J. Embryol. exp. Morph. Vol. 43, pp. 185-194, 1978 \ g 5 Printed in Great Britain © Company of Biologists Limited 1978

The ultrastructure of the mesenchymal element of the palatal shelves of the fetal mouse

By PETER B. INNES 1

From the Department of Anatomy, University of Saskatchewan, Saskatoon, Canada

SUMMARY

The ultrastructure of the mesenchymal element of the palatal shelves of C3H mouse embryos aged 13 days 18 h and 14 days 18 h m utero was studied. At 13 days 18 h the mesen­chyme showed a high density of cells. The cells contained a well developed system of rough endoplasmic reticulum and Golgi complex; many were ciliated and multi-vesicular structures were common. By 14 days 18 h, many of the mesenchymal cells contained large numbers of glycogen particles. These cells also possessed long cytoplasmic processes which sometimes were seen to contain many filaments 5-7 nmin diameter:. Some: of: the* cettssalsocontained a fine filamentous network just below the plasma membrane. Developing mononuclear and binucleate skeletal muscle cells containing myofibrils were present in the posterior region of the palate, with groups of cells which consisted of typical peripheral neurons and their surrounding satellite cells. Both the muscle and nerve cells were only observed in the 14 day 18 h material. It is suggested that both the myofibrils in the skeletal muscle cells and the filamentous network in the mesenchymal cells may play a role in shelf reorientation.

INTRODUCTION

An important step in the development of the secondary palate is the process of shelf reorientation which results in the palatal shelves moving from a lateral position beside the tongue to a horizontal position where they are situated above the tongue.

Although the details of this process of reorientation are well known, no convincing explanation exists for the mechanism that is responsible for this change in orientation even though many mechanisms have been suggested (Fraser, 1967; Greene & Pratt, 1976).

A knowledge of the ultrastructure of the palatal shelves is very important in understanding the mechanism of reorientation, yet information in this area is rather sparse. The ultrastructure of the palatal shelves in 7-to-17-day-old mouse embryos was studied by Walker (1961) while Babiarz, Allenspach & Zimmerman (1975) studied the ultrastructure of 14-5-day-old mouse embryo palatal shelves.

Because of the lack of detailed knowledge of the ultrastructure of the palatal shelves at the time or reorientation, it was decided to study the mesenchymal

1 Author's address: Department of Anatomy and Histology, The University of Adelaide, Adelaide, Australia.

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component of the shelves at this stage in their development in the hope that such a study would provide more information about the structures present and any changes that occur in them that could be related to the process of reorientation.

MATERIALS AND METHODS

C3H mice were used in this study. Adult mice were placed together for a period of 1 h for mating and the end of this hour was designated the beginning of day 0. Pregnant mice were killed by cervical dislocation and the gravid uteri were removed immediately and placed on crushed ice. Palatal shelves were then dissected under a dissecting microscope from embryos aged 13 days 18 h and 14 days 18 h. Care was taken to ensure that no shelves were removed from embryos in which shelf rotation had begun. Upon removal, the shelves were placed in ice-cold phosphate-buffered 3 % glutaraldehyde (Millonig, 1962) for 2 h. They were then rinsed and post-fixed in phosphate-buffered 1 % osmium tetroxide for 1 h. The specimens were then dehydrated in graded ethanol and embedded in Epon. One micron-thick sections were cut on a Porter-Blum II ultramicrotome and stained with aqueous toluidine blue for light microscopy. Thin sections were stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and examined in a Philips 200 electron microscope.

RESULTS 13 days 18 h

The mesenchyme of the shelves at this stage contained many cells. The cells were fairly uniform in appearance throughout the whole of the shelf. Their cytoplasm contained many free ribosomes which were often present in a rosette arrangement. They also contained a well developed system of rough endoplasmic reticulum which contained considerable amounts of material within its cisternae. The Golgi complex was also well developed.

Many of the cells were found to be ciliated and in cross-section the cilia were seen to contain nine peripheral doublet microtubules but no central microtubules.

A prominent feature of the mesenchymal cells at this stage was the presence of vesicular structures protruding from the surface of the cells. The structures consisted of localized blebbings of the plasma membrane which contained many microvesicles or myelin figures (Fig. 1).

The extracellular spaces contained very few collagen fibres, but numerous bundles of naked axons were seen throughout the mesenchyme of the shelf.

14 days 18 h

At this stage, the mesenchyme still showed a high cell density. The cells were very loosely arranged, but some junctional complexes were present between them.

Although some of the cells were still ciliated, the frequency of cilia was

Palatal shelf ultrastructure 187

Fig. 1. An electron micrograph of mesenchymal tissue from a 13/18 (13 day 18 h) embryo. A mesenchymal cell containing a large nucleus, numerous free ribosomes and some rough endoplasmic reticulum is seen. The cell also possesses a cilium (C) and a myelin figure within an outpouching of the plasma membrane (M), x 14500.

Fig. 2. An electron micrograph of a mesenchymal cell from a 14/18 embryo. The cell contains large numbers of glycogen granules (G) and a collection of microvesicles (V). x 18200. Fig. 3. An electron micrograph from the shelf of a 14/18 embryo. Cytoplasmic processes from mesenchymal cells containing microfilaments (F) are seen, x 85000. Fig. 4. An electron micrograph of cells in the shelf of a 14/18 embryo. A fine fila­mentous network (N) is seen just below the plasma membrane of the cell, x 70000.

considerably reduced compared with the early stage. Most of the cells also contained large numbers of glycogen particles in their cytoplasm (Fig. 2). The cells at this stage also possessed many long thin cytoplasmic processes which contained an amorphous material. In many of the processes fine filaments approximately 5-7 nm in diameter were visible, especially in the peripheral

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Fig. 5. An electron micrograph of an immature skeletal muscle cell from an 14/18 embryo. It contains a developing myofibril (M) composed of thick and thin filaments. Developing Z bands (Z) are also present, x 31000. Fig. 6. An electron micrograph of two immature skeletal muscle cells from a 14/18 in the process of fusing, x 28000. Fig. 7. An electron micrograph of an area of a ganglion found in the posterior region of the shelf from a 14/18 embryo. Numerous neurons (N) and satellite cells (S) are present. (A) is a bundle of axonal processes, x 4300. Fig. 8. An area from a ganglion showing part of two neurons (N) and the process from a satellite cell (S). One of the neurons contains a collection of small vesicles (V). x 28000.

region of the processes (Fig. 3). In a few of the cells a fine filamentous network was visible in the cytoplasm just below the plasma membrane (Fig. 4). The filaments in this network were 5-7 nm in diameter.

The vesicular structures were still present in the cells, but at this stage they were often closely associated with areas of glycogen particles (Fig. 2).

The amount of collagen fibres present in the extracellular space was increased

Palatal shelf ultrastructure 189

Fig. 10. An area from a ganglion in a 14/18 embryo showing neurons (N) surrounded by satellite cell processes (S). x 11000. Fig. 11. An area from a ganglion showing multivesicular structures present in satellite cell processes (S) and in a neuron (N). x 58000. Fig. 12. An electron micrograph from the shelf of a 14/18 embryo showing Schwann cell processes (S) beginning to surround bundles of axonal processes (A), x 13000.

when compared with the earlier stage. The fibres were especially prominent close to the basal lamina.

In the upper posterior region of the shelf, numerous immature skeletal muscle cells were present, both mononucleate and binucleate. The mononucleate cells differed from the mesenchymal cells in that they contained fewer ribosomes and two types of filaments were present in their cytoplasm, approximately 5 nm and 12 nm in diameter. The filaments were aligned in groups parallel to each other and numerous immature Z bands were present. Well defined A and

13 E MB 43

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1 bands were not seen since the thick filaments appeared to extend right along to the Z bands (Fig. 5. Most of the immature myofibrils appeared to be aligned at right angles to the anteroposterior axis of the shelf. Some of the mononuclear cells appeared to be in the process of fusing (Fig. 6). In the regions where the cells were fusing, the plasma membrane of the cells seemed to disappear and be replaced by a series of membranous vesicles which themselves soon disappeared.

Also present in the upper posterior region of the shelf was a large circum­scribed collection of cells which, when examined in the light microscope, was seen to consist of two morphologically distinct types of cells. The most numerous type had a large pale-staining nucleus and a large amount of pale-staining cyto­plasm, whereas the other type was much smaller with dark-staining nucleus and cytoplasm.

The larger cells, when examined in the electron microscope, were seen to contain large nuclei with little condensed chromatin. The cytoplasm of these cells contained large numbers of free ribosomes, almost all arranged in rosettes. Considerable amounts of rough endoplasmic reticulum and Golgi apparatus were also present and the rough endoplasmic reticulum was dispersed fairly evenly throughout the cytoplasm (Figs. 7-10). Vesicular structures, similar to those seen in the mesenchymal cells were also present (Fig. 8). Some of these cells gave rise to cytoplasmic processes resembling axons. The processes contained many microtubules 25-30 nm in diameter and numerous mitochondria (Fig. 9). The bodies of these cells were surrounded by thin cytoplasmic processes from the second type of cell (Fig. 10).

The second type of cell possessed smaller nuclei containing more condensed chromatin than the larger cells. The most outstanding morphological character­istic of these cells was the fact that they gave rise to long thin processes containing few cytoplasmic organelles apart from free ribosomes which sur­rounded both the cell bodies of the large cells and bundles of axonal processes. The gap between these processes and the large cell bodies varied but at times was as narrow as 20 nm. In some instances the processes of these cells contained vesicular structures similar to those seen in the mesenchymal cells (Fig. 11).

The axons present in the shelf at this stage of development showed the beginning of neurolemmal sheath formation (Fig. 12).

DISCUSSION

Although many theories of palatal shelf rotation involving both intrinsic and extrinsic factors have been proposed there is at present no convincing expla­nation of the mechanism involved.

Walker & Fraser (1956) first proposed that there was some kind of 'internal shelf force' which caused the reorientation to occur. Initially they postulated that this force was produced either by a network of elastic fibres or by acid mucopolysaccharides. The possibility that elastic fibres are important has since

Palatal shelf ultrastructure 191

been dismissed since it has been demonstrated (Frommer, 1968; Frommer & Monroe, 1969) that no such network exists at the time of shelf reorientation. The evidence with regard to the importance of acid mucopolysaccharides, however, is much more conflicting. Studies (Larsson, 1961 ; Walker, 1961 ; Pratt, Goggins, Wilk & King, 1973) have shown that palatal shelves actively synthesize sulphated proteoglycans and that the total amount of these substances present in the shelves increases just prior to the time of shelf reorientation. There is also evidence (Larsson, 1962; Pratt et al. 1973) that some teratogens which induce cleft palate by interference with shelf reorientation alter the metabolism of acid mucopolysaccharides in the palate. However, experimental work by Walker (1961) and Nanda (1970) cast doubts on the importance of acid mucopoly­saccharides in shelf elevation. Another possible source of an 'internal shelf force' is mitotic activity in the mesenchymal cells of the palate. Jelinek & Dostal (1973) found that the peak of mesenchymal cell proliferation in the shelves occurs 24-48 h prior to elevation while Hassell, Pratt & King (1974) found that the incidence of cleft palates is increased by substances which decrease DNA synthesis. Similar circumstantial evidence (Pratt & King, 1971; Pratt & King, 1972) suggests that collagen synthesis may be related to shelf elevation.

Finally, there is evidence (Lessard, Wee & Zimmerman, 1974; Wee, Wolfson & Zimmerman, 1976) which suggests that contractile proteins within the shelves may be important in generating an internal force.

Numerous proposals have also been made as to external factors which could be important in producing shelf elevation. It has been suggested (Verrusio, 1970) that straightening of the cranial base may play a role in shelf elevation. However, this is also a controversial question since Diewert (1974) failed to show any significant change in cranial base angle at the time of shelf elevation and Brinkley, Basehoar, Branch & Avery (1975) have shown that elevation occurs in vitro in the absence of an intact cranial base. The role of the tongue in the elevation process is also uncertain. Holt (1974) provided histochemical evidence that the tongue is functional at the time of elevation, but experiments (Brinkley et al. 1975; Wee et al. 1976) in which elevation has been found to occur in vitro in the absence of the tongue demonstrate that the action of the tongue musculature is not necessary for elevation to occur.

Although the present study does not clarify the controversy which prevails concerning the force that causes shelf rotation, it does provide ultrastructure evidence for a number of factors that might be involved.

The findings reported confirm the results of Babiarz et al. (1975) regarding the presence of skeletal muscle cells and neurons in the mesenchyme of the palatal shelves of mice at the time of their elevation. By including an examination of shelves aged 13 days 18 h the present study has demonstrated that both of these cell types first appear within the palatal shelves during the period 13 days 18 h and 14 days 18 h, just prior to the time that the reorientation of the shelves occurs in mice (Walker & Fraser, 1956).

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There is no doubt that the groups of cells in the posterior region of the palate in 14/18 embryos are peripheral nerve ganglia, since the two types of cell found in them closely resemble neurons and satellite cells as described by Peters, Palay & Webster (1970). The nerve cells are characterized by the presence of large pale-staining nuclei containing very little concentrated chromatin. The ribosomes in the cytoplasm tend to be arranged in rosettes of five to six granules surrounding a central granule and the cell body is surrounded by cytoplasmic extensions of satellite cells. Finally, dendritic processes containing microtubules and microfilaments were found arising from the cell bodies. The satellite cells contained smaller nuclei in which the chromatin was much more condensed than that found in the neurons. These cells also possessed long thin cytoplasmic processes which surrounded the bodies of the neurons. The function of these neurons is at present unknown but they are probably parasympathetic since these are the only neurons believed to be present in the head region outside the central nervous system. It seems very unlikely that they are innervating the developing skeletal muscle in the shelf as suggested by Baviarz et al. (1975), since the idea that skeletal muscle fibres are innervated by nerve fibres from sympathetic ganglia has been disproved (Hinsey, 1927).

The presence of myoblasts and myotubes containing myofibrils at the time of palatal shelf closure suggests that skeletal muscle contraction may be important in shelf reorientation, especially since Zimmerman, Patel & Chang (1974) have shown that D-tubocurarine inhibits palatal shelf reorientation in mice in vitro. The orientation of the myofibrils seen at 14 days 18 h is not as random as that reported by Babiarz et al. (1975), but the material used by these workers was slightly younger than that used in the present study. It is possible that a con­siderable alignment of the myofibrils occurred during the 6-12 h just prior to 14 days 18 h.

The fine filaments present within some of the mesenchymal cells at 14 days 18 h may also be important in producing the force necessary for shelf orientation, since it has been suggested by Wessells et al. (1971) that many morphogenetic movements may result from the presence of intracellular microfilaments. The arrangement of filaments seen in this study is similar to that which Luduena & Wessells (1973) believe to be responsible for the extension and movement of the cell surface in migrating cells. The experimental manipulation of these filaments should provide interesting data relating to shelf leorientation.

The cilia that are so prevalent in the mesenchymal cells at this stage in develop­ment may also play a role in shelf reorientation. Although cilia which have a 9 + 0 arrangement of microtubules are often considered to be non-motile, there is evidence (Satir, 1974) that such cilia are capable of movement.

Vesicular structures were found in the mesenchymal cells, neurons and satellite cells. Similar structures have been reported in fibroblasts in rabbit ovaries (Espey, 1971) and in glial and neuronal processes in vitro (Guillery, Sobkowicz & Scott, 1970). Espey (1971) considers that they could contain

Palatal shelf ultrastructure 193

a substance important in decomposing collagenous connective tissue, but there is no histochemical evidence that this is so. Guillery et al. (1970) believe that they are evidence of growing cytoplasmic processes in neurons and glial cells. Since it is believed (Spooner, Yamada & Wessells et al. 1971) that the addition of new membrane at the anterior edge of the cell may be a normal component of locomotory activity, the microvesicular structures may represent areas of membrane addition associated with cell migration or possibly with membrane removal, since some membrane resorption must occur elsewhere on the cell to compensate for the addition of new membrane at the front.

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(Received 5 May 1977, revised 5 August 1977)