ABSTRACT
We report the characterization of the ortholog of the Xenopus XlHbox8 ParaHox gene from the sea urchin Strongylocentrotus purpuratus, SpLox. It is expressed during embryogenesis, first appearing at late gastrulation in the posterior-most region of the endodermal tube, becoming progressively restricted to the constriction between the mid- and hindgut. The physiological effects of the absence of the activity of this gene have been analyzed through knockdown experiments using gene-specific morpholino antisense oligonucleotides. We show that blocking the translation of the SpLox mRNA reduces the capacity of the digestive tract to process food, as well as eliminating the morphological constriction normally present between the mid- and hindgut. Genetic interactions of the SpLox gene are revealed by the analysis of the expression of a set of genes involved in endoderm specification. Two such interactions have been analyzed in more detail: one involving the midgut marker gene Endo16, and another involving the other endodermally expressed ParaHox gene, SpCdx. We find that SpLox is able to bind Endo16 cis-regulatory DNA, suggesting direct repression of Endo16 expression in presumptive hindgut territories. More significantly, we provide the first evidence of interaction between ParaHox genes in establishing hindgut identity, and present a model of gene regulation involving a negative-feedback loop.
Subject(s)
Body Patterning/genetics , Digestive System/embryology , Endoderm/embryology , Homeodomain Proteins/genetics , Strongylocentrotus purpuratus/embryology , Animals , Base Sequence , Biomarkers/metabolism , Blastula/cytology , Blastula/drug effects , Blastula/metabolism , Body Patterning/drug effects , Cell Adhesion Molecules/metabolism , Digestive System/cytology , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Endoderm/cytology , Endoderm/drug effects , Endoderm/metabolism , Gene Expression Regulation, Developmental/drug effects , Gene Silencing/drug effects , Homeodomain Proteins/metabolism , Larva/cytology , Larva/drug effects , Larva/metabolism , Models, Biological , Molecular Sequence Data , Oligonucleotides, Antisense/pharmacology , Phenotype , Regulatory Sequences, Nucleic Acid/genetics , Repressor Proteins/metabolism , Strongylocentrotus purpuratus/drug effects , Strongylocentrotus purpuratus/geneticsABSTRACT
A systematic search in the available scaffolds of the Strongylocentrotus purpuratus genome has revealed that this sea urchin has 11 members of the ets gene family. A phylogenetic analysis of these genes showed that almost all vertebrate ets subfamilies, with the exception of one, so far found only in mammals, are each represented by one orthologous sea urchin gene. The temporal and spatial expression of the identified ETS factors was also analyzed during embryogenesis. Five ets genes (Sp-Ets1/2, Sp-Tel, Sp-Pea, Sp-Ets4, Sp-Erf) are also maternally expressed. Three genes (Sp-Elk, Sp-Elf, Sp-Erf) are ubiquitously expressed during embryogenesis, while two others (Sp-Gabp, Sp-Pu.1) are not transcribed until late larval stages. Remarkably, five of the nine sea urchin ets genes expressed during embryogenesis are exclusively (Sp-Ets1/2, Sp-Erg, Sp-Ese) or additionally (Sp-Tel, Sp-Pea) expressed in mesenchyme cells and/or their progenitors. Functional analysis of Sp-Ets1/2 has previously demonstrated an essential role of this gene in the specification of the skeletogenic mesenchyme lineage. The dynamic, and in some cases overlapping and/or unique, developmental expression pattern of the latter five genes suggests a complex, non-redundant function for ETS factors in sea urchin mesenchyme formation and differentiation.
Subject(s)
Biological Evolution , Genes, Regulator , Proto-Oncogene Proteins c-ets/genetics , Sea Urchins/embryology , Sea Urchins/genetics , Animals , Ectoderm/physiology , Embryo, Nonmammalian/physiology , Endoderm/physiology , Gene Expression Regulation, Developmental , Sea Urchins/growth & development , Transcription, GeneticABSTRACT
Mesoderm and mesodermal structures in the sea urchin embryo are entirely generated by two embryologically distinct populations of mesenchyme cells: the primary (PMC) and the secondary (SMC) mesenchyme cells. We have identified the extracellular signal-regulated kinase (ERK) as a key component of the regulatory machinery that controls the formation of both these cell types. ERK is activated in a spatial-temporal manner, which coincides with the epithelial-mesenchyme transition (EMT) of the prospective PMCs and SMCs. Here, we show that ERK controls EMT of both primary and secondary mesenchyme cells. Loss and gain of function experiments demonstrate that ERK signaling is not required for the early specification of either PMCs or SMCs, but controls the maintenance and/or the enhancement of expression levels of regulatory genes which participate in the process of specification of these cell types. In addition, ERK-mediated signaling is essential for the transcription of terminal differentiation genes encoding proteins that define the final structures generated by PMCs and SMCs. Our findings suggest that ERK has a central pan-mesodermal role in coupling EMT and terminal differentiation of all mesenchymal cell types in the sea urchin embryo.
