HE6

Function

HE6 codes for a hatching enzyme, which is synthesized and secreted by the blastula stage embryo to digest the fertilization envelope (Ghiglione et al., 1993).
This enzyme belongs to the matrix metalloproteinase family, which includes the vertebrate collagenases and stromelysins (Alexander and Werb, 1989; Matrisian, 1990).

Protein

The HE6 1761 bp open reading frame codes for a preprohatching enzyme with an 18 amino acid signal sequence, a 148 amino acid activation peptide and a 421 amino acid mature enzyme which has homologies with the mammalian collagenases (Lepage et al., 1990).
SWISS_PROT: P22757

Subcellular location

Immunofluorescent labeling showed that in the youngest blastula, the enzyme is present in a diffuse or punctate circular pattern about the cell nuclei. A division cycle later, the enzyme is present in a small area above the nucleus, on the apical side of the cell.
Close to the time of hatching, the major part of the label is punctate and confined to a narrow region beneath the apical surface of the cell (Lepage et al., 1992a).

Expression Pattern

HE6 transcripts are not maternal but are the products of new transcription, which is limited to a few hours during the prehatching blastula stage. HE6 is expressed coordinately with another sea urchin gene BP10.
Immunolocalization experiments detected no labeling above background up to 64-cell stage. The same was true for the 12 h embryos that had hatched. Between these two stages blastulas were clearly labeled.
The domain in which the hatching enzyme is expressed has such features: (a) The area comprises about two-thirds of the blastula cells. (b) The boundary of this domain is sharp and very narrow. This boundary is perpendicular to the animal-vegetal axis and thus is parallel to the horizontal planes that separate blastomere tiers during early cleavage. (c) This area corresponds to the animal-most two-thirds of the blastula: all the animal hemisphere and a subequatorial part of the vegetal hemisphere.
In situ hybridization showed that the HE6 mRNA and protein are expressed at the same area of the embryo.

mRNA level

Temporal accumulation
Method 1: Northern blot analysis
Reference: Lepage et al., 1992b (data are taken from Lepage et al., 1990).

Stage Egg 2 hr 4 hr 6.5 hr 7.5 hr 8.5 hr 9.5 hr 10.5 hr 11.5 hr 12.5 hr 15 hr 30 hr 60 hr

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

Protein level

Temporal accumulation
Method: Immunofluorescent labeling
Reference: Lepage et al., 1992a

Stage 5-hr blastula (50 cells) 6-hr blastula (about 120 cells) 7-hr blastula (about 220 cells) 9-hr blastula (about 350 cells) 13-hr hatching blastula (about 500 cells)

Level - + + + -

Spatial localization
Method: Immunofluorescent labeling
Reference: Lepage et al., 1992a

Stage 10-hr hatching blastula (about 350 cells)

Tissue The animal-most two-thirds of the blastula: all the animal hemisphere and a subequatorial part of the vegetal hemisphere

Ectopic expression

Dissociated blastomeres
Expression of HE6 transcripts
Slot-blot hybridization showed that the pattern seen in intact embryos was identical with the pattern described above.
In blastomers raised permanently in isolation, the mRNA level increased with the same time course as intact embryos and reached the ame peak value. However, where in normal embryos the transcripts disappear rapidly, in isolated blastomeres their level begins to decrease but then remains constant for many hours.
The abnormal pattern observed is independent of the association state of blastomeres during the early stages and is not due to a prolonged Ca²⁺ deprivation and cannot be corrected by Ca²⁺ addition.
Thus the increase in transcripts level is indepenent of contacts and short-range interactions between blastomeres.

Synthesis of HE6 protein
Blastomers were raised in isolation for ~ 10 h and stained with antibodies directed against HE6. The immunolabeling of individual blastomers resembled that observed in whole blastulae: a single spot was visible on one side of the nucleus.
Furthermore, only two-thirds of the blastomeres were labeled. This corresponds to the fraction of cells which synthesises the protein in intact embryos, suggesting thta the same cells were labelled in both cases. Thus, dissociation does not affect the inherent spatial control of this gene (Ghiglione et al., 1993)

Action of animalizing agents
Expression of HE6 transcripts
Slot-blot hybrydization showed that in embryos treated with Zn²⁺, the time course of accumulation and decay as well as the maximal abundance were almost identical to those in the control embryos.
Thus Zn²⁺ does not affect the control of the mRNA level.

HE6 protein localization
Immunolocalization of HE6 protein in prehatching blastulae embryos raised in the presence of Zn²⁺ could not be distinguished from that of the control embryos. The labeling is restricted to the animal two-thirds of the embryo (Ghiglione et al., 1993).

Action of vegetalizing agents
Expression of HE6 transcripts
Embryos were exposed continuously to various concentrations of LiCl ranging from 0 to 90 mM and the the maximal level of mRNA accumulation for each Li⁺ concentration was detected by densitometric scanning of the slot-blots. Li⁺ clearly decreased the amount of mRNA accumulation.

Transcription activity of the HE6 gene
The intron-RNase protection method was used to assess the effect of Li+ on the HE transcription rate. At 30mM LiCl, the variation of primary transcript level, and thus the transcription rate variation, followed the same time course as the control, but the transcription rate was at all times markedly reduced.
This reduction runs parallel to the reduction in transcript accumulation which occured under the same conditions. Thus, Li⁺ depressed the transcription of the HE6 gene.

HE6 protein localization
Immunolocalization of HE6 protein in prehatching blastulae of embryos raised in the presence of Li⁺ shows that the size of the territory in which protein is detected is dramatically reduced. This effect is concentration - dependent, as the Li⁺ concentration increases, the size of the territory decreases.
The effect of Li⁺ is time dependent. A maximal effect was obtained when the Li⁺ incubation began within 30 min after fertilization. This effect decreased if Li⁺ was added later, and completely disappeared if lithium was added later than 6 h after fertilization, which is the time when the transcription rate of the gene had nearly reached its peak value in normal embryos (Ghiglione et al., 1993).

Sequences

GenBank:

2880 bp mRNA

Regulatory Regions

Regions

Regulatory Connections

Upstream Genes

HE6

Downstream Genes

Evolutionary Homologues

Links

Urchin Web

Bibliography

UrchiNet

Search the GeNet

Stage	5-hr blastula (50 cells)	6-hr blastula (about 120 cells)	7-hr blastula (about 220 cells)	9-hr blastula (about 350 cells)	13-hr hatching blastula (about 500 cells)
Level	-	+	+	+	-

Stage	10-hr hatching blastula (about 350 cells)
Tissue	The animal-most two-thirds of the blastula: all the animal hemisphere and a subequatorial part of the vegetal hemisphere