Gene Networks Database


Lytechinus variegatus Genes in Development: Primary mesenchyme-specific genes


LvSM30


Function

LvSM30 is a PMC marker gene (Guss et al., 1997).
SM30 was previously characterized as an acidic glycoprotein of the spicule matrix. The specific role of matrix proteins have not been elucidated, although they may function in the nucleation or orientation of crystal growth (George et al., 1991; Livingston et al., 1991; Katoh-Fukui et al., 1991, 1992; Harkey et al., 1995).

Protein

Subcellular location


Expression Pattern

Nothern blot analysis recognized a transcript of approximately 2 kb, which was first detectable at low levels at the mesenchyme blastula stage. Expression increase during gastrulation and through the late pluteus larva stage.
Whole-mount in situ hybridization detected that LvSM30 transcripts accumulate specifically in PMCs located in the ventrolateral clusters of the forming ring. By the late gastrula stage cells located in the ventrolateral clusters exhibit the highest level of LvSM30 expression, whereas cells along the dorsal branch of the ring exhibit the intermediate levels. Expression is undetectable in the cells of the ventral chain and at the tips of the longitudinal chains that extend toward the animal pole from the ventrolateral clusters.
At later stages, cells at the tips of the longitudinal chains begin to express LvSM30 as they migrate toward the animal pole. By the pluteus larva stage, strong expression is exhibited by all PMCs in the embryo except those located along the ventral transverse rods (Guss et al., 1997).

mRNA level

Temporal accumulation (persent maximum abundance*)

Method 1: Nothern blot hybridization
Reference: Guss et al., 1997

Stage
Hatched blastula
Early mesenchyme blastula
Late mesenchyme blastula
Midgastrula
Late gastrula
Prism
Early pluteus
Late pluteus
Level
8
10
15
19
35
53
70
100

*Values were corrected for relative amounts of hybridizable RNA based on ubiquitin expression levels.

Spatial localization

Method 1: Whole-mount in situ hybridization
Reference: Guss et al., 1997

Stage
Early gastrula
Late gastrula
Prism
Early pluteus
Pluteus larva
Tissue
PMCs that are forming in the ventrolateral clusters
PMCs in the ventrolateral clusters (highest level), PMCS in the dorsal chain (intermediate level)
PMCs that are forming in the ventrolateral clusters, especially along anonymous rods (highest level)
PMCs at the tips of postoral arms (highest level), most PMCs except those along ventral transverse rods (increased level)
all PMCs except those along ventral transverse rods (highest level)


Ectopic expression

Alteration of cell number

In PMC-deficient embryos (containing 3-30 PMCs, n=30), the highest expression levels were seen in the ventrolateral clusters, despite the reduced numbers of cells at these sites. In some embryos with very few PMCs, the positions of the ventrolateral clusters could not be determined. Regardless, the LvSM30 expression was observed in aggregates of as few as three cells. These observations indicate that clustering of PMCs is not necessary for the activation of LvSM30 expression, at least to moderate levels.
In PMC-supplemented embryos (containing 2-3 times the normal complement of PMCs, n=7), despite the greatly increased density of cells along the ventral chain, these cells did not express detectable levels of LvSM30. Therefore, high cell density is not sufficient to induce LvSM30 expression (Guss et al., 1997).

Action of agents ventralizing ectoderm

NiCl2 ventralizes the ectoderm; as a consequence, the arrangement of PMCs in the blastocoel is altered and the number of spicule rudiments is increased (Hardin et al., 1992; Armstrong et al., 1993).
When embryos were raised continuously from fertilization in 0.05 mM NiCl2, both the patterning of the PMC ring and the expression of LvSM30 were altered. In most NiCl2-treated embryos, LvSM30 expression was relatively uniform throughout the PMC ring. In some embryos, multiple, discrete clusters of cells expressing high levels of LvSM30 were visible, with lower transcript levels exhibited by cells connecting these clusters. This pattern of expression was more common in embryos cultured in higher concentrations of nickel (e.g. 0.2 mM).
To determine whether the aberrant LvSM30 expression pattern was due to an effect of nickel on the ectoderm or the PMCs, we examined LvSM30 expression after transplanting nickel-treated PMCs into untreated, PMC-depleted host embryos. In such embryos, nickel-treated PMCs formed a ring and exhibited a pattern of LvSM30 expression similar to that observed in control embryos. In 6/7 cases, the PMCs formed a ring in which two clusters and either the ventral and/or the dorsal chain were distinguishable. PMCs in the two ventrolateral clusters exhibited the highest expression of LvSM30. The dorsal chain, when present, exhibited faint to strong staining, whereas the ventral chain showed consistently low expression. As a control, a sample of nickel-treated donor embryos was allowed to continue development in IO in parallel with the experimental embryos. These donor embryos were ventralized and showed an aberrant pattern of LvSM30 expression. These results indicate that nickel causes a misregulation of LvSM30 expression by altering the ectoderm (Guss et al., 1997).

Cell transplantations

Transplantation of PMCs into recipient embryos of different developmental stages has been used to provide evidence that aspects of PMC behavior are regulated by extrinsic cues (Ettensohn and McClay, 1986). Here the PMCs from mesenchyme blastulae were transferred into younger embryos (pre-hatching blastulae) and LvSM30 expression was examined after 5- 5,5 hours, when sibling donor embryos had reached the mid-late gastrula stage and were expressing high levels of LvSM30 mRNA. As a control for the transplantation procedure, PMCs were removed from mesenchyme blastulae and microinjected into recipient embryos of the same developmental stage from which all endogenous PMCs had been removed (isochronic controls).
In the combined results of three series of heterochronic transplants, in 23 of 40 experimental embryos, the PMCs showed no significant signal above background The remaining embryos contained 1-2 cells exhibiting signal above background (6/40) or more than two cells above background (11/40).
In contrast to the low levels of LvSM30 expression observed following heterochronic transplantations, 26/27 isochronic control embryos exhibited more than two positive cells, although signal intensity ranged from just above background to very strong. In 17 of those embryos, a ring was distinguishable and the PMCs exhibited some degree of spatial regulation of LvSM30 expression, e.g. the highest degree of signal was localized to cells in at least one cluster. In the remaining cases, the presence of a ring could not be confirmed because of the orientation of the embryos (five cases), or the ring was incompletely formed and/or there were no differences in levels of staining in cells in different regions of the ring (four cases). Thus, the delay in LvSM30 expression following heterochronic PMC transplantation cannot be attributed to the effects of microsurgery alone, since almost all of the isochronic controls showed detectable levels of LvSM30 expression after the same period of time (Guss et al., 1997).


Sequences


Regulatory Regions


Regulatory Connections

Upstream Genes

LvSM30

Downstream Genes


Evolutionary Homologues


Links


Bibliography


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