HpEts

Function

HpEts is a member of ets family of transcription factors. The ets family is characterized by homology to the v-ets oncogene (LePrince et al., 1983; Nunn et al., 1983) and shares a conserved ets-domain that is responsible for sequence-specific DNA binding. About 30 ets-related genes have now been found in diverse species and have been implicated to play a role in the regulation of gene expression during a variety of biological processes, including cell proliferation, transformation, T-cell activation and development.
The comparison of the HpEts ets-domain with that of the other ets family members revealed that HpEts is the sea urchin homologue of the c-ets-1/-2.
HpEts plays a key role in the development of sceletogenic mesenchyme cells during sea urchin development (Kurokawa et al., 1999).

Protein

HpEts is the ets-related protein which consists of 555 amino acids (Kurokawa et al., 1999).

Subcellular location

Expression Pattern

Nothern blot analysis indicated that the HpEts mRNA is stored as maternal mRNA. Intense expression was found in unfertilized eggs and early stage embryos until unhatched blastula stage; thereafter, it diminished rapidly.
The whole mount in situ hybridization demonstrated the presence of the HpEts mRNA at nearly the same level in all cells of the embryos before hatching.
At the hatched blastula stage, the signal was restricted to the area of the presumptive PMCs, which formed a ring around the vegetal pole. The HpEts positive cells then migrated into the blastocoel. No detectable levels of HpEts mRNA were found in cells other than the PMCs after hatching. In addition, after the early mesenchyme blastula stage, the signal was no longer detectable by whole mount in situ hybridization even in PMCs (Kurokawa et al., 1999).

mRNA level

Temporal accumulation
Method: Nothern blot analysis
Reference: Kurokawa et al., 1999

Stage Unfertilized egg 16 cells Morula Unhatched blastula Hatched blastula Mesenchyme blastula Gastrula Prism

Level + + + + + + + + + + - + - + -

Spatial localization
Method: Whole mount in situ hybridization
Reference: Kurokawa et al., 1999

Stage Unhatched blastula Hatched blastula Early mesenchyme blastula

Tissue Transcripts are ubiquitously distributed in the embryo The signal was restricted to the area of the presumptive PMCs, which form a ring around the vegetal pole Sceletogenic mesenchyme cells migrating into the blastocoel

Ectopic expression

Gene overexpression

H. pulcherrimus fertilized eggs were injected with approximately 1, 2, 4 or 8 pg/egg of FLAG tagged HpEts mRNA.
No effects were apparent until unhatched blastula stage (12 hours after fertilization). However, at 24 h after fertilization the blastula wall of the injected embryos thickened. The cells around the vegetal pole then migrated out apically into the seawater and the blastocoel lost its integrity. The transformation of blastomeres into migrating cells propagated from the vegetal pole to the animal pole, and the number of migrating cells increased with time. Finally, at 36 h after fertiization almost all cells of the embryos had been transformed into migrating cells.
About 60% of the injected embryos showed this extensive transformation with 8 pg/egg HpEts mRNA. Although with one 1 pg/egg HpEts mRNA, 80% of the injected embryos developed almost normally to pluteus larvae.
The nature of migrating cells is determined by examination of expression of PMC-specific HpSM50 protein with anti-HpSM50 monoclonal antibody.
In the embryos injected with HpEts mRNA, the cells around the vegetal pole that were migrating out apically were intensely stained with the HpSM50 antibody at 24 hours after fertilization. At 36 h after fertilization almost all the cells in injected embryos expressed the HpSM50 protein. The SM50 staining progressed from vegetal to animal pole just like the migratory property. Cells of the blastula wall that retained an epithelial character, such as those in the animal pole of less severely affected embryos, were not stained by the anti-HpSM50 antibody.
Quantative RT-PCR also demonstrated a marked suppression of aboral ectoderm-specific HpArs and endoderm-specific HpEndo16, but a great elevation of PMC-specific HpSM50 in the embryos injected with HpEts mRNA. The level of ubiquitously expressed ubiquitin mRNA was unaffected in these embryos (Kurokawa et al., 1999).

Tissue-specific gene expression in control and HpEts injected embryos
Method: RT-PCR analysis, southern blotting of RP-PCR products
Reference: Kurokawa et al., 1999

Gene Control embryos Injected embryos

HpARS + + + -

HpEndo16 + + + -

HpSM50 + - + +

Ubiquitin + +

Animal cap (mesomere) assay

Eight mesomeres from a normal 16-cell stage embryo and a single mesomere from an embryo injected with HpEts mRNA (8 pg) were combined and allowed to develop in culture. The mesomere from the injected embryos was marked by the co-injection of 10 pg of Texas red dextran.
Six of nine such chimeric embryos generated spicules after 3 days in culture, and the PMCs forming the spicules were solely composed of Texas red positive cells. Control chimeras composed of nine normal mesomeres, one of which was similarly marked by Texas red, never developed spicules (Henry et al., 1989; Khaner and Wilt, 1991). Thus, this assay confirmed the transformation of mesomeres into sceletogenic PMCs by HpEts overexpression (Kurokawa et al., 1999).