It appears that hyaluronate is associated with cell migration and the chondroitin sulphates with differentiation during morphogenesis of the chick embryo. The aim of this study was to see if such a correlation could be made for chondrocranium morphogenesis. Specifically, the purpose of this study was (1) to determine the proportion of extracellular matrix (ECM) to cell area and total head mesenchymal area during chondrocranium morphogenesis; and (2) to identify the location, types, and relative amounts of glycosaminoglycans (GAG) being synthesized in the presumptive chondrocranium at the onset of chondrogenesis and prior to this time.

Morphometric analyses were made on median and parasagittal sections of heads of stage-24 and -33 embryos in order to determine relative contributions of cells and ECM to the total area of head mesenchyme at these stages. Presumptive chondrocrania (heads minus eyes) of these stage embryos were also analysed histochemically and biochemically in order to identify the GAGs present in the ECM. Sections of whole heads were stained with alcian blue at low and high pH as well as digested prior to staining with hyaluronidase (Streptomyces and testicular). Identification of GAGs was done by pulse labelling embryos with [3H]glucosamine, digesting homogenates with hyaluronidase (Streptomyces or testicular), precipitating the undigested GAGs with cetylpyridinium chloride and counting the dissolved precipitates using scintillation spectrophotometry. The types and relative amounts of GAGs present in the presumptive chondrocranium were determined by comparing the amount of radioactivity in the precipitates of the non-digested GAG with the counts in the precipitates of the predigested GAGs.

This study reports that chondrogenesis begins in the presumptive chondrocranium of the chick embryo at stage 33 and that the area of the head mesenchyme increases 60-fold between stages 24 and 33. Little change in cell density and individual cell area as well as in the relative proportion of total area allocated to cells and ECM occurs. GAGs are localized exclusively in the presumptive chondrocranium. These GAGs are restricted to the ventral half of the presumptive chondrocranium. Within this region, the GAGs are further localized to the presumptive facial area, perichordal region, ethmoid, sphenoid and periotic regions.

The types of GAG being synthesized in the head mesenchyme of both stage-24 and -33 embryos are hyaluronate, the chondroitins and unidentified sulphated GAGs (dermatan, keratan, heparin and heparan sulphate). At stage 24, hyaluronate, chondroitin and the unidentified sulphated GAGs constitute about 33 % each of the GAG being synthesized. At stage 33, the level of hyaluronate synthesis drops to 2%, the chondroitins to 24% and the unidentified sulphated GAGs increase to 74 %. There is an 18·5-fold decrease in the percentage of hyaluronate, a 1·5-fold decrease in the amount of chondroitins and a 2·7-fold increase in the percentage of unidentified sulphated GAGs being synthesized as chondrocranium morphogenesis proceeds.

Studies of morphogenesis of the cornea, kidney, presumptive spinal column, and limb suggest that there is an accumulation of hyaluronate along the pathways of migrating mesenchymal cells, followed by a reduction in the amount of hyaluronate and a rise in sulphated GAG concurrent with differentiation of the migrated cells (Fig. 1). This pattern suggests that glycosaminoglycans (GAG) present in the immediate environment may play a role in these developmental sequences.

Fig. 1.

A schema depicting changes in GAG synthesis and hyaluronidase activity associated with cell migration and differentiation during morphogenesis. Cell migration appears to be correlated with a high concentration of hyaluronate whereas cell differentiation occurs with a decrease in hyaluronate and concomitant increase in chondroitin sulphate. An increase in hyaluronidase activity parallels a decrease in hyaluronate. This diagram is a composite based on data primarily generated by the laboratory of Toole (1981).

Fig. 1.

A schema depicting changes in GAG synthesis and hyaluronidase activity associated with cell migration and differentiation during morphogenesis. Cell migration appears to be correlated with a high concentration of hyaluronate whereas cell differentiation occurs with a decrease in hyaluronate and concomitant increase in chondroitin sulphate. An increase in hyaluronidase activity parallels a decrease in hyaluronate. This diagram is a composite based on data primarily generated by the laboratory of Toole (1981).

Predominant macromolecules in the extracellular matrix (ECM) include glycoproteins and proteoglycans (Manasek, 1975; Hay, 1981). Glycosaminoglycans are the carbohydrate moiety of proteoglycans and are composed of long-chained, poly anionic, repeating disaccharide units covalently attached to a core protein that exists in conjunction with counterions, normally Na+ (Manasek, 1975). There are eight types of GAGs of which six are sulphated. Based on numerous developmental biology studies, these GAGs can be divided into three functional groups: (1) hyaluronate, one of the non-sulphated GAGs; (2) chondroitin and chondroitin sulphate; and (3) dermatan sulphate, keratan sulphate, heparin, and heparan sulphate (Toole, 1976).

To date, most of what is known about the morphogenetic role of GAGs is their function in the formation of the chick embryo cornea. It appears that the corneal endothelial cells produce hyaluronate that causes the cornea stroma to swell providing space into which mesenchymal cells can migrate (Toole & Trelstad, 1971; Meier & Hay, 1973). Cessation of cell migration and the initiation of differentiation of these mesenchymal cells into corneal fibroblasts occurs at the same time as do a significant decrease in hyaluronate coupled with a rise in hyaluronidase activity plus an increase in the amount of chondroitin sulphates (Toole & Trelstad, 1971; Meier & Hay, 1973). The newly differentiated corneal fibroblasts appear to secrete the chondroitin sulphate (Meier & Hay, 1973).

A similar pattern of cell migration associated with a hyaluronate-rich environment and cell differentiation associated with a low hyaluronate, high chondroitin sulphate environment has been reported for the morphogenesis of the metanephric kidney (Belsky & Toole, 1983); for the regenerating newt limb (Toole & Gross, 1971); and for the developing vertebral column from sclerotomal cells (Kvist & Finnegan, 1970a; Toole, 1976; Derby, 1978). Other studies that support the idea that hyaluronate creates cell-free spaces into which mesenchymal cells may migrate have been done by Solursh (1976) on chick gastrulae.

Not only has hyaluronate been implicated in cell migration but also in the prevention of cell differentiation. Cell contact is thought necessary for differentiation of mesenchymal cells of the limb and it is suggested that hyaluronate may inhibit distal limb differentiation by preventing cell contact (Kosher & Savage, 1981).

Another mesenchymal area that involves cell migration prior to cell differentiation during morphogenesis is the presumptive chondrocranium. Several studies have focused on the migration of neural crest into the skull region (Johnston, 1966; Noden, 1975; Pratt, Larsen & Johnston, 1975; and LeLievre, 1978) and some studies have noted the biochemical milieu of the ECM of the developing skull (Hall, 1968). No study has documented the GAG content of the ECM in the presumptive chondrocranium at a time preceding chondrogenesis compared to the time chondrogenesis begins. Furthermore, no study has documented the GAG content of the chondrocranium during neural crest migration. Nor has any study shown the proportion of ECM to cell area and total head mesenchymal area during chondrocranium morphogenesis.

Although we did not determine the amount of GAG synthesis during neural crest migration in this study, we did determine (1) the proportion of ECM to cell area and total head mesenchymal area during chondrocranium morphogenesis and (2) the location, types, and relative amounts of GAG being synthesized in the presumptive chondrocranium at the onset of chondrogenesis and prior to this time (Fig. 2).

Fig. 2.

A,B. Photomicrographs using bright-field optics of median sections of heads of stage-24 and -33 chick embryos. At stage 24 (A) the head mesenchyme (presumptive chondrocranium) is relatively undifferentiated, consisting of mesenchymal cells and extracellular matrix (ECM). Likewise, the presumptive skin ectoderm and neuroepithelium both consist of one layer of cells. There are five major brain vesicles at this stage and the ventricles are the more prominent component of the brain. By stage 33 (B) many morphogenetic changes have occurred in the head, particularly in the head mesenchyme. The area of the presumptive skull has increased many fold but the cell density and cell size have not. The mesenchymal cells have aggregated into discrete foci. The cells within these foci have begun differentiating into chondrocytes as evidenced by the deposition of alcian blue material and blueish coloration upon tri chrome staining. The foci of chondrogenesis are restricted to the ventral half of the head which will become face and the ethmoid, sphenoid, temporal and periotic regions of the chondrocranium, ne, neuroepithelium; bv, brain ventricle; hm, head mesenchyme; se, skin ectoderm. Magnification: ×17 (stage 24) and ×9 (stage 33).

Fig. 2.

A,B. Photomicrographs using bright-field optics of median sections of heads of stage-24 and -33 chick embryos. At stage 24 (A) the head mesenchyme (presumptive chondrocranium) is relatively undifferentiated, consisting of mesenchymal cells and extracellular matrix (ECM). Likewise, the presumptive skin ectoderm and neuroepithelium both consist of one layer of cells. There are five major brain vesicles at this stage and the ventricles are the more prominent component of the brain. By stage 33 (B) many morphogenetic changes have occurred in the head, particularly in the head mesenchyme. The area of the presumptive skull has increased many fold but the cell density and cell size have not. The mesenchymal cells have aggregated into discrete foci. The cells within these foci have begun differentiating into chondrocytes as evidenced by the deposition of alcian blue material and blueish coloration upon tri chrome staining. The foci of chondrogenesis are restricted to the ventral half of the head which will become face and the ethmoid, sphenoid, temporal and periotic regions of the chondrocranium, ne, neuroepithelium; bv, brain ventricle; hm, head mesenchyme; se, skin ectoderm. Magnification: ×17 (stage 24) and ×9 (stage 33).

Morphometric analyses were made on median and parasagittal sections of skulls of stage-24 and -33 embryos in order to determine relative contributions of cells and ECM to the total area of head mesenchyme at these stages. Presumptive chondrocrania (heads minus eyes) of these stage embryos were also analysed histochemically and biochemically in order to identify the GAGs present in the ECM. This study reports that chondrogenesis begins in the presumptive chondrocranium of the chick embryo at stage 33 (Hamburger & Hamilton, 1951) and that the area of the head mesenchyme increases 60-fold between stages 24 and 33. Cell density and individual cell area as well as the relative proportion of total area allocated to cells and ECM exhibit little change between these two stages. GAGs are localized exclusively in the presumptive chondrocranium (head mesenchyme plus basement membrane of the neuroepithelium). These GAGs are not uniformly distributed throughout the head mesenchyme but are restricted to the ventral half of the presumptive skull. Within the ventral half, the GAG is further localized to specific areas such as the presumptive facial area, perichordal region, and presumptive ethmoid, sphenoid and periotic regions.

The types of GAG being synthesized in the presumptive chondrocranium of both stage-24 and -33 embryos are hyaluronate, the chondroitins and unidentified sulphated GAG (dermatan sulphate, keratan sulphate, heparin and heparan sulphate). Hyaluronate, chondroitin and the unidentified sulphated GAG constitute about 33 % each of the GAG being synthesized in the stage-24 presumptive skull. At stage 33, the level of hyaluronate synthesis drops to 2 %, the chondroitins to 24% and the unidentified sulphated GAG increases to 74%. There is an 18×5-fold decrease in the percentage of hyaluronate, a 1×5-fold decrease in the amount of chondroitins and a 2×7-fold increase in the percentage of unidentified sulphated GAG being synthesized as skull morphogenesis proceeds.

(A) Culture of embryos

(1) In ovo

White Leghorn chick embryos (stage 0 after Hamburger & Hamilton, 1951) were incubated in a forced air humidified incubator maintained at 37·5°C and 99·9% humidity. The incubator rotated the eggs every hour to prevent embryos from sticking to the shell. Embryos were cultured in ovo for 2 days then either were transferred to in vitro culture or continued incubation in ovo for a total of 10 days.

(2) In vitro cultures

Stage-12 (2-day-old) embryos were cultured outside the shell via the method of Auerbach, Kubai, Knighton & Folkman (1974). The fertilized egg was cracked open and the yolk plus a small amount of white was placed in a 60×15 mm sterile glass Petri dish. A maximum of three such cultures were then put into a sterile plastic 150× 25 mm Petri dish, sterile water was poured into the dish to ensure humidity, then the dish was covered. The cultures were incubated at 37·5°C, 99·5 % humidity in a BOD type incubator for the appropriate time necessary for the embryos to develop to stage 24 (4 days).

(B) Morphometric analyses

Mesenchymal areas, total number of cells within this area and individual cell areas were measured using the SMI Unicomp. The area of mesenchymal cells was calculated based on the formula for the area of an ellipsoid, πa·b, where a is the long axis and b is the short axis of the ellipse. Areas were measured for the median section plus the two sections on either side of the median section for three different embryos at stages 24 and 33 (Fig. 2). Cell densities were measured for nine different areas of each of the sections used for area measurements and the average used.

(C) Histochemical techniques

(1) Enzymatic treatment and alcian blue staining

Embryos of stages 24 and 33 were excised from the yolk. Both vitelline and amniotic membranes were removed before the embryos were fixed with formalin containing 4% cetylpyridinium chloride (CPC) for one week (Williams & Jackson, 1956). The embryos were then dehydrated and cleared using a series of ethanol and butanol solutions. Processing included exposing the embryos sequentially to 70% ethanol, 70% ENBA, 80% ENBA, 95 % ENBA plus eosin, 100 % ENBA, and 1:1100 % ENBA: paraplast. ENBA is a mixture of ethanol and n-butyl alcohol where 95% ENBA contains 5 parts 95% ethanol: 95 parts n-butyl alcohol and 100% ENBA is 5 parts absolute ethanol: 95 parts n-butyl alcohol. Times of exposure of the tissue to these solutions was 1 h for all solutions except those containing paraplast which were for 2h each (Humason, 1972).

Serial sections of stage-24 and -33 heads were made with an AO microtome at 5 μm section thickness. Sections of five heads at each stage were wet mounted onto precleaned slides and placed on a slide warmer to dry overnight. The sections were then hydrated by placing the slides in xylene followed by a series of ethanol solutions for 5 min each, and finally distilled water (Humason, 1972). After hydration, the sections were treated with the following enzymes: Streptomyces hyaluronidase, 100OTRUml-1, and testicular hyaluronidase type IV, 500 units ml-1 (both enzymes were obtained from Sigma Chemical Co.). Streptomyces and testicular hyaluronidases were dissolved in a 0·02M-sodium acetate, 0·lM-sodium chloride solution, pH5·0 (Table 1). Three to four drops of the appropriate enzyme solutions were placed directly atop the hydrated sections and the slides were incubated in a humidified chamber at 37·5°C for 4h. Controls were prepared by placing buffer only atop sections and then subjecting the slides to the same conditions as the enzyme-treated slides. After 4h, the slides were rinsed three times in double-distilled water and placed in a 3% glacial acetic acid solution for 3min. The sections were then stained with 1 % alcian blue 8GX at pH2·5 or 1·0 for 2h, dehydrated with ethanol, cleared and mounted with Permount. Both enzyme-treated and control sections were stained at the low and high pH in order to distinguish between sulphated and non-sulphated GAG (Lev & Spicer, 1964) (Table 2).

Table 1.

Summary of enzyme specificity for GAG1

Summary of enzyme specificity for GAG1
Summary of enzyme specificity for GAG1
Table 2.

Summary of differential staining of GAG1 by alcian blue

Summary of differential staining of GAG1 by alcian blue
Summary of differential staining of GAG1 by alcian blue

(2) Colorimetric and histochemical assays for hyaluronidase activity

Both a quantitative colorimetric assay and a less quantitative histochemical assay were used to assess the activity of the hyaluronidases prior to using them to treat embryo sections. The colorimetric method used was originally devised by Aminoff, Morgan & Morgan (1952), refined by Reissig, Strominger & Leloir (1955) and further refined by Hatae & Makita (1975).

Four materials were used in this assay: a buffer (0·8M-potassium borate, pH9-1); a chromagen (p-dimethylaminobenzaldehyde (DMAB); the substrate (hyaluronate); and the enzyme (Streptomyces hyaluronidase). The chromagen was made by placing 10 g of DMAB in 100 ml of glacial acetic acid that contained 12·5 % (v/v) of 10 N-HC1. This stock solution was diluted to 1:9 (v/v) with glacial acetic acid prior to use in the assay. The substrate, hyaluronate, was made in the concentrations of 50mgml-1, 40mgml-1, 30mgml-1, 20mgml-1, and lOmgml-1 of hyaluronate in 1·0ml distilled water. All concentrations of substrate were done in triplicate. The enzyme, Streptomyces hyaluronidase, had a concentration of 40TRU per 1·0 ml of distilled water.

The assay was performed in the following manner: to 200 μl of solutions of the various hyaluronate concentrations was added 50 μl (2TRU) of hyaluronidase solution. All tubes were incubated at 60°C for 2 h, after which time the tubes were placed in a boiling water bath for 3 min and then cooled to room temperature with running tap water. Next, 2· 0 ml of diluted DMAB solution was added to each tube with immediate shaking. The tubes were left standing at 37°C for 20 min to allow for full colour development; the tubes were then cooled again to room temperature with running tap water. Using a Bausch and Lomb Spectronic 21 and quartz cuvettes, all tubes were read at 585 nm. A linear regression analysis was done with O.D. values and corresponding concentrations in order to construct a standard curve. This colorimetric assay was done for every new batch of enzyme.

The routine assay for determining hyaluronidase activity was to treat hydrated sections of human umbilical cord with the enzyme. Umbilical cord was chosen because of its high content of hyaluronate (Wharton’s Jelly). Following incubation at conditions identical to those cited previously for the embryo sections, the umbilical sections were stained with alcian blue at pH 2·5. The intensity of blue was compared between treated and control sections. A 50 to 75 % loss in alcian blue staining was used as the criterion that the hyaluronidase was active and could be used for the embryonic sections.

(D) Identification of GAG using selective enzymatic degradation procedure

(1) Incorporation of [3H]glucosamine

A precursor of all GAGs, glucosamine hydrochloride, D-(1,6-H(N)) - specific activity of 38·9 Ci mmol-1, NEN)), (Kim & Conrad, 1976), was applied directly atop stage-24 and -33 embryos. Stage-24 embryos were in whole embryo culture at the time of pulse labelling. Since it is difficult for large numbers of embryos to survive to stage 33 in whole embryo cultures, these older embryos were left in their shells and a small rectangular opening was made through which the isotope was applied. Stage-24 embryos received 0·5 ml [3H]glucosamine (6·25 μCi 0·5 ml-1) and stage-33 embryos were pulsed with 1·0 ml [3H]gluco-samine (12·5μCiml-1). After 4h of incubation (37·5°C) in the presence of the isotope, the embryos were excised from the yolk and rinsed with chick saline solution. Excess yolk and the vitelline membranes were removed, followed by extirpation of the eyes. The heads were transected from the embryo axis at the posterior border of the metencephalon, pooled, and blotted as dry as possible on filter paper to prevent excess water being included in the weight of each pool. The number of heads and pool weight of the stages were kept consistent, with a pool of stage-24 heads averaging 13 heads weighing about 200 mg. Because the heads of the older embryos were so large, only one head was used per assay so that it was not a pool. Individual heads of these stage-33 embryos averaged 364 mg. All weighed samples were frozen at –60°C.

(2) Selective enzyme degradation of GAG

Each sample (pool or individual) of chick embryo heads was thawed and homogenized in 1·0 ml of 0·2M-Tris (pH7·8) buffer using a Brinkman Instruments polytron for 5 s at the maximum setting (Solursh, 1976). After homogenization, the polytron was rinsed with 1·0 ml of buffer and the rinse was added to the homogenate to bring the total volume of each test pool to 2·0 ml. A 0·1 ml aliquot was taken from the homogenate and put into a test tube containing 0·9ml of 1 N-NaOH and stored at 4°C for future protein determination (Fig. 3).

Fig. 3.

A synopsis of the biochemical protocol used to identify hyaluronate, chondroitin sulphate and the rest of the sulphated GAGs. The tissue homogenate consisted of heads minus eyes.

Fig. 3.

A synopsis of the biochemical protocol used to identify hyaluronate, chondroitin sulphate and the rest of the sulphated GAGs. The tissue homogenate consisted of heads minus eyes.

To ensure as complete digestion of GAG as possible, linker and core proteins were enzymatically digested using a non-specific bacterial protease made from Streptomyces griseas (Sigma). The concentration of protease used for digestion was 1·0 mg ml-1 of a solid with 5·8 units of proteolytic activity per mg. Protease of the same concentration was again added to the homogenate 12 h after the first treatment. All tubes were incubated at 37·5 °C and continually shaken for a total of 24 h at which time the tubes were placed in a boiling water bath for 10 min to stop enzymatic activity.

The enzymes used for the degradation of GAG were Streptomyces hyaluronidase (500 i.u.) and bovine testicular hyaluronidase (3000 i.u.) (Table 1). Selective enzyme degradation of GAG was performed by taking three 0·5 ml aliquots from the homogenate and placing them into 15 ml glass centrifuge tubes. Two aliquots were enzymatically treated: one with Streptomyces hyaluronidase and the other with bovine testicular hyaluronidase. A 1·0 ml quantity of 0·02 M-sodium acetate, 0·1M-sodium chloride buffer (pH5·0) with enzyme was added to bring the volume to 1·5ml in each tube. To the third aliquot (control), 1·0 ml of buffer only was added (Toole & Gross, 1971; Solursh, 1976). A 1·0 ml volume was used to optimize the pH to enhance optimal activity of the hyaluronidases (Ohya & Kaneko, 1970; Toole, 1973). Preliminary experimentation showed that two volumes of acetate buffer (pH 5·0) to one volume of Tris buffer (pH 7·8) properly corrected the pH. The final pH was approximately 5·5. After 4h of incubation (37·5 °C) the tubes were placed in a boiling water bath for 10 min to arrest enzyme activity.

After letting the tubes cool to room temperature (20°C), 1·0 mg each of carrier hyaluronate and chondroitin sulphate was added to the tubes. This was done to increase the yield of precipitate of non-digested GAG, specifically hyaluronate and the chondroitin sulphates. Precipitation of undigested GAG was done by adding 0·5 ml of a 4·0% CPC solution to give each tube a final volume of 2·0 ml and a CPC concentration of 1· 0 %. The precipitates were then centrifuged (Sorvall RC-5) for 30 min at 12000 g. The pellet was washed three times by suspending it in 95 % ethanol and then centrifuging as before. After the third rinse, the pellet was suspended in 1·0 ml of methanol. The 1·0 ml of methanol with the precipitate was dissolved in 10ml of Aquasol (New England Nuclear). Scintillation vials were counted twice for 10min using a liquid scintillation counter (Packard C-2425).

(3) Determination of protein

Protein content per pool of homogenated chick embryo heads was determined by using the colorimetric assay of Lowry, Rosebrough, Farr & Randall (1951). A standard curve was plotted by preparing a standard protein solution containing crystalline bovine serum albumin at a concentration of 0·05 g 100ml-1. From this standard solution, aliquots of 0·10, 0·20, 0·25, 0·30, 0·40, 0·50, and 0·70 ml were taken and put into separate test tubes. Each amount was measured out in triplicate. Double-distilled water was added to all the tubes to bring the volume up to 1·0 ml.

Solutions of 2·0% sodium carbonate (w/v) in 0·1N-NaOH and 0·5% copper sulphate pentahydrate (w/v) in 1·0 % potassium tartrate (w/v) were made. These solutions were mixed together 50:1 respectively. Then 5·0 ml of this freshly mixed reagent was added to every tube that contained the dilutions of the standard proteins. The tubes were mixed with a vortex mixer and let stand to react at room temperature (20°C) for 10min. After that time, 0·5ml of 1·0N-Folin-Ciocalteau phenol reagent was added to every tube. The tubes were shaken and placed in an incubator (37°C) for at least 30 min to allow for full colour development. The tubes were then read using a spectrophotometer at 500 nm. After the O.D. values were recorded, a standard curve was plotted using linear regression.

The unknown samples, saved after homogenization, were placed in a 90°C water bath for 30 min. Then a 0·1 ml aliquot was taken from each unknown test sample and placed in tubes containing 0·9 ml of double-distilled water. Experimental samples were run through the assay the same way as the ones in determining the standard curve. From the O.D. values recorded for the test samples, an accurate determination of the amount of protein in each homogenated pool of chick embryo heads was made by referring to the standard curve and allowing for the dilution factor of the original homogenate concentration.

(A) Morphometric and histological findings

Histological examination of comparable sagittal sections of embryonic heads from stage-24 and -33 chicks showed at stage 24, the skin ectoderm was lifted away from the neuroepithelium throughout the entire head due to the thickness of the head mesenchyme. Foci of chondrogenesis were first apparent in the head mesenchyme at stage 33 based on assessment of analogous sections from stages 24 to 33. Morphometric analysis of the head mesenchyme region for these two stages showed a 60-fold enlargement of the mesenchymal area between these two stages while cell density and cell area of individual cells remain unchanged (Table 3) suggesting that the increase in mesenchymal area is primarily due to the increase in ECM. Measurements of the ECM support the idea that most of the increase in mesenchymal area is due to an increase in ECM. Roughly 65 % of the total mesenchymal area is ECM whereas 35 % is cellular (Table 3). These increases in ECM are primarily in the ventral half of the head corresponding to the presumptive facial, ethmoid, sphenoid and periotic regions.

Table 3.

Proportion of total mesenchyme area of the presumptive skull allocated to cell and ECM1 areas for stage-24 and -33 chick embryos

Proportion of total mesenchyme area of the presumptive skull allocated to cell and ECM1 areas for stage-24 and -33 chick embryos
Proportion of total mesenchyme area of the presumptive skull allocated to cell and ECM1 areas for stage-24 and -33 chick embryos

(B) Histochemical findings

(1) Localization and identification of GAG in stage-24 presumptive skulls

Sections stained with alcian blue and examined with bright-field optics showed distinct presence or absence of blue material in specific regions. Most notable was that GAGs were present only in the head mesenchyme (presumptive chondrocranium) and basement membrane of the neuroepithelium and not either the neuroepithelium proper or skin ectoderm. Dark-field optics were employed since photomicrographs utilizing appropriate filters with bright-field optics did not clearly show alcian blue material as distinct from unstained regions, especially in comparing controls with enzyme-treated sections. In order to maximize localization of alcian blue material in sections of whole heads, low magnification was used and thus resolution was sacrificed. Nonetheless, dark-field optics consistently enhanced the presence of alcian blue material when compared to bright-field optics of the same material. The bright-field image was the kind of alcian blue result previously published by one of us (Schoenwolf & Desmond, 1984). The photographs do show distinct differences between alcian blue material differentially stained and on sections pretreated with enzymes (Figs 4, 5). The neuroepithelium appears brighter than any other material in the head with darkfield optics. This degree of brightness was never blue under bright field nor magenta under dark field, and thus was never interpreted to be due to the presence of alcian blue material. On the other hand, the head mesenchyme was blue under bright field, magenta under dark field and less bright compared to the neuroepithelium on photographs with dark-field optics.

Fig. 4.

A–D. Photomicrographs using dark-field optics of median and near median sections of whole heads from stage-24 chick embryos. Sections A,B were stained with alcian blue at pH2·5 (A) and pH 1·0 (B). Section C was treated with Streptomyces hyaluronidase prior to alcian blue staining at pH 2·5, and section D was pretreated with testicular hyaluronidase. Alcian blue material within the head and upper neck mesenchyme is most notable in Fig. 4A indicated by arrows. Lack of alcian-blue-stained material in B,C,D suggest that the majority of GAGs stained with alcian blue is hyaluronate and the chondroitins. Magnification: ×4.

Fig. 4.

A–D. Photomicrographs using dark-field optics of median and near median sections of whole heads from stage-24 chick embryos. Sections A,B were stained with alcian blue at pH2·5 (A) and pH 1·0 (B). Section C was treated with Streptomyces hyaluronidase prior to alcian blue staining at pH 2·5, and section D was pretreated with testicular hyaluronidase. Alcian blue material within the head and upper neck mesenchyme is most notable in Fig. 4A indicated by arrows. Lack of alcian-blue-stained material in B,C,D suggest that the majority of GAGs stained with alcian blue is hyaluronate and the chondroitins. Magnification: ×4.

Fig. 5.

A–C. Dark-field photomicrographs of sections of stage-33 chick embryo heads. Section A was stained with alcian blue at pH2·5; B,C were stained with alcian blue at pH2·5 after pretreatment with Streptomyces hyaluronidase (B) and testicular hyaluronidase (C). Foci of chondrogenesis are identified within the arrows. The fact that the staining profile is the same for all three treatments suggests that the predominant GAGs present in the head mesenchyme at this time are GAGs other than hyaluronate and the chondroitins. The arrow indicates the area containing the alcian blue material. Magnification: ×4.

Fig. 5.

A–C. Dark-field photomicrographs of sections of stage-33 chick embryo heads. Section A was stained with alcian blue at pH2·5; B,C were stained with alcian blue at pH2·5 after pretreatment with Streptomyces hyaluronidase (B) and testicular hyaluronidase (C). Foci of chondrogenesis are identified within the arrows. The fact that the staining profile is the same for all three treatments suggests that the predominant GAGs present in the head mesenchyme at this time are GAGs other than hyaluronate and the chondroitins. The arrow indicates the area containing the alcian blue material. Magnification: ×4.

In the head mesenchyme there is a striking difference in the intensity of alcian blue material of stage-24 embryos stained at pH 1·0 and 2·5. Sections from the same embryo exhibited almost no alcian blue material at pH 1·0 in contrast to a high intensity of material at pH2·5 (Fig. 4A,B). The stained regions were most prominent in the ventral half of the head mesenchyme in the region of the mesencephalon and prosencephalon and in the perinotochordal area. These regions would correspond to the future face as well as the ethmoid, presphenoid, basisphenoid, alisphenoid, squamosal and periotic portions of the future chondrocranium. The dorsal half of the skull in the same brain regions was faintly stained.

Stage-24 sections stained with alcian blue after Streptomyces hyaluronidase treatment exhibited much less alcian blue material in the ventral region compared to the control section stained at the same pH (2·5) (Fig. 4A,C). Likewise, similar sections treated with testicular hyaluronidase showed identical staining with alcian blue as those sections treated with Streptomyces hyaluronidase (Fig. 4C,D). Both results of the differential pH staining and enzyme treatments suggest that the majority of GAGs present in the skull of stage-24 embryos is hyaluronate and the chondroitins.

(2) Localization and identification of GAG in stage-33 presumptive skulls

Alcian-blue-stained sections of stage-33 presumptive skulls like the stage-24 sections exhibited a positive reaction only in the ventral region of the presumptive skull at pH 2·5. The staining was not uniformly distributed throughout the ventral region, but was evident in specific areas, such as the perichordal region, presumptive facial area and the presumptive ethmoid, sphenoid, temporal and periotic regions of the chondrocranium (Fig. 5A). Furthermore, the alcian blue material was condensed into specific foci (Fig. 5A).

Sections treated with both Streptomyces and testicular hyaluronidase prior to alcian blue staining exhibited no reduction in alcian-blue-positive material when compared to the control sections (Fig. 5A,B,C). This clearly shows that the GAGs contributing to alcian blue staining are other than hyaluronate or the chondroitin sulphates.

(C) Biochemical findings: identification of GAG via selective enzyme degradation

The histochemical findings localized all GAGs in the heads of both age groups to the presumptive chondrocranium, i.e. head mesenchyme plus neuroepithelial basement membrane. This means that all of the [3H]glucosamine that was incorporated into the head at the time of the pulse-labelling experiments was utilized by GAGs in only the presumptive chondrocranium region. For stage-24 embryos, the presumptive chondrocranium exhibits approximately equal amounts of synthesis of hyaluronate, chondroitins and all other GAGs (Table 4). Based on the c.p.m. of CPC precipitates, hyaluronate synthesis accounts for 37·34 % of the GAG, the chondroitins for 35·33%, while 27·33% is one or more of all other GAGs, i.e. dermatan sulphate, keratan sulphate, heparan sulphate and/or heparin (Table 5). The average number of heads per pool for this biochemical assay was 13, the average weight of each pool was 210 mg and the average amount of protein was 9·86 mg (Table 5).

Table 4.

Percentage of GAG1 in pooled heads (stage 24) and individual heads (stage 33) ofchick embryos

Percentage of GAG1 in pooled heads (stage 24) and individual heads (stage 33) ofchick embryos
Percentage of GAG1 in pooled heads (stage 24) and individual heads (stage 33) ofchick embryos
Table 5.

CPM of GAG1 per mg protein for heads of chick embryos at stages 24 and 33

CPM of GAG1 per mg protein for heads of chick embryos at stages 24 and 33
CPM of GAG1 per mg protein for heads of chick embryos at stages 24 and 33

Synthesis of GAG in the presumptive chondrocrania of stage-33 embryos consists of 2 % hyaluronate, 24·25 % chondroitins and 73·75 % dermatan sulphate, keratan sulphate, heparin and heparan sulphate (Tables 3,4). The average weight per stage-33 head was 364 mg and the average amount of protein per head was 22·18 mg.

A comparison of the relative amounts of GAG being synthesized at the two developmental stages, 24 and 33, based on the amount of GAG per mg protein indicates there is an 18·5-fold decrease in the percentage of hyaluronate, only a 1·5-fold decrease in the percentage of chondroitins and a 2·7-fold increase in the percentage of unidentified sulphated GAG as skull morphogenesis proceeds (Fig. 6).

Fig. 6.

This bar graph represents the percentages of hyaluronate (HA) and the chondroitins, i.e. chondroitin (CH) and chondroitin sulphate (CH-S) being synthesized by the head mesenchyme at two developmental periods. Stages 24 and 33 represent times prior to and at the onset of chondrogenesis of the head mesenchyme.

Fig. 6.

This bar graph represents the percentages of hyaluronate (HA) and the chondroitins, i.e. chondroitin (CH) and chondroitin sulphate (CH-S) being synthesized by the head mesenchyme at two developmental periods. Stages 24 and 33 represent times prior to and at the onset of chondrogenesis of the head mesenchyme.

The histochemical and biochemical data of this study must be considered together in order to make any correlation of GAG synthesis with chondrocranium morphogenesis. Since it was impossible to isolate the presumptive chondrocranium tissue from the brain and skin ectoderm, it was necessary to show that all of the GAG in the head was restricted to the presumptive chondrocranium. The alcian blue staining clearly demonstrates this, to be the case for the heads from embryos at stages 24 and 33. Similarly, it was impossible to separate the facial area from the neurocranium proper of the presumptive skull for the biochemical assay. Based on the histochemistry, the alcian blue material appeared to be distributed equally between the presumptive face and neurocranium.

In order to determine the relative proportions of GAG present in the chondrocranium during development, it was necessary to quantify the amount of alcian blue material in sections using microspectrophotometry or densitometry, or to use an entirely different approach. We chose to use the pulse-labelling method followed by selective enzyme degradation. The major drawback of this pulselabelling method is that one is limited to measuring the amount of GAG being synthesized at the time of adding the label with no knowledge of the rates of degradation of GAGs during the time periods of labelling. In other words, this methodology does not provide identification of the total amount of a specific GAG present. Consequently, correlating GAG type with specific morphogenetic events must be limited to correlating the synthesis of specific GAGs with morphogenesis. Whether it is the synthesis or total amount of GAG type that may influence a morphogenetic event remains to be examined. Thus far, developmental biologists have limited morphogenetic correlations to synthesis of a specific GAG (Toole, 1981). Likewise, this study correlates skull morphogenesis with synthesis of specific GAG types and has the same limitations.

The histochemical findings that no GAG was detected in the neuroepithelium of the heads examined in this study contradict the report of Toole (1976), which cited an increase of GAG in the brain as the brain developed. It is not clear from that report by Toole whether the brains were isolated from the head mesenchyme. So based on our study the question remains whether this reported increase in GAG synthesis was really for the head not the brain.

Localization of GAG in the basal lamina of the neuroepithelium parallels the finding of others who have shown that the neuroepithelium secretes GAG (Hay & Meier, 1974). The absence of GAG in the basal lamina of the skin ectoderm differs from what is reported by Fisher & Solursh (1977), for younger embryos. Such an absence may indicate that the GAG present in the basal lamina of the ectoderm has migrated into the ECM of the head mesenchyme as shown in other developing systems such as the teeth (Thesleff & Pratt, 1980) and heart (Markwald & Funderburg, 1983). Although this study did not identify specifically the GAG located in the basal lamina, the differential staining with alcian blue suggests a high concentration of sulphated GAG in the basement membrane. Fisher & Solursh (1977) showed that the predominant GAG in the basal lamina of the ectoderm is hyaluronic acid. Others showed that chondroitin sulphate is most prominent in the neuroepithelium (Hay & Meier, 1974; Trelsted, Hayashi & Toole, 1974). These differences in type of GAG being synthesized by the two different epithelia could suggest that the roles of the two GAG are different.

It may be that the chondroitin sulphate from the neuroepithelium serves as a template onto which chondrogenesis and/or osteogenesis shape the skull. In other words, the chondroitin sulphates might serve as an epithelial-mesenchymal interface transferring the structural nuances of the brain onto the developing skull. Such an epithelial-mesenchymal interface could explain the highly refined morphological similarities between the brain and skull. The GAG interface could be instructive like an inductor or merely serve as a template. Schowing (1968) used selective deletion of the prosencephalon and mesencephalon to show that the frontal bone derived in part from the prosencephalic crest received inductive signals from both the prosencephalon and mesencephalon.

The histochemistry results show that most of the GAGs are located in the ventral half of the presumptive chondrocranium at the embryonic stages studied. This half of the chondrocranium consists primarily of endochondral bones, i.e. bones formed via cartilage model precursors. The correlation of different proportions of sulphated to non-sulphated GAGs in the ventral skull over time may suggest that GAGs function as environmental regulators of endochondral bone formation. Hall (1968, 1970) has reported that formation of secondary cartilage at sites of articulation or attachment of muscle, ligaments or tendons occurs in response to a deposition of GAG. On the other hand, the correlation may have no causal effect on bone morphogenesis whatsoever but simply represent the GAGs present in the head mesenchyme during the period that skull morphogenesis occurs.

The chondroitin sulphates present in the ECM once chondrogenesis has begun are probably secreted by the chondrocytes as has been shown for other developmental systems (Lash, Holtzer & Whitehouse, 1960; Franco-Browder, DeRydt & Dorfman, 1963; Kvist & Finnegan, 1970a,b; O’Hare, 1973; Fisher & Solursh, 1977, 1979; Solursh, 1976; Solursh & Morriss, 1977; Morriss & Solursh, 1978). Such a chondroitin-sulphate-rich matrix appears to be necessary for maintaining chondrocytes in the differentiated state (Holtzer, 1964; Lash, 1968). Not only is chondroitin sulphate necessary for maintenance of chondrocytes in the differentiated state but very low concentrations of hyaluronate or even a tetrasaccharide product of hyaluronidase digestion totally prevents chondrogenesis (Toole, Jackson & Gross, 1972; Toole, 1973).

The fact that there is a higher proportion of chondroitin sulphate being synthesized prior to than at the onset of chondrogenesis seems somewhat contradictory and could indicate several things. As stated above, it appears that the drop in hyaluronate synthesis serves more as a stimulator of chondrogenesis than does the increase in synthesis of chondroitin. Furthermore, it may be that the proportional amounts of these GAGs present in the particular site of differentiation are more critical to regulating differentiation than is the proportional synthesis of these GAGs. This study only measured the actual synthesis at the two times. Finally, chondrogenesis may be initiated and/or maintained by one of the other GAGs not identified in this study such as keratan sulphate. Keratan sulphate chains are usually present with and covalently linked to the same protein as chondroitin sulphate (Hay, 1981).

Hyaluronate from the basal laminae of both of the epithelia could increase the extracellular space. Such an increase in extracellular space is in agreement with other studies reported in the literature of developmental systems at a time of undifferentiated cell migration. Mesenchymal cell migration in the presumptive skull at stage 24 occurs at the same time that cell migration is occurring in the corneal stroma and from the sclerotome (Kvist & Finnegan, 1970a; Toole & Trelsted, 1971). Hyaluronate is the major GAG present at this time and probably initiates cell migration because of its ability to create cell-free spaces and/or prevent cell-cell interaction. It is thought that hyaluronate which is highly polyanionic due to the presence of numerous carboxyl moieties creates cell-free spaces by entrapping water. As a result a space highly disproportionate to the concentration of hyaluronate is formed.

Additionally, there is evidence that cells may have GAG receptors on their plasma membranes that facilitate cell-to-cell surface binding (Toole, 1981). GAG receptor binding may occur via plasma membrane proteins that covalently bind to the GAGs or by a reaction between the GAG and cell surface receptor sites. An area rich in hyaluronate would effectively bind up all hyaluronate receptors, negating any chance for cell-to-cell contact. Therefore, cells would be more free to move. Studies have shown that cell lines in suspension aggregate when a moderate amount of hyaluronate is present. When hyaluronidase or excess hyaluronate is added, aggregation of cells does not occur (Underhill & Toole, 1981). Hyaluronate and chondroitin sulphate are also implicated as weakening the initial cell-substratum attachments of cells grown in vitro (Culp, Murray & Rollins, 1979). These investigators report that heparan sulphate on the cell surface, along with fibronectin, initiates binding to the substratum. A weakening of such adhesion promotes movement. Just how these in vitro findings relate to cell migrations in vivo is not known but hyaluronate synthesis is elevated during migration in many morphogenetic systems as discussed in the Introduction. However, a very direct test of the effect of hyaluronate on neural crest migration in vivo was reported by Anderson & Meier (1982). They treated embryos with Streptomyces hyaluronidase in concentrations that significantly reduced normal amounts of hyaluronate in the ECM and prevented neural tube fusion but not neural crest migration.

This work was supported by a Villanova University Summer Grant to M.E.D. and a grant from Sigma Xi to C.D.G. We acknowledge Optical Apparatus, Ardmore, PA for their generous loan of a dark-field base for use with the Nikon SMZ-10, Mary Pacheco for doing the morphometric measurements and Dr Russell Gardner for his critical evaluation of the biochemical methodology.

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