Gene Networks Database

Lytechinus variegatus Genes in Development: Extracellular matrix (ECM) proteins

ECM 18

Function

ECM 18 gene codes for a new molecule of the extracellular matrix in Lytechinus variegatus.
ECM 18 appears to be important in the morphogenesis of PMCs and vegetal plate descendants during gastrulation (Berg et al., 1996).

Protein

ECM 18 does not appear to be similar to any known ECM proteins in sea urchin embryo, including Endo16 (Nocente-McGraph et al., 1989), ECM 1 (Ingersoll and Ettensohn, 1994) and ECM 3 (Wessel and and Berg, 1995).
One region of ECM 18 sequence has three repeating motifs, each 67 amino acids in length, containing nine absolutely conserved cysteins. This segment of cysteine repeats has 42% sequence identity to the domain D of the von Willebrand sequence (Berg et al., 1996).
GenBank: 1100979

Subcellular location

Immunofluorescence assay showed that during invagination of the endoderm, ECM 18 accumulates in the basal lamina underlying the ectoderm and endoderm cells and no signal is detected in the cytoplasm or in the apical lamina of these cells.
Ultrastructural immunolabaling shows that ECM 18 accumulates within the basal lamina and within nearby fibers of the blastocoel in plutei. No consistent immunolabaling was found within the cytoplasm of the epithelial cells, within the apical lamina of the embryo, or surrounding the cell bodies of the primary mesenchyme cells (PMCs)(Berg et al., 1996).

Expression Pattern

The pattern of ECM 18 expression is unique among the extracellular matrix molecules known in this embryo.
The RNase protection assay shows that ECM 18 mRNA is present in eggs and early embryos and increases in abundance approximately 3-fold at mesenchyme blastula and gastrula followed by a slight decrease at pluteus.
ECM 18 appears as a transcript approximately 6.5 kb, detection of a higher molecular weight ECM 18 mRNA, of up to 7 kb, occurs at gastrulation.
In situ hybridization show that ECM 18 mRNA is evenly dispersed throughout the embryo in eggs and early blastula. During gastrulation, however, ECM 18 mRNA accumulates to the highest levels in endoderm cells with approximately half as much signal in the ectoderm. Primary mesenchyme cells have no detectable signal. The accumulation of ECM 18 mRNA in endoderm is uniform along the digestive tract, beginning with primary invagination and continuing through development to the larval stage.
Immunolocalization experiments detected no ECM 18 protein in eggs and early blastula. At mesenchyme blastula ECM 18 begins to accumulate along the blastocoel wall, but very little signal is associated with mesenchyme cells throughout the blastocoel. During gastrulation signal is detected underlying both the ectoderm and the invaginating endoderm. This accumulation pattern continues through pluteus. In larvae, ECM 18 accumulates along the blastocoel wall underlying all epithelial tissues.
In an attempt to understand the cause of the delayed onset in accumulation of ECM 18 protein, the association of ECM 18 mRNA with polysomes was examined. ECM 18 mRNA is detected in polysomes isolated from eggs, and a small but detectable signal is present in early embryos. However, during gastrulation, when protein is first detected by immunolocalization and western blots, substantially more ECM 18 is polysome-associated. The increased association of ECM 18 mRNA with polysomes from cleavage to mesenchyme blastula is approximately fivefold. Thus, one explanation for the lack of ECM 18 protein in early embryos is that the maternal ECM 18 mRNA does not associate with polysomes and thus, is not translated (Berg et al., 1996).

mRNA level

Temporal accumulation
Method 1: RNase protection assay
Reference: Berg et al., 1996
Method 2: RNA blot hybridization
Reference: Berg et al., 1996

Stage Egg Early blastula Mesenchyme blastula Gastrula Pluteus

Level + + + + +

Protein level

Temporal accumulation
Method: Western blot analysis
Reference: Berg et al., 1996

Stage Egg Blastula Early gastrula Late gastrula Early pluteus

Level - - + + +

mRNA spatial localization
Method 1: in situ hybridization
Reference: Berg et al., 1996

Stage Egg Early blastula Early gastrula Late gastrula Pluteus

Tissue Evenly distributed through the embryo Evenly distributed through the embryo Endoderm (highest levels), ectoderm Endoderm (highest levels), ectoderm Endoderm (highest levels), ectoderm

Protein spatial localization
Method 1: Immunolocalization assay
Reference: Berg et al., 1996

Stage Egg Early blastula Mesenchyme blastula Gastrula Pluteus

Tissue - - Along the blastocoel wall, little signal in mesenchyme cells throughout the blastocoel Underlying both ectoderm and invaginating endoderm Underlying both ectoderm and invaginating endoderm

Polysomal associated RNA level

Temporal accumulation
Method 1: RNase protection assay
Reference: Berg et al., 1996

Stage Egg Cleavage Mesenchyme blastula Gastrula

Level - - + + +

Sequences

GenBank:

1515 bp mRNA

Regulatory Regions

Regions

Regulatory Connections

Upstream Genes

ECM 18

Downstream Genes

Evolutionary Homologues

von Willebrand sequence

Links

Urchin Web

Bibliography

UrchiNet

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Stage	Egg	Early blastula	Mesenchyme blastula	Gastrula	Pluteus
Level	+	+	+	+	+

Stage	Egg	Blastula	Early gastrula	Late gastrula	Early pluteus
Level	-	-	+	+	+

Stage	Egg	Cleavage	Mesenchyme blastula	Gastrula
Level	-	- +	+	+