SpKrox1

Function

SpKrox1 is a member of the Kruppel/Krox family of genes which encodes an important class of zink-finger transcription factors essential for many developmental events in insects and vertebrates (Schuh et al., 1986).
The Southern blot analysis indicated that most likely SpKrox1 is a single-copy gene in the S. purpuratus genome (Wang et al., 1996).

Protein

In vitro translation of SpKrox1 yielded a protein of 95 kDa.
SpKrox1 protein has four contiguous zink-finger motifs between amino acids 410 and 514 with each finger containing 28 amino acids. Four zink fingers of SpKrox1 are of the C₂H₂ type; all four fingers perfectly match the consensus sequence.
The inspection of the SpKrox1 sequence revealed an aspartic acid and glutamic acid-reach region between amino acid residues 569 and 576, which might serve as a transcriptional activation domain; two arginine-rich regions between residues 524 and 529, which are potential nuclear localization signals; and leucine zipper motif between residues 707 and 751 (Wang et al., 1996).

Subcellular location

Expression Pattern

SpKrox1 is expressed at low levels in unfertilized eggs.
RNase protection analysis showed that SpKrox1 transcripts begin accumulating as early as 6 h after fertilization. At this point embryos were at the 16- and 32- cell stage. Relative accumulation reached a peak by the early blastula stage (19 h after fertilization) and transcripts remain at that level until at least the early gastrula stage (30 h after fertilization). By the mid gastrula stage (44 h after fertilization), SpKrox1 RNA levels had decreased to 50% of peak values, and they continued to decline to 25% of peak values at the pluteus stage (72 h after fertilization).
Whole mount in situ hybridization showed that SpKrox1 transcripts are localized to vegetal plate cells and their precursors. Transcripts were first detected at the 16-cell stage in macromeres, whose descendants are the veg1 and veg2 blastomeres, but not in micromeres and mesomeres.
By the mesenchyme blastula stage, when SpKrox1 RNA levels were maximal, localization was restricted to cells of the vegetal plate. At the mid and late gastrula stages, expression was again limited to the vegetal pole and was seen as a ring of cells encircling the blastopore.
Very few if any of the cells that invaginated into the blastocoel were labeled at later stages, when endoderm had differentiated fully and SpKrox1 transcripts were much less prevalent, expression was found mostly in a ring of cells around the anal pore. So, as cells invaginated into the blastocoel, they apparentlly lost their ability to express SpKrox1 (Wang et al., 1996).

mRNA level

Relative temporal accumulation*
Method: RNase protection assay
Reference: Wang et al., 1996

Stage	Egg	3 hr	6 hr	9 hr	12 hr	19 hr	24 hr	30 hr	44 hr	72 hr
Level	5	5	7	25	57	95	97	100	46	29

*Data were quantifired by phosphorimager analysis normalized to the endogenous 18S RNA.

Spatial localization
Method: Whole mount in situ hybridization
Reference: Wang et al., 1996

Stage	16 cells	Mesenchyme blastula	Mid gastrula	Late gastrula	Pluteus
Tissue	macromeres, whose descendants are veg1 and veg2 blastomeres	vegetal plate	ring of cells encircling the blastopore	ring of cells encircling the blastopore	mostly in a ring of cells around the anal pore