Gene Networks Database
Lytechinus variegatus Genes in Development: Sea urchin Notch homologue

LvNotch

Function

LvNotch is the first echinoderm Notch homolog. The Notch intercellular signaling pathway mediates the specification of numerous cell fates in both invertebrate and vertebrate development.
The experiments suggest roles for the Notch pathway in sea urchin in secondary mesoderm and endoderm lineage segregation, and in the establishment of dorsoventral polarity in the endoderm (Sherwood et al., 1997).

Protein

The identified 7,646 bp cDNA sequence predicts open reading frame of 2,531 amino acids. LvNotch contains all conserved domains shared by Notch proteins. LvNotch has multiple EGF-like repeats, three Notch/Lin-12 repeats, a transmembrane domain, six Ankyrin repeats and a putative PEST domain.
However, LvNotch contains only 35 EGF-like repeats, in contrast to the 36 repeats found in Drosophila Notch and vertebrate Notch1 and 2. An alignment of Notch family members, including at least one of the four vertebrate Notch proteins and Drosophila Notch revealed a deletion of EGF-repeat 14 in LvNotch.
Examination of western blots of gastrula protein extracts revealed prominent immunoreactive bands at 116*103 Mr and 260*103 Mr, respectively, as well as additional lower abundance fragments. This banding pattern suggested that the majority of endogenous LvNotch product is present as two fragments, corresponding roughly in size to the intracellular and extracellular domains. The functional significance of this processing or degradation of Notch is unknown (Sherwood et al., 1997).
GenBank: 2570351

Subcellular location

While western analysis suggested that LvNotch may exist predominantly as an extracellular and intracellular fragment, whole-mount immunofluorescence using these extracellular-and intracellular-specific antibodies revealed no differences in localization pattern.
Whole-mount immunofluorescent analysis showed that LvNotch protein was dynamically expressed at the cellular and subcellular level after cleavage stages. During cleavage stages, expression was uniform on the surfaces of all blastomeres.
In the early blastula embryo (6 hours in development) an increase in basolateral membrane staining was observed in all cells except the sector of the embryo that later becomes the center of the vegetal plate.
At mid to late-mesenchyme blastula stage LvNotch was strongly upregulated on the apical surface of cells surrounding the central region of the vegetal plate, low levels were observed in the cytoplasm of the eight small micromere descendants.
At this same time, LvNotch, which previously was localized predominantly basolaterally in cells of the animal half of the embryo, was now distributed uniformly in cell membranes. Examination of cell surfaces in the early mesenchyme blastula vegetal plate revealed a slight upregulation of LvNotch along the apical surface of cells bordering the central vegetal plate. Midway through the mesenchyme blastula stage (approx. 11.5 hours) a subset of cells began to express high levels of apical LvNotch in a striking cell-by-cell manner. By the end of this stage (12- 12.5 hours), cells with high levels of apical LvNotch formed an asymmetric ring, with one side of the ring having both increased levels of apical expression and more cells expressing apical LvNotch.
In the mid-gastrula (14 hours), apical LvNotch was detected in presumptive endoderm cells. By the late gastrula (16 hours), apical LvNotch extended to the tip of archenteron (Sherwood et al., 1997).

Expression Pattern

LvNotch mRNA was present during all stages of sea urchin embryogenesis. Low levels of maternal mRNA were found in the egg. After fertilization the amount of LvNotch mRNA appeared to increase, peaking in abundance at the gastrula stage, and then decreasing to lower levels in the pluteus larva.
Whole-mount immunofluorescent analysis showed that during cleavage stages, LvNotch protein expression was uniform on the surfaces of all blastomeres.
In the early blastula embryo (6 hours in development), loss of LvNotch expression was seen in a sector of the embryo that later becomes the center of the vegetal plate.
This pattern of expression persisted through the thickened vegetal plate stage (10 hours), when the area lacking detectable LvNotch was identified as the central thickened vegetal plate.
Only 1 to 2 hours later (mid to late-mesenchyme blastula stage), LvNotch was strongly upregulated on the apical surface of cells surrounding the central region of the vegetal plate. Low levels were also observed inconsistently in the cytoplasm of the eight small micromere descendants at the center of the vegetal plate.
In the mid-gastrula (14 hours), high levels of apical LvNotch expression were maintained in cells now identifiable as presumptive endoderm cells, while the SMCs at the tip of the archenteron lacked detectable LvNotch expression.
By the late gastrula (16 hours), when most of the SMCs have migrated away from the tip of the archenteron, apical LvNotch extended to the tip of this structure. Notably, throughout most of gastrulation the highest levels of apical LvNotch were distributed along one side of the archenteron, except in the last presumptive endoderm cells to invaginate, where LvNotch was expressed at high levels in a symmetric manner.
To examine wheather this polarity correlated with the dorsoventral axis of the embryo, the position of the asymmetric ring of apical LvNotch in the early gastrula stage embryo was compared to the orientation of the ventrolateral clusters of primary mesenchyme cells (PMCs), one of the first morphological features distinguishing the ventral from the dorsal side of the embryo. Double staining with a PMC-specific monoclonal antibody, IG8, and LvNotch showed that 86% of embryos had a clear dorsal polarity.
At late gastrula all of these embryos had higher levels of apical LvNotch along the dorsal side of the presumptive endoderm.
Apical LvNotch thus both delineates a boundary between the presumptive endoderm and mesoderm of the vegetal plate, and has a consistent dorsal polarity (Sherwood et al., 1997).

mRNA level

Temporal accumulation

Method: Nothern blot analysis
Reference: Sherwood et al., 1997
Stage Egg 7th cleavage Thickened vegetal plate blastula Early gastrula Late gastrula Prism Pluteus larva
Level
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Protein level

Temporal accumulation

Method: Western blot analysis
Reference: Sherwood et al., 1997
Stage Egg 16 cells 7th cleavage Thickened vegetal plate blastula Early gastrula Late gastrula Pluteus larva
Level
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Protein spatial localization

Method: Whole-mount immunofluorescent analysis
Reference: Sherwood et al., 1997
Stage
Cleavage stages
 Early blastula (6 hours)
Thickened vegetal plate stage (10 hours)
Mid late-mesenchyme blastula (11-12 hours)
Mid-gastrula (14 hours)
 Late gastrula (16 hours)
Tissue
The surfaces of all blastomeres
Loss of expression in a sector of the embryo that later becomes the center of the vegetal plate
Area lacking detectable expression in the central thickened vegetal plate
Apical surface of cells surrounding the central region of the vegetal plate. Low levels were also observed inconsistently in the cytoplasm of the eight small micromere descendants at the center of the vegetal plate. In contrast to the vegetal plate, the cells of the animal half of the embryo express LvNotch at lower levels with a nonpolar distribution in membranes
High levels of apical LvNotch expression are maintained in presumptive endoderm cells, while the SMCs at the tip of the archenteron lacks detectable LvNotch expression
Apical LvNotch extends to the tip of archenteron and is asymmetrically distributed along the sides (has dorsal polarity), except in the last presumptive endoderm cells to invaginate, where LvNotch was expressed at high levels in a symmetric manner

Ectopic expression

Conversion of cell types (endoderm to SMCs)

Previous studies have shown that, after microsurgical removal of the presumptive SMCs early in gastrulation, a new SMCs population is established via conversion of presumptive endoderm cells to SMCs at the tip of the recovering archenteron (McClay and Logan, 1996).
Immediately after removing the presumptive SMCs, apical LvNotch extended to the tip of the archenteron. In embryos allowed to recover for only 1.5 hours, however, apical LvNotch was no longer present at the tip of archenteron.
Apical LvNotch thus appears to be rapidly downregulated in responce to the lineage conversion of presumptive endoderm to SMCs (Sherwood et al., 1997).

Action of vegetalizing agents

To test the possibility that the specific pattern of the expression of apical LvNotch along the presumptive SMC-endoderm boundary is tied to animal/vegetal axis of the embryo, embryos were treated with LiCl, which increases the amount of vegetally derived tissues (Hostradius, 1973).
LiCl treatment caused an expansion in the number of presumptive SMCs that lacked LvNotch and in the number of presumptive endoderm cells that express apical LvNotch in mesenchyme blastula embryos.
LiCl also reduced the assymmetry of apical LvNotch, consistent with its less well-characterized, but known radializing effect.
The expansion of cells lacking and expressing apical LvNotch in LiCl-treated embryos provides further evidence that apical LvNotch is specifically expressed in the presumptive endoderm and is excluded from the presumptive SMCs (Sherwood et al., 1997).

Action of ventralizing agents

To determine if the dorsal bias in LvNotch expression changes with experimental perturbation of the dorsoventral axis, this axis was disrupted by treatment with NiCl2, which increases ventral ectoderm at the expence of dorsal ectoderm, and alters the patterning of mesodermal structures associated with the endoderm and ectoderm (Hardin et al., 1992). NiCl2 treatment did not affect the number of cells lacking LvNotch expression in the center of the mesenchyme blastula vegetal plate. However, disruption of the dorsoventral axis did cause a marked reduction in the overall intensity, asymmetry and number of cells expressing apical LvNotch in the vegetal plate. This decrease in apical LvNotch continued through most of the gastrula stage; protein extractions revealed that mid-gastrula embryos treated with NiCl2 had an approximately 30% decrease of LvNotch protein.
Thus, the presence of a dorsoventral axis appears to be required for the asymmetric expression of apical LvNotch (Sherwood et al., 1997). 

Sequences

GenBank:

Regulatory Regions

Regulatory Connections

Upstream Genes

LvNotch

Downstream Genes

Evolutionary Homologues

Links

Bibliography

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