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/ . Embryol. exp. Morph. Vol. 58, pp. 131-142, 1980 Printed in Great Britain © Company of Biologists Limited 1980 Cell behaviour and cleft palate in the mutant mouse, amputated By O. P. FLINT 1 From the Department of Zoology, University of Glasgow SUMMARY Cleft palate with a genetic origin normally arises because of a failure of the palatal shelves to elevate or fuse. Until now attention in studies of palatal development has been focused on two critical phases, those of elevation and fusion. In the mutant mouse, amputated, however, cleft palate arises because of a failure of the palatal shelves to make any significant outgrowth between the 12th day after conception when the palatal shelves are first observed and the 14th day when elevation and fusion normally occur. When cell proliferation (mitotic index) was measured in the palatal shelves on days 12, 13 and 14 no difference was found between mutant and normal. The failure of the mutant palate to grow cannot be accounted for on grounds of reduced cell proliferation. For this reason the palatal mesenchyme in 12-5-day and 14-5-day normal and amputated mice has been studied with the scanning electron microscope. This work shows that the mesenchymal cells in the mutant palate are clumped together and have much greater areas of cell contact than are observed in the normal palate. The abnormal cell behaviour described in mutant palatal mesenchyme is typical of amputated embryonic mesenchyme in general, and in other cases has been shown to cause abnormal morphogenesis. We propose that aberrant cell behaviour causing an aggregation through increased cell adhesion inhibits palatal outgrowth in the mutant, and for this reason the palatal shelves subsequently fail to elevate and fuse. INTRODUCTION Abnormal cell behaviour causing the cells to clump together and inhibiting cell movement affects the development of the mouse mutant, amputated, at all stages involving morphogenetic cell movements. The length of the embryo is reduced because node and streak regression is retarded (Flint, Ede, Wilby & Proctor, 1978). Clumping of the somite sclerotome cells may partly contribute to fusions and distortions later found among the vertebral cartilages (Flint, \911a). Snout outgrowth is strongly reduced because increased cell adhesion and cell clumping retards a critical early stage of facial development when the facial mesenchyme grows by expansion of the extracellular volume, which normally forces the cells to move apart (Flint, 1977b; Flint & Ede, 1978a). In in vitro culture, amputated cells clump together and cell movement can be observed in time-lapse films to have been retarded (Flint, 1977a and Flint, in preparation). 1 Author's address; ICI Pharmaceuticals Division Mereside Alderley Park Macclesfield Cheshire SK10 4TG, U.K. 132 O.P.FLINT Another developmental system involving cell movement is the palate. Cleft palate is found in all mouse embryos homozygous for the single recessive gene amputated. Its development is described as part of a programme of research into the cellular basis of abnormalities in mutant development. We find that amputated palates fail to rotate and therefore to fuse because palatal shelf outgrowth is retarded through anomalous cell behaviour similar to that already described as affecting development in other parts of the embryo. Palate development has been reviewed by Greene & Pratt (1976) who describe two phases of palate development: (1) elevation (including rotation) of the palatal shelves and (2) palatal fusion. A failure of elevation or fusion can cause cleft palate. In the mouse mutant shorthead (Fitch, 1961) elevation of the palatal shelves is arrested, by a tongue which is too large for the abnormally small buccal cavity. In the chick, on the other hand, rotation occurs but there is no fusion when the palatal shelves meet, resulting in the type of cleft palate which is normal in avian embryos (Greene & Pratt, 1976). Cleft palate arising out of the earliest stage of palatal shelf outgrowth, as in amputated, has not so far been described, and this earliest stage of palatal morphogenesis has been neglected. METHODS The mice are a CBA/101 hybrid intercross carrying the single recessive gene amputated. Conception was taken to coincide with the midpoint of the dark period just prior to noticing a vaginal plug (Snell, Fekete, Hummel & Law, 1940). Matings were designed to produce litters with homozygous amputated embryos. The techniques of fixation and critical-point drying of mouse embryos for the scanning electron microscope have been described by Flint & Ede (19786). After fixation and while in 70 % ethanol the heads were dissected under the dissecting microscope with micro-surgery scalpels (Moria-France) to remove the mandibles and tongue, revealing the roof of the buccal cavity. In some cases the heads were then cut transversely to produce sections of the palatal shelves. It was found that the effect of cutting like this was not to crush the tissue but to fracture it in the plane of cutting. The stages of palatal development we describe are not so highly resolved as those described by Walker & Fraser (1956) or Walker & Crain (1960) since we were not specifically interested in palatal fusion and also since the earliest stage we describe (12-5 days) is two days younger than the embryos described by these authors. Cell proliferation and cell density were measured in normal and amputated palatal shelves and in the mesenchyme to one side of the base of the palatal shelf (control area) at 12-5, 13-5 and 14-5 days. Cell proliferation was recorded as mitotic index; the number of mitotic figures as a percentage of total cell number. Statistical analysis of the figures was carried out by the standard analysis of variance procedure. Cell behaviour and palatal morphogenesis 133 Palatal development Gross development changes: Normal. The earliest stage at which the palatal shelves can be seen is at 12-5 days, when they appear as a pair of parallel ridges growing down from either side of the roof of the buccal cavity (Fig. 1 a). That these are ridges can be seen at their posterior end where the knife has cut a glancing transverse section through the tissue. By the 14th day of development (14-5 days) the ridges have grown well out and away from the palatal roof (Figs. lc,3a, c) and rotation can be seen to have begun in the anterior half (Fig. 3 a) of at least one shelf. The palatal rugae (Figs. \c, 3a, c) begin to make their appearance in an anteroposterior sequence at this stage before the palatal shelves meet and fuse. Each pair of rugae appears at the same level of the palate so that after palatal fusion (Fig. 3 b) they will form almost continuous ridges across the roof of the mouth. At the end of the 14th day of development (approximately 15-0 days) fusion has begun along the meeting central portions of the two palatal shelves (Figs. 2 a, 3 b). Amputated. As in the normal embryo, mutant palatal shelves begin their outgrowth during the 12th day of development (Fig. Ib). There are two anatomical features which differ from the normal embryo. First, the width of the head is greater and the snout shorter (Flint & Ede, 1978 a), so that the palatal shelves form at a greater distance apart. Secondly, the opening to Rathke's pouch persists to this late stage in amputated, but not in the normal embryo (Fig. 1 a, b). By 14-5 days, though, the overall shape of the head in amputated remains blunter anteriorly and broader than normal, the distance between the palatal shelves being the same in normal and mutant (Fig. 1 c, d). There is little or no downgrowth of the palatal shelves away from the roof of the mouth (Fig. 1 d, 3d), when compared to the normal (Figs. 1 c, 3a, c) at the same stage, or the amputated embryo at 12-5 days (Fig. \b). As in the normal embryo, the first palatal rugae make their appearance at 14-5 days (Fig. 1 d) and by the end of the 14th day of development several more have appeared (Fig. 2b), but there is no further outgrowth of the mutant palatal shelves. In the normal embryo growth of the mandibles and the cranial base enlarges the buccal cavity so that the tongue drops out of the way of the elevating palatal shelves. In amputated, snout outgrowth is retarded (Flint, 19776 and Flint & Ede, 1978a). But growth of the tongue is not so strongly inhibited and its impression can be seen in the palatal shelves in Fig. 2b. The epithelial surface of the palate Normal. The aboral epithelial surface of the palate shows some interesting regional variation at 15-0 days associated with the appearance of the rugae and the point of fusion. Between the rugae numerous microvilli are seen on the surfaces of the epithelial cells (Fig. 4 c). These are especially dense at the borders 134 O. P. FLINT FIGURE 1 Scanning electron micrographs of the heads of normal (o, c) and amputated (b, d) mice at 12-5 {a, b) and 14-5 (c, d) days of development. The mandibles and tongue have been dissected away so that a view of the roof of the buccal cavity is obtained in each case, in, internal nares; pp, primary palate including median process; pr, palatal ruga; ps, palatal shelf; rp, opening to Rathke's pouch. Cell behaviour and palatal morphogenesis 135 FIGURE 2 Scanning electron micrographs of normal (a) and amputated (b) mouse heads at 150 days. The preparation is the same as that described in Fig. l.fp, point of fusion between the palatal shelves; pp, primary palate and median process; /?r, palatal ruga; ps, palatal shelf; tg, tooth germ (upper incisor) exposed in amputated because the broadening and foreshortening of the cranium draws the lips apart. between cells. This is typical of an epithelial surface in contact with a fluid medium, in this case the amniotic fluid, and has been observed on the chick limb epidermis (Ede, Bellairs & Bancroft, 1974) and on the naso-frontal region of the hamster face (Waterman & Meller, 1973), as well as on the mouse (Waterman, Ross & Meller, 1973) and human (Waterman & Meller, 1974) palate. There is a transition between inter-rugal epithelium and epithelium associated with the rugae. Rugal epithelium is comparatively bare of microvilli and the cell surfaces are much rougher (Fig. 4a). At the point of palatal fusion all boundary distinction between cells is lost and numerous elongate cell projections are produced. A large quantity of debris also accumulates on the surface. Amputated. Late in the 14th day of development the inter-rugal epithelium is very similar in mutant and normal palates (cf. Fig. 4c, d). But the rugae, far from lacking microvilli, are even more profusely covered, with many more microvilli on the surface as well as at the junctions between cells (Fig. 4 b). 136 O. P. FLINT FIGURE 3 Low-power scanning electron micrographs of normal embryos at 145 (a) and 150 (6) days to show the palatal shelves elevating (a) and fusing (b). Higher powers of sections, at 14-5 days, of the right palatal shelf in a normal embryo (c) and the left palatal shelf in an amputated embryo (d) are also shown to compare the outgrowth of the palate in mutant and normal, in, internal nares; pp, primary palate and median process; pr, palatal ruga; ps, palatal shelf. Sectioned palatal shelves Normal. The section through the raised palatal shelf (Fig. 3 c) is reminiscent of a section through a developing limb bud, but there is no apical ectodermal ridge specialization of the distal ectoderm. At 12-5 days cells are spaced well apart and are connected by numerous fine and very fine filopodia (Fig. 5 a). By 14-5 days the cells are still well spaced but, in addition to the filopodia, they are coated with a dense fibrous and globular matrix of intercellular materials (Fig. 5 c, e). On the basis of the work of Hassell & Orkin (1976), who studied the production of extracellular materials in the palate, we would identify the long Cell behaviour and palatal morphogenesis 137 FIGURE 4 The epithelial surface of the palatal shelves at 150 days in normal {a, c, e) and amputated {b, d) mice. The normal series shows a, the surface of a ruga and the transition from rugal to inter-rugal surface (at the bottom of the figure); c, the inter-rugal surface and e, the point of fusion between two palatal shelves. The mutant series shows b, the rugal surface and d, the inter-rugal surface. A section through the fusing palatal shelves of a normal mouse at 150 days is shown in/. 138 O. P. F L I N T FIGURE 5 The mesenchyme of the palatal shelves in normal (a, c, e) and amputated (b, dj) mice at 12-5 days (a, b) and 14-5 days (c, d, e,f) just prior to elevation. Cell behaviour and palatal morphogenesis 139 Table 1. Mitotic index {mitoses per 100 cells) in the palatal shelves and adjacent control areas of normal and amputated mouse embryos Values are given as mean plus or minus the standard deviation. Tissue Palate nbryo N am Control N am 12-5 days 13-5 days 14-5 days Cells counted l-94±013 210±014 1-44 ±012 2041015 l-81±014 3-21 ±0-85 l-38±012 l-47±013 O-98±O13 l-86±O13 0-21 ±008 0-72 ±003 9836 4029 10061 5947 fibrous intercellular material as collagen and the globular material as a product of the basement membrane which can extend far into the palate matrix. At the point where the two palatal shelves meet a zone of continuity is formed, where the breakdown of the epithelial interface occurs and the two palatal mesenchymes become one (Fig. 4/). The scanning electron microscope evidence is consistent with transmission electron microscope studies on the palatal mesenchyme (Babiarz, Allenspach & Zimmerman, 1975 and Innes, 1978). Amputated. Whereas normal cells at 12-5 days give a well-spaced structure to the palatal tissue, amputated cells appear to have aggregated together (Fig. 5 b). The filopodia adhere in a tangled web over all the cell surfaces. This is exactly similar to preparations of the somite sclerotome in the mutant embryo at 9*5 days (Flint & Ede, 1978&). By 14-5 days there has been no overall change to this aggregated or clumped appearance (Fig. 5d). But the intercellular spaces, as in the normal palatal tissue, have become filled with globular and filamentous extracellular material (Fig. 5d,f). Cell proliferation in the palatal shelves Values of mitotic index measured from sections of palatal shelves on 12-5, 13-5 and 14-5 days after conception are given in Table 1. There is no significant overall difference between normal and amputated (P > 0-10). But there is a significant overall difference in mitotic index measured in the palatal shelves and in the adjacent control areas (0-05 > P > 0-025). This difference largely rests on a drop in mitotic index in the control area between 13-5 and 14-5 days. The only other significant difference occurs at 14-5 days when in palate and adjacent control areas mitotic indices are higher in amputated than in normal (P < 0-05). No explanation for this difference emerges from the current work. Cell density Values of cell density measured at the same time as mitotic index are given in Table 2. There is no significant overall difference between normal and amputated (P > 0-10). But there is a significant overall difference in the cell IO EMB 58 140 O. P. FLINT Table 2. Cell density (cells per 1OZ ju,m2) in the palatal shelves and adjacent control areas of normal and amputated mouse embryos (same number of cells counted as in Table 1) Values are given as mean plus or minus the standard deviation. Tissue Embryo 12-5 days 13-5 days 14-5 days Palate N am Control N 1408 ±1-52 15-44±l-56 l5-24±3-12 15-24 ±1-72 16-08 ±1-44 15-28 ±1-60 15-40 ±1-96 15-68 ±1-22 ll-92±l-44 1116±l-8O 21-76±l-54 18-68 ±0-98 am density measured in the palatal shelves and adjacent control areas. This difference rests largely on a drop in cell density in the palatal shelves between 13-5 and 14-5 days. DISCUSSION Wherever genetically-caused cleft palate has been described in the mouse the critical phase at which palate development has been inhibited has been that of elevation and rotation (i.e. during the 14th day of development). But there is no significant change in the outgrowth of mutant palatal shelves from the time when they arefirstobserved at 12-5 days up to early in the 14th day of development (Figs. 1 b, 3 d). Normal specimens show considerable downgrowth of the palatal shelves on either side of the tongue and away from the roof of the buccal cavity during this time (Figs. \a, c, 3 a, c). In spite of abnormal morphogenesis the differentiation of palatal shelf cells proceeds normally, so that not only is the extracellular matrix material secreted at the appropriate time accompanied by a parallel reduction in mutant and normal cell density (see Table 2) but also the palatal rugae make their appearance on cue (Fig. 1 d, 2b). Between the rugae the normal palatal epithelium is covered with microvilli (Fig. 4 c), but the rugae have lost their coating of microvilli (Fig. 4a). Microvilli are most probably a mechanism of conserving cell membrane (O'Neill & Follett, 1970). The development of the elevated ridge-like rugae in the normal embryo on the expanding surface of the palatal shelves involves a stretching of the epithelial surfaces and consequently a loss of the microvilli. In the mutant there is no expansion of the palatal shelves. The formation of the rugae involves a concentration of the available epithelium, and consequently an increase in the number of microvilli (Fig. 4 b), rather than a decrease. Outgrowth of the naso-frontal region, like that of the palatal shelves, is inhibited at the earliest stage in the amputated mouse (Flint, 19776; Flint & Ede, 1978 a). This inhibition cannot be accounted for on the grounds that cell proliferation is reduced. Similarly mitotic index is the same in mutant and Cell behaviour and palatal morphogenesis 141 normal palatal shelves throughout the earliest stages from the 12th to the 14th day of development. In the case of the naso-frontal region we have found that increased cell adhesion between the cells in the mutant causes clumping similar to that found elsewhere in the embryo and this retards the tissue expansion which occurs in the normal embryo by secretion of extracellular material, and therefore retards morphogenesis from the 10th day of development. This clumping of cells is typical of the mutant mesenchyme both in vivo (Flint, 1977 a, b; Flint & Ede, 1978/7, b) and in vitro (Flint, 1977 a; Flint, in preparation). Increased cell contacts and areas of cell-cell adhesion causing an aggregation of the palatal mesenchyme exactly similar to that described in other parts of the embryo persist from the 12th (Fig. 5 b) to the 14th (Fig. 5d) day of development. Since it is during this very early stage of palatal morphogenesis prior to elevation that palatal outgrowth is inhibited, and since there is such a high correlation in other parts of the embryo between abnormal cell behaviour and abnormal morphogenesis (see also Introduction), we ascribe the cleft palate in amputated to an anomaly of cell behaviour causing increased cell adhesion which inhibits downward growth of the palatal shelves. The increased cell adhesion does not increase cell density in the mutant mesenchyme. There are the same number of cells per unit volume as in normal palatal shelves, but because of increased cell adhesion these cells tend to clump together; they are not so well dispersed as in the normal palate. Our measurements of the somite sclerotome in the mutant produced the same result (Flint & Ede, 1978 b). Matrix secretion causing a drop in cell density in the palatal shelves of both mutant and normal embryos between the 13th and 14th day of development comes too late to help the mutant palatal shelf catch up with the growth already made by normal shelves. Comparing Figs 1 and 2, however, it can be seen that some expansion of mutant palatal shelves occurs late in the 14th day of development, probably as a result of the secretion of matrix material. The development of the palatal shelves between the 12th and the 14th day, when elevation occurs, has hitherto received little attention, but it appears that this is a critical phase in palatal development and that disturbance of normal growth during this stage may produce abnormal cleft palate at a later one. A comparison of the effect on cell behaviour of amputated with work on other mutant genes can be found in Flint & Ede (1978 a, b). REFERENCES B. S., ALLENSPACH, A. L., ZIMMERMAN, E. F. & (1975). Ultrastructural evidence of contractile systems in mouse palates prior to rotation. Devi Bio I. 47, 32-44. EDE, D. A., BELLAIRS, R. & BANCROFT, M. (1974). A scanning electron microscope study of the early limb-bud in normal and talpid3 mutant chick embryos. /. Embryol. exp. Morph. 31, 761-785. FITCH, N. (1961). Development of cleft palate in mice homozygous for the shorthead mutation. /. Morph. 109, 151-157. BABIARZ, 142 O. P. FLINT O. P. (1977a). Cell interactions in the developing axial skeleton in normal and mutant mouse embryos. In: Vertebrate Limb and Somite Morphogenesis (ed. D. A. Ede, J. R. Hinchliffe & M. Balls), pp. 464-484. Brit. Soc. Devi. Biol. Symp. 3, Cambridge University Press. FLINT O. P. (19776). Cell interactions in facial development in the mouse. / . Anat. 124 225-226. FLINT, O. P. & EDE, D. A. (1978a). Facial development in the mouse: a comparison between normal and mutant (amputated) mouse embryos. J. Embryol. exp. Morph. 48, 249-267. FLINT, O. P. & EDE, D. A. (19786). Cell interactions in the developing somite: in vivo comparisons between amputated (am/am) and normal mouse embryos. / . Cell Sci. 31, 275-292. FLINT, O. P., EDE, D. A., WILBY, O. K. & PROCTOR, J. (1978). Control of somite number in normal and amputated mutant mouse embryos: an experimental and theoretical analysis. / . Embryol. exp. Morph. 45, 189-202. GREENE, R. M. & PRATT, R. M. (1976). Developmental aspects of secondary palate formation. / . Embryol. exp. Morph. 36, 225-245. HASSELL, J. R. & ORKIN, R. W. (1976). Synthesis and distribution of collagen in the rat palate during shelf elevation. Devi Biol. 49, 80-88. INNES, P. B. (1978). The ultrastructure of the mesenchymal element of the palatal shelves of the fetal mouse. / . Embryol. exp. Morph. 43, 185-194. O'NEILL, C. H. & FOLLETT, A. C. (1970). An inverse relation between cell density and the number of microvilli in cultures of BHK21 hamster fibroblasts. J. Cell Sci. 7, 695-709. FLINT, SNELL, C. D., FEKETE, E., HUMMEL, K. P. & LAW, L. W. (1940). The relation of mating, ovulation, and the estrus smear in the house mouse to the time of day. Anat. Rec. 76, 39-54. WALKER, B. E. & FRASER, F. C. (1956). Closure of the secondary palate in three strains of mice. J. Embryol. exp. Morph. 4, 176-189. WALKER, B. E. & CRAIN, B. (1960). Effects of hypervitaminosis A on palate development in two strains of mice. Am. J. Anat. 107, 49-58. WATERMAN, R. E. & MELLER, S. M. (1973). Nasal pit formation in the hamster. A transmission and scanning electron microscope study. Devi Biol. 34, 255-266. WATERMAN, R. E. & MELLER, S. M. (1974). Alterations in the epithelial surface of human palatal shelves prior to and during fusion: a scanning electron microscope study. Anat Rec. 180, 111-136. WATERMAN, R. E., ROSS, L. M. & MELLER, S. M. (1973). Alterations in the epithelial surface of A/Jax mouse palatal shelves prior to and during fusion: a scanning electron microscopic study. Anat. Rec. 176, 361-376. (Received 11 October 1979, revised 30 January 1980)