Glycosaminoglycan localization and role in maintenance of tissue spaces in the early chick embryo¹

It has become increasingly apparent during recent years that molecules present in the matrix surrounding cells may have profound influences on the morphogenetic activity and cytodifferentiation of those cells. Specific examples include the influence of collagen on corneal differentiation (Dodson & Hay, 1973; Meier & Hay, 1974), the influence of glycosaminoglycans (GAG) on organogenesis (Bernfield, Cohn & Banerjee, 1973), and the effect of noto-chordal collagen and GAG on somite chondrogenesis (Strudel, 1973; Kosher & Lash, 1975). One such molecule, hyaluronic acid (HA), is a GAG commonly found associated with extracellular matrix (ECM) in adult and embryonic tissues (see Toole, 1976, for review). In chick embryos, for example, HA has been shown to be present in the matrix of migrating sclerotome (Kvist & Finnegan, 1970) and neural crest cells (Pratt, Larsen & Johnston, 1975) as well as in the developing heart (Manasek et al. 1973) and limb (Toole & Trelstad, 1971; Toole, 1972). Furthermore, HA has been shown to be synthesized in high proportions relative to other GAG by gastrula-stage chick (Manasek, 1975; Solursh, 1976) and rat (Solursh & Morriss, 1977) embryos.

The work presented here demonstrates that HA is also a constituent of the ECM of chick primary mesenchyme and that its continued presence is necessary for maintenance of the extensive intercellular spaces which are characteristic of the mesenchyme in the head region of the embryo. Three experimental approaches have been used: (1) paraffin sections of normal embryos were stained with Alcian blue at pH 1·0 or 2·5 to illustrate the distribution of sulfated and non-sulfated GAG; (2) tissue sections were treated with specific hyaluronidases prior to Alcian blue staining to localize HA specifically; and (3) tissue sections prepared from embryos incubated in ovo with hyaluronidase for 3 h before fixation were examined for morphological abnormalities which might result from disruption of the integrity of the ECM by HA removal.

Chick embryos [White Leghorn (Welp Inc., Bancroft, la.) or Hubbard Golden Comet (Johnson Co. Feed & Hatchery, Iowa City, la.)] to be used only for histochemical examination were incubated undisturbed to stage 8 (Hamburger & Hamilton, 1951). Embryos to receive in ovo enzyme treatment were incubated and allowed to reach late head-process or early head-fold stage (stage 5 or 6 respectively) and then candled to determine the position of the embryo so that a hole could be cut in the shell directly over the embryo. These embryos then received a sub-blastodisc injection of 20 μl of testicular hyaluronidase, 400 u/ml (Worthington, HSEP), Streptomyces hyaluronidase, 200 u/ml (Calbiochem), or Howard Ringer. The eggs were then resealed with Parafilm and returned to the incubator for an additional 3 h incubation.

Embryos for scanning EM observation were fixed in 2% glutaraldehyde in saline G for 1 – 20 h and postfixed by treatment with 1% OsO₄ in 04 M cacodylate buffer at pH 7·4 and thiocarbohydrazide as described by Kelley, Dekker & Bluemink (1973). The fixed specimens were then dried by the critical point method, gold-coated and examined in a Cambridge S4 stereoscan micro-scope.

Some injected embryos and the control embryos which were to be prepared for Alcian-blue staining and enzyme treatment of sectioned material were fixed for 1 h in Carnoy’s fixative containing 0-5 % cetylpyridinium chloride, then dehydrated through an alcohol series and embedded in paraffin.

Deparaffinized, hydrated sections were incubated for 2 h in the presence of 100 u/ml Streptomyces hyaluronidase, 400 u/ml testicular hyaluronidase, or 0·1 M phosphate buffer, pH 5·0 (Yamada, 1971). Following enzyme treatment, sections were stained with 1 % Alcian blue at pH 2·5 or 1·0 (Humason, 1972), then dehydrated, cleared in xylene and mounted in permount.

Streptomyces hyaluronidase was chosen because of its reported specificity for hyaluronate (Ohya & Kaneko, 1970). Testicular hyaluronidase degrades chondroitin and chondroitin sulfates A and C as well as hyaluronate. Both enzymes were tested for possible contamination with protease (Davis & Smith, 1955) and no protease activity was found in either enzyme. In addition, it is unlikely that the effects of hyaluronidase treatment reported here are due to toxic effects of the digestion products. Hughes, Freeman & Fadem (1974) studied the teratogenic effects of a number of sugars including mono-, di-, and trisaccharides. While all sugars tested were found to be teratogenic, the abnormalities reported did not include those reported in this paper.

When sections stained with Alcian blue at pH 2·5 (stains all GAG) are examined, one sees in the head region that there is an extensive basement membrane underlying the neural and non-neural ectoderm and surrounding the notochord. The basement membrane appears to stain more intensely under the neural ectoderm (particularly the lateral curved surfaces) and around the notochord than under the non-neural ectoderm (Fig. 1 A). Underlying the endoderm in the region where the foregut is closed, intensely staining basement membrane is associated only with the ventral endodermal wall (not shown). Posterior to the anterior intestinal portal there is staining basement membrane associated with the endoderm only in the region of the lateral body fold, where the splanchnic mesoderm appears organized as a columnar epithelium. In addition to the basement membrane, Alcian-blue-staining material is abundant in association with the head mesenchyme. The material appears precipitated on the surfaces of cells as small granules and as more extensive aggregates (Fig. 1D).

In sections stained at pH 1·0 (stains only sulfated GAG), the basement membrane is still visible although reduced somewhat in staining intensity (Fig. 1B). This is particularly true of the endodermal basement membrane which stains only as patches, if at all, in the ventral wall of the closed foregut (not shown). The Alcian-blue-staining material present in association with the head mesenchyme is virtually eliminated at this pH. It may be concluded from these results that there is a considerable contribution by both sulfated and non-sulfated GAG to the basement membranes in the head region, while material associated with the mesenchyme is predominantly non-sulfated.

More posteriorly in the embryo, at the region where somites have formed, staining at pH 2·5 reveals a prominent basement membrane underlying the ectoderm (particularly the neural ectoderm and adjacent non-neural ectoderm over the somites) and surrounding the notochord (Fig. 3 A). The endoderm, again, appears to lack a prominently staining basement membrane. If present at all, it seems to be confined to the region underlying the intermediate mesoderm. The somites themselves are seen to be surrounded by a basement membrane which contains Alcian-blue-staining material, as are the dorsal surface and, to a lesser extent, the ventral surface of the lateral plate mesoderm. Between the dorsal surface of the lateral plate mesoderm and the ectoderm, as between the ventral surface and the endoderm, are seen heavily staining granules associated with cell processes. Granules are increasingly abundant more laterally. The lateral plate mesoderm cells at this level are quite closely packed and there is no significant stainable material within the cell mass.

When the somite region is stained at pH 1·0, the same distribution of ECM as seen at pH 2·5 is evident. Stainable material is present in basement membranes and as granules between the mesoderm and ectoderm or endoderm as well as within the more disperse lateral mesoderm, but the staining intensity is somewhat reduced (Fig. 3B). These results show that in this region the ECM contains predominantly sulfated GAG.

In the yet unsegmented region between the somites and the primitive streak, nearly the same distribution of Alcian-blue-staining ECM is seen as in the somite region. Closer to the region of Hensen’s node there is a progressive change in the amount of staining matrix until, at the node, there is a slight basement membrane underlying the lateral ectoderm only. This is visible when stained at either pH (Fig. 4A, B). At this level there appears to be occasional staining material associated with the mesoderm, particularly in the more lateral portions. This material stains less intensely at pH 1·0. These results indicate that in the axial region of the embryo from the level of the somites through the primitive streak, Alcian-blue-staining ECM is predominantly composed of sulfated GAG with a small contribution by non-sulfated GAG. Non-sulfated GAG becomes increasingly evident in the more lateral extremities of the embryo at this level.

The presence of HA is demonstrated by comparison of sections incubated in buffer with and without Streptomyces or testicular hyaluronidase and then stained with Alcian-blue at pH 2·5. Streptomyces hyaluronidase specifically degrades HA while testicular hyaluronidase degrades chondroitin and chondroitin sulfates as well as hyaluronate, yet treatment with either enzyme yields similar results.

In the head region all Alcian-blue-stainable material visible in buffer-treated sections (as described above) appears to have been removed by enzyme treatment (Fig. 1C). Some residual patches of stainable basement membrane may be seen associated with the neural fold ectoderm in enzyme-treated sections stained at pH 1·0 (not shown). This is probably visible because of the reduced intensity of background staining at this pH. The amount of stainable basement-membrane material seen under these conditions is slight compared to non-enzyme-treated sections stained at pH TO. These results show that HA is present in the basement membrane as well as in the ECM associated with the head mesenchyme. Furthermore, removal of hyaluronate by Streptomyces hyaluronidase digestion results in loss of most sulfated GAG, which must be intimately associated with HA in the ECM.

In the somite region, following enzyme treatment some stainable basementmembrane material is still present underlying the neural ectoderm and, to a lesser extent, on the dorsal surfaces of the somite and lateral plate mesoderm.

However, the granular material present associated with cell processes between the mesoderm and ectoderm or endoderm is no longer visible (Fig. 3 C) nor is the granular material associated with the more disperse lateral mesoderm. This is true also of the ECM material in the unsegmented mesoderm posterior to the somites after enzyme treatment, and is not unexpected in light of our previous observations that most stainable material in these regions is sulfated GAG. However, some HA is also present here, predominantly associated with the more lateral mesoderm and the spaces separating the mesoderm from ectoderm and endoderm.

Finally, in Hensen’s node area, where very little was present initially, enzyme-treated sections are virtually devoid of Alcian-blue-staining ECM. Faint traces of basement membrane remain under the lateral ectoderm (Fig. 4C).

The above results demonstrate that HA is associated with the ECM in all regions of the young embryo, and is particularly abundant in cell-free spaces associated with mesoderm. More non-sulfated, Alcian-blue-staining material sensitive to Streptomyces hyaluronidase is found in association with the head mesenchyme than anywhere else in the embryo. In an attempt to understand the developmental significance of this molecule, embryos were incubated in ovo for 3 h after the injection of hyaluronidase and then fixed and examined histologically to determine any morphological abnormalities which might result from this treatment. Similar results are obtained when either Streptomyces or testicular hyaluronidase is injected. The most striking result is the loss of cell-free spaces associated with the mesoderm. This effect of HA removal is most obvious in the head mesenchyme (Fig. 2). After enzyme treatment the mesenchyme cells appear clumped and resting on the endoderm. The mesenchyme only rarely retains connection with either the neural or non-neural ectoderm. This tissue separation is demonstrated consistently in both light microscopic and SEM (Fig. 6) preparations of enzyme-treated embryos only, and is therefore not attributable to an artifact of either Carnoy fixation or paraffin embedding. The space under the neural groove, between the neural ectoderm and the endoderm, which normally is at least two cell diameters in width, is now reduced to as little as one third of its former size with barely enough room for a single flattened cell, and space between mesenchymal cells appears by light microscopy to be completely gone (Fig. 2). The notochord is accordingly flattened and somewhat disorganized in appearance. In addition, there is a conspicuous decrease in ECM which can be seen by SEM (Figs. 5 – 8) or by Alcian-blue staining of sections from enzyme-treated embryos (Fig. 2). The SEM micrographs show that there is ECM material present in control embryos (Figs. 5, 7) which is absent from enzyme-treated embryos (Figs. 6, 8). The reduction of Alcian-blue-stainable matrix is seen throughout most embryos. In these embryos (Fig. 2), stainable matrix appears to be as completely removed as it is when sections are incubated with hyaluronidase. Some embryos appear to have no significant reduction of Alcian-blue-staining matrix, but show clumped mesenchyme. This probably reflects variation in the effective enzyme concentration in the embryo through inconsistency in making the injection, so that the amount of enzyme present may be sufficient to alter the hydrostatic properties of the matrix but not its stainability. These observations indicate that HA is required for the maintenance of the extensive intercellular spaces characteristic of head mesenchyme, and that the removal of HA from the ectodermal basement membrane results in a less stable interaction between mesenchyme cells and the ectoderm as evidenced by the absence of connections between these two tissues in sections from enzyme-treated embryos.

Solursh (1976) demonstrated that chick embryos at the stage studied here are synthesizing GAG in the following relative proportions of HA/chondroitin sulfate/heparan sulfate: 22/1·5/1. The results reported here provide histochemical evidence for the presence and location of both sulfated and nonsulfated GAG as constituents of the ECM throughout the early chick embryo. Both types of GAG are associated with epithelial basement membranes at all levels of the embryo to varying degrees, while hyaluronate predominates in intercellular spaces. In these young embryos the basement membranes which are most prominently stained by Alcian blue are those underlying epithelia which are actively involved in morphogenesis -i.e. the folding neural ectoderm and the endoderm in the region of the lateral body fold, or, where the foregut is already closed, the ventral wall of the foregut. Others have demonstrated the presence of GAG, including HA, in embryonic epithelial basement membranes from older embryos and have implicated these molecules in the morphogenetic events occurring in the epithelia associated with these basement membranes (Bernfield, Banerjee & Cohn, 1972; Cohn, Banerjee & Bernfield, 1977; Hay & Meier, 1974; Manasek, 1975; O’Hare, 1973).

Our observations indicate one important role that basement membranes may play in tissue interactions. Basement membranes act as substrata for the maintenance of intimate association of two tissues. In embryos injected with hyaluronidase, the association of the head mesenchyme with head ectoderm and neural ectoderm is affected, presumably due to the structural alteration of the basement membrane. As shown in Figs. 2 and 6, contacts between the mesenchyme and ectoderm are lost, leaving spaces adjacent to the neural ectoderm and subjacent to the head ectoderm. While exaggerated in size, perhaps due to the additional clumping effect, these spaces are similar in location to those which form normally during development to provide paths for neural-crest cell migration. Some alteration in the composition of the basement membranes could possibly account for the normal loss of attachments between mesenchyme and ectoderm in these regions.

HA is present not only in the basement membranes of epithelia undergoing morphogenesis, but also in the ECM associated with many embryonic mesenchymes. Our results show that HA is present in primary mesenchyme matrix in the chick as in the rat (Morriss & Solursh, 1978). HA is particularly apparent in the head mesenchyme where greater intercellular space is found.

To explain the function of HA in ECM of mesenchymes (many of which are actively migrating), Toole (1972, 1976) has postulated that HA may inhibit extensive intercellular interactions by physically separating cells. This physical separation could allow the cells to migrate and/or temporarily inhibit cyto-differentiation. Inhibition of cytodifferentiation by HA has so far been demonstrated only for chondrocytes (Toole, Jackson & Gross, 1972; Solursh, Vaerewyck & Reiter, 1974), and evidence concerning the role of HA in cell migration is still only circumstantial. In contrast, there is an increasing body of direct evidence for the role of HA in establishing and maintaining spaces during embryonic development. We have shown here that when young embryos are injected with hyaluronidase and incubated in ovo for a short period of time subsequent to the injection, a marked loss of intercellular space results. Earlier, Toole & Trelstad (1971) showed the importance of HA in forming space in the developing corneal stroma, and Pratt et al. (1975) showed that HA is present in the newly formed space underlying the ectoderm through which neural crest cells will migrate. In the rat embryo, Solursh & Morriss (1977) have correlated the production of HA with the appearance of mesenchyme and its associated spaces in the primitive streak stage, and have shown HA to be present in the matrix underlying the neural folds (Morriss & Solursh, 1978). Experiments are in progress to study further the importance of HA in forming and maintaining spaces as well as the importance of the spaces themselves in development of the chick embryo.

This investigation was supported by NIH grant HDO5505 to M. S. and USPHS training grant no. HD-00152 from the National Institute of Child Health and Human Development, while M.F. was a predoctoral trainee.