Hyalin

Function

Hyaline encodes a protein, which is the major constituent of the hyaline layer, an extraembryonic matrix that functions as a substrate for cell adhesion through early development. At the beginning of gastrulation primary mesenchyme cells lose contact with the hyaline layer and ingress into the blastocoel. At that time, these cells lose an affinity for hyalin and increase their adhesion toward other substrates (McClay and Fink, 1982; Fink and McClay, 1985).
Later, the vegetal plate invaginates to form the endoderm. Concurrent with this morphogenesis, the ectoderm and endoderm change in their relative affinities for hyalin; ectoderm cells retain an affinity while endoderm cells lose their affinity (Burdsal et al., 1991).
The hyaline matrix was shown to be sensitive to calcium ions by Herbst (1900), and this property was later used by investigators to devise isolation schemes, alternating calcium-free solubilization with calcium-induced precipitation (Faust et al., 1959; Vacquier, 1969; Kane, 1970; Citkowitz, 1971).
Genomic DNA blots showed that hyalin gene is present as a single copy (Wessel et al., 1998).

Protein

Hyalin is a fibrillar glycoprotein of approximately 330 kDa that multimerizes in the presence of calcium. Hyalin contains approximately 25% acidic residues, only 3.5% basic residues, and 2ñ3% carbohydrate (Stephens and Kane, 1970; Citkowitz, 1971). In addition, it has distinct heterogeneity. On SDSñPAGE gels hyalin appears as a smear, suggesting that the molecule is either differentially glycosylated and/or is of a heterogeneous length.
Physical studies of purified hyalin have also suggested that the protein is heterogeneous with different sedimentation properties (Gray et al., 1986; Justice et al., 1992). The ultrastructure of the protein was identified as a filamentous molecule about 75 nm long with a globular ëëheadíí about 12 nm in diameter (Adelson et al., 1992). Though most of the filaments of the protein are 75 nm in length, other filament regions are 25 ñ 50 nm longer than the majority, supporting the heterogeneous nature of the protein. In the presence of calcium, the hyalin monomers aggregate with their globular regions to a high-molecular-weight core particle that remains associated with hyalin throughout purification.
Sequence from both species (S. purpuratutus and L. variegatus) contains a large region of an extended tandem repeat averaging 84 amino acids. The predicted amino acid sequence is strongly conserved among the repeats both within each species (ranging from 38 to 97% identical) and between species (64% identical) suggesting a strong functional conservation. Analysis of this repeat shows that it is a unique sequence within the GenBank database with only weak similarity to mucoid protein sequences.
The tandemly repeated sequence resides within the long filamentous portion of the molecule and not in the globular head region. Repeat region of hyalin appears to contain the ligand for the hyalin cell surface receptor. These data help explain some of the classic functions ascribed to the hyalin protein in early development (Wessel et al., 1998).
GenBank: 3420721

Subcellular location

Hyalin is exocytosed at fertilization from cortical granules, within which the hyalin is selectively packaged into the electron-lucent, homogeneous region (Hylander and Summers, 1982). At fertilization, the hyaline layer forms rapidly and swells to approximately 2ñ3 mm thick within 10ñ15 min postinsemination (Harvey, 1956). The intracellular distribution of the hyalin message is consistent with its encoding hyalin since it is confined to the region of the rough endoplasmic reticulum around the nucleus as expected for a protein targeted to the secretory pathway. The flattened squamous cells of the aboral ectoderm enable this visualization (Wessel et al., 1998).

Expression Pattern

RNA gel blot analysis showed that the mRNA of 12 kb is present in ovaries but not in eggs or early development. The same sized mRNA reappears in gastrulation and accumulates in larvae.
The new hyalin mRNA appears only in the ectoderm in a pattern that is consistent initially with an enriched distribution in aboral ectoderm relative to oral ectoderm. A sharp boundary of hyalin message is present in a ring around the vegetal plate, the diameter of which decreases as gastrulation progresses. Based on other lineage markers, these data suggest that hyalin is excluded from endoderm at the ectodermal/endodermal boundary (Ruffins and Ettensohn, 1996; Logan and McClay, 1997).
As gastrulation proceeds the RNA becomes further enriched in the aboral ectoderm, and in early plutei the hyalin mRNA appears almost exclusively in the aboral ectoderm.
The hyalin mRNA pattern in larvae is unusual. First, the oral ectoderm has no signal by in situ RNA hybridization analysis except for a thin strip of ectoderm adjacent to the ciliary band. The entire ciliary band is negative for hyalin RNA. Since the ciliary band is thought to represent the boundary between the oral and the aboral ectoderm (Cameron et al., 1990), the pattern of hyalin message distribution does not strictly adhere to this boundary since a stripe several cells thick on the oral side of the ciliary band contains hyalin mRNA. The oral surface area beyond these cells is negative for hyalin mRNA.
As larvae age, a progressive loss of signal is apparent within the aboral ectoderm, beginning in the apex of the embryo and moving progressively toward the oral surface.
Significant mRNA is retained at the boundary between the ciliary band and the oral ectoderm except at the very tips of the arms, where no label is detectable, and at the apex of the oral hood.
This pattern of hyalin mRNA and protein accumulation is very similar between L. variegatus and S. purpuratus. The only significant differences are that the clear band around the ciliary band in S. purpuratus is not well defined and the apex of the larva is not stained even late in gastrulation. Immunoblot analysis showed that the smear of immunolabeling characteristics of hyalin protein in whole eggs and embryos is present at similar levels throughout development. It is apparent that the protein present from fertilization until gastrulation is of maternal origin.
The hyalin protein is abundant in the lumen of the invaginating gut and is visible in live embryos, in isolated hyalin bags (Citkowitz, 1971), and in fixed sections of embryos where it appears thicker than the hyalin over the ectoderm. This endodermal-associated hyalin appears to be maternally derived, from the original cortical granule exocytosis, since cells of the early embryo and the endodermal lineage of the postgastrula embryo do not accumulate detectable hyalin mRNA.
Thus, the lumenal hyalin is appparently drawn into the archenteron during invagination and results from a strong adhesion of the endodermal precursor cells to the hyaline layer.
Following formation of the mouth and feeding by the larvae, the lumenal hyalin is removed in the midgut and hindgut regions (Wessel et al., 1998).

mRNA level

Temporal accumulation
Method: RNA gel blot analysis
Reference: Wessel et al., 1998

Stage Developing oocytes Egg Blastula Mesenchymal blastula Gastrula Pluteus

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