HpOtx

Function

HpOtx gene is present as a single copy in the Hemicentrotus pulcherrimus genome (Sakamoto et al., 1997).
HpOtx gene encodes two distinct types of orthodentical-related proteins (early type: HpOtxE, late type: HpOtxL), which have been implicated as enhancer element binding factors of the aboral ectoderm-specific HpArs gene (Mitsunaga-Nakatsubo et al., 1998)

Protein

The HpOtx open reading frames encode proteins of 295 amino acids (HpOtxL) and 371 amino acids (HpOtxE).
The amino acid sequence of HpOtxE is almost identical to that of SpOtx, suggesting that HpOtxE is a homologue of SpOtx.
The amino acid sequence of HpOtxL from its N-terminal to the 5` end of the homeodomain is different from HpOtxE and SpOtx, though the amino acid sequences of the homeodomain and the region downstream to the C terminal are almost identical (Sakamoto et al., 1997).

GenBank: 3176391 (HpOtxE)

Subcellular location

Expression Pattern

The mRNAs of HpOtxE and HpOtxL are transcribed from a single HpOtx gene by altering the transcription start sites and by alternative splicing (Kiyama et al., 1998). HpOtxE transcript is present in the unfertilized egg and gradually decreases in amount after hatching. HpOtxL transcript first appears after hatching, increases gradually until the gastrula stage, and then decreases to a very low level in the pluteus (Sakamoto et al., 1997).
Whole-mount in situ hybridization showed that HpOtxE mRNA is maternally stored and exists apparently in a non-localized manner by the blastula stage.
After hatching, hybridization signals became gradually intense at the vegetal half and detectable at the small central region of the vegetal plate where primary mesenchyme precursors reside.
At the mesenchyme blastula stage, the HpOtxE mRNA was expressed at high levels throughout most of the vegetal plate. Primary mesenchyme cells (PMCs) that had ingressed into the blastocoel did not express detectable levels of HpOtxE mRNA.
As the primary invagination was initiated, intence hybridization signals appeared in a ring of presumptive endodermal cells, though signals were detected neither in invaginating archenteron nor in PMCs in the blastocoel.
HpOtxE transcripts gradually decreased by the prism stage, a positive signal for HpOtxE was retained only by presumptive endodermal cells surrounding the blastopore. At the prism stage, expression of HpOtxE transcripts were under detectable level.
HpOtxL mRNA was first detectable in hatched blastula stage embryos with the intence signal at the vegetal half. Subsequently, HpOtxL transcripts were intensely distributed at the vegetal plate cells prior to PMC ingression.
At the late blastula stage, HpOtxL transcripts were concentrated in micromere-derived cells and were found in PMCs that had just ingressed into the blastocoel. Thereafter, hybridization signals were observed at the vegetal plate cells including precursors of secondary mesenchyme cells and endoderm, though PMCs in the blastocoel became negative.
During gastrulation, HpOtxL transcripts were exclusively expressed within the ectoderm; appreciable levels of HpOtxL expression were not detected in the invaginating archenteron or in the mesenchyme cells located in the blastocoel.
At the prism stage, however, signals were detectable at the oral ectoderm and endoderm (Mitsunaga-Nakatsubo et al., 1998)

mRNA level

Temporal accumulation of HpOtxE
Method: Nothern blot analysis
Reference: Sakamoto et al., 1997

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

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

mRNA level

Temporal accumulation of HpOtxL
Method: Nothern blot analysis
Reference: Sakamoto et al., 1997

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

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

Spatial localization of HpOtxE
Method: Whole mount in situ hybridization
Reference: (Mitsunaga-Nakatsubo et al., 1998)

Stage Unhatched blastula Hatched blastula Late hatched blastula Early mesenchyme blastula Early gastrula Mid-late gastrula Prism

Tissue Transcripts are distributed throughout embryos The hybridization signals become intence in the vegetal half Vegetal plate, prior to PMC ingression Vegetal plate Intence hybridization signals appear in a ring of presumptive endodermal cells, no staining in invaginating archenteron and in PMCs in the blastocoel Vegetal plate cells especially surrounding blastopore stain intensely, invaginating archenteron does not show signals above background Expression is under detectable level

Spatial localization of HpOtxL
Method: Whole mount in situ hybridization
Reference: (Mitsunaga-Nakatsubo et al., 1998)

Stage Hatched blastula Late blastula Mesenchyme blastula Late mesenchyme blastula Early-mid gastrula Prism

Tissue Transcripts are distributed throughout embryo with intense signals at the vegetal half Intense hybridization signals are localized at the vegetal plate (micromere-derived cells) PMCs that have just ingressed are positive Transcripts are localized at the vegetal plate cells Transcripts are primarily detected in ectoderm, mesenchyme cells and invaginating archenteron are negative Hybridization signals are detectable in gut and oral ectoderm

Ectopic expression

mRNA injection

Morphological effects
Capped mRNA were injected into fertilized eggs and embryos were developed for 45h at 15^oC.
Overexpression of HpOtxE or HpOtxL caused similar morphological effects on sea urchin embryogenesis in spite of difference in their N-terminal regions. More than 90% of the eggs injected with 11pg mRNA formed radialized spherical structures without PMC ingression and archenteron invagination. No recognizable stomodea were formed and no ciliary band was evident. These embryos lacked the morphological features that distinguished the oral from aboral ectoderm.
As the amount of injected mRNA decreased to 3 pg/egg, about 35% of embryos developed into embryos with partial effect.
Thus, specification of positional identity along the oral-aboral axis did not occur in the HpOtx mRNA-injected embryos (Mitsunaga-Nakatsubo et al., 1998).

Expression of cell-type-specific proteins and genes in injected embryos
Overexpression of HpOtxE or HpOtxL caused both the aboral ectoderm-specific protein HpArs (Mitsunaga-Nakatsubo et al., 1998) and the oral ectoderm-specific Hpoe (Yoshikawa, 1997) to be expressed in all ectoderm cells. Expression of endoderm-specific protein HpEndo16 (Akasaka et al., 1997) and PMC-specific protein were markedly reduced compared to the uninjected control.
Expression of cell-type specific genes in HpOtxE/L mRNA- injected embryos was examined by quantative RT-PCR using specific primers. An excessive expression of HpOtxE and HpOtxL was detected in each of the mRNA-injected embryos at the prism stage.
The embryos injected with HpOtxE/L mRNA showed significally lower levels of HpEndo16 (a marker for endoderm; Akasaka et al., 1997) and HpSM50 (PMC; Katoh-Fukui et al., 1992) mRNA in comparison to the uninjected ones.
In contrast, the expression of aboral ectoderm-specific HpArs (Yang et al., 1989; Akasaka et al., 1990) and HpHbox (Angerer et al., 1989) were slightly affected by injections of HpOtxE/L mRNA (Mitsunaga-Nakatsubo et al., 1998).

Sequences

GenBank:

GenBank (HpOtxE)

Regulatory Regions

Regions

Regulatory Connections

Upstream Genes

HpOtxE, HpOtxL

HpArs

Downstream Genes

Evolutionary Homologues

SpOtx S. purpuratus (for HpOtxE)

Bibliography

UrchiNet

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Stage	Unfertilized egg	16 cells	Morula	Unhatched blastula	Hatched blastula	Mesenchyme blastula	Gastrula	Pluteus
Level	+	+	+	+ +	+ +	+	+ -	+ -

Stage	Hatched blastula	Late blastula	Mesenchyme blastula	Late mesenchyme blastula	Early-mid gastrula	Prism
Tissue	Transcripts are distributed throughout embryo with intense signals at the vegetal half	Intense hybridization signals are localized at the vegetal plate (micromere-derived cells)	PMCs that have just ingressed are positive	Transcripts are localized at the vegetal plate cells	Transcripts are primarily detected in ectoderm, mesenchyme cells and invaginating archenteron are negative	Hybridization signals are detectable in gut and oral ectoderm