: 106- facial and laterofacial clefts. J. max.-fac. Surg., 4: 69-92, 130, 1975. 1976. O'Raniccy, R., The early development of the eye in TinkEtEnBERG, J., Graphic reconstruction, micro-anat- staged human embryos. Contr. Embr., 38: 1-42, 1966. omy with a pencil. /. Audiovisual Media in Medicine, 2: O'RaniLLy, R., The early development of the nasal pit 102-106, 1979. in staged human embryos. Anat. Rec., 157: 380, 1967. Van per MruLEn, J. C., Mazzora, R. F., VermEj-KEeEeRs, R. and GARDNER, E., The timing and se- CiHrR., STtrickE®Rr, M. and Rapnar1, B., A morphogenet- quence of events in the development of the human ical classification of craniofacial malformations. ]. nervous system during the embryonic period proper. Plast. Reconstr. Surg., in press. Z. Anat. Entwickl. Gesch., 134: 1-12, 1971. Van Oostrom, C. G., De initiele regionale ectodermon- PaTtTEN, B. M., Human Embryology. London, J. and A. twikkeling in het kopgebied bij de muis. Thesis, Am- Churchill, Ltd., 2nd. ed., 1953. sterdam, 1972. PatTTEN, B. M., The Normal Development of the Facial CnR., De facialis musculatuur en trans- Region. Ed. S. Pruzansky. Congenital Anomalies of formaties in het kopgebied. Thesis, Leiden, 1967. the Face and Associated Structures. Springfield, Ill., CHER., Transformations in the facial re- Charles C Thomas Co., 1961. gion of the human embryo. Erg. Anat. Entwickl., 46, 5: PETER, K., Atlas der Entwicklung der Nase und des 1-30, 1972 a. Gaumens beim Menschen. Jena, G. Fischer Verlag, VrErmEIj-KrErErRrs, Cnur., Degeneration in the epithelial 1913. ' plate of Hochstetter in the mouse: a light and electron PrEirER, G., Die Entwicklungsstorungen des Gesi- microscopic study. Acta Morphol. Neerl.-Scand., 9: 386- chtsschadels als Klassifikationsproblem. Deutsch. Zahn- 387, 1972 b. ~ ‘ Mund-Kieferhielk., 48: 22-40, 1967. VrermEe1j-Kerrs, CinR., The development of the nose, with PrEirER, G., Systematik und Morphologie der kraniofa- the naso-lacrimal duct of the human embryo. Acta zialen Anomalien. Fortschr. Kiefer - u. Gesichtschir., 18: Morphol. Neerl.-Scand., 13: 126-127, 1975 a. 1-14, 1974. Cinr., Primary congenital aphakia and PorrmaNNn, R. E., Differential mitosis and degeneration the rubella syndrome. Teratology, 11: 257-265, 1975 b. patterns in relation to alterations in shape of the Vrermrj-KEerErs, Cnr. and PoELMANN, R. E., The neural embryonic ectoderm of early postimplantation mouse crest: a study on cell degeneration and the improba- embryos. /. Embryol. exp. Morphol., 55: 33-51, 1980. bility of cell migration in mouse embryos. NetA. J. Zool., PoErmaANN, R. E., The formation of the embryonic mes- 30: 74-81, 1980. oderm in the early postimplantation mouse embryo. Warrkany, J., Congenital Malformations. Chicago, Year Anat. Embryol., 162: 29-40, 1981. Book Medical Publishers, Inc., 1975. Porrmann, R. E. and VrermE1j-Kerrs, CHrR., Cell degen- Weston, J. A., A radioautographic analysis of the migra- eration in the mouse embryo: a prerequisite for normal tion and localization of trunk neural crest cells in the development. Ed. N. Muller-Bérat et al. Progress in chick. Dev. Biol. 6: 279-310, 1963. Differentiation Research, Amsterdam, North-Holland