Gene Networks Database

Paracentrotus lividus Genes in Development: Homeobox class genes

PlHbox12

Function

PlHbox12 is a homeobox-containing gene. Di Bernardo et al. (1995) suggest a possible role of the PlHbox12 gene in the early events of cell specification of the presumptive ectodermal territories (Di Bernardo et al., 1995).

Protein

PlHbox12 cDNA contains the coding information for 227 amino acids. The homeodomain is close to the N terminus of the protein, and the C terminus is rich in threonine and serine residues, suggesting that the activity of this homeoprotein might be modulated by phosphorylation and dephosphorylation events. The paired box is absent in this protein. Unlike the paired class proteins that contain serine in position 50 of the homeodomain, this residue, which gives sequence specificity of binding of the protein (Hanes and Brent, 1989), is substituted by glutamine in PlHbox12, suggesting that the encoded homeodomain could specifically bind Antennapedia class target sequences. Sequence analysis of a genomic clone revealed that the coding sequence of the helix III of the homeodomain is interrupted by an intron. The intron-exon boundary lies at valine 47. The interruption f the coding sequence at this amino acid residue is conserved in otp, otd and other homeogenes (Di Bernardo et al., 1995).

Subcellular location

Expression Pattern

PlHbox12 is one of the earliest transcribed zygotic homeogenes identified in lower deuterostomes.
The PlHbox mRNA is absent in unfertilized eggs. It is first detected by RNase protection assay in the 4- to 8- stage embryos. The transcripts increase in abundance in until the 64- to 128-cell stage of development, abruptly decline thereafter and return to an undetectable level by the gastrula stage. Thus, PlHbox12 mRNA derives from transcription of the zygotic genome and its expression is transient and occurs during early/mid cleavage.
Whole mount in situ hybridization showed the prevalent localization of the PlHbox12 transcripts in some but not all of the animal blastomeres and in a fraction of the macromere and vegetal tiers and the absence of staining in the micromeres at the vegetal pole. This spatially restricted pattern of expression is already evident in embryos at the 16-cell stage. Preferential hybridization seemed also to occur on the blastomeres located on one side of the embryo. Two animal 2 tier blastomeres and two macromeres, respectively, belonging to the animal 2 and macromere tiers of a 32-cell stage embryo, are clearly stained on one side of the embryo and unstained on the opposite side. At the 64-cell stage no expression of the PlHbox12 gene seems to occur in the autonomously specified micromeres at the vegetal pole, whereas the transcripts are spatially restricted toward blastomers of the animal cap and the veg1 tier, located on one side of the embryo.
The observed staining pattern suggests that the transcripts are asymmetrically distributed along the embryonic animal-vegetal axis. according to the cell lineage chart of S. purpuratus, the fate of the blastomeres expressing PlHbox12 is to give rise to the presumptive ectoderm territories (Cameron et al., 1987, Cameron et al., 1990).
PlHbox12 expression immediately precedes the specification of the first lineage founder cell (the Na blastomere) of the aboral ectoderm lineage that occurs at the 8-cell stage. In addition, maximal expression of PlHbox12 was observed concommitantly with segregation of the lineage founder cells of the five embryonic territories at the 64-cell stage of development. The results of in situ hybridization in whole-mount embryos seem consistent with the possible involvement of PlHbox12 in the initial specification of cell fate (Di Bernardo et al., 1995).

mRNA level

Temporal accumulation

Method: RNase protection assay
Reference: Di Bernardo et al., 1995

Stage
Egg
3 h (4-8-cells)
4.5 h (16-32-cells)
5.5 h (64-128-cells)
8 h (blastula)
11 h (hatching blastula)
24 h (gastrula)
48 h (pluteus)
Level
-
+
+ +
+ +
+
+
-
-

Spatial localization

Method: whole mount in situ hybridization
Reference: Di Bernardo et al., 1995

Stage
16 cells
32 cells
64 cells
Tissue
Some but not all of the animal blastomeres and in a fraction of the macromere and vegetal tiers (preferential hybridization on the blastomeres located on one side of the embryo), no staining in the micromeres at the vegetal pole
Two animal 2 tier blastomeres and two macromeres, respectively, belonging to the animal 2 and macromere tiers are clearly stained on one side of the embryo and unstained on the opposite side
Transcripts are spatially restricted toward blastomers of the animal cap and the veg1 tier, located on one side of the embryo, no expression seems to occur in the autonomously specified micromeres at the vegetal pole

Ectopic expression

Dissociated blastomeres
Fertilized eggs were cultured for severel hours in Ca+ - free sea water. This method prevented cells from reassociating without interfering with cell division.
The expression of hatching enzyme (HE) was monitored in control and dissociated embryos (this gene is expressed autonomously from the zygotic genome). In P. lividus embryos the abundance of HE transcripts reaches a peak at the prehatching blastula stage. The results of RNA blot hybridization confirmed this prediction and at 6 h of development the high level of transcripts was detected both in control and dissociated embryos.
In contrast, the expression of the PlHbox12 gene is severely affected in dissociated embryos. Thus, the maximal expression of the PlHbox12 gene is dependent on intercellular interactions (Di Bernardo et al., 1995).

mRNA level

Temporal accumulation in control (C) and dissociated (D) blastomeres

Method 1: RNA blot hybridization (for both genes)
Reference: Di Bernardo et al., 1995
Method 2: RNase protection assay (only for PlHbox12)
Reference: Di Bernardo et al., 1995

Stage
C 4 h
D 4 h
C 6 h
D 6 h
Level (PlHbox12)
+
+ -
+ +
+ -
Level (hatching enzyme)
-
-
+ +
+ +

Sequences

GenBank:

Regulatory Regions

Regions

Regulatory Connections

Upstream Genes

PlHbox12

Downstream Genes

Evolutionary Homologies

Links

Urchin Web

Bibliography
[Previous] UrchiNet[Up] Search the GeNet
Comments are welcome to Sveta Surkova
Copyright © 1997 GeNet Team