Cofactor CLIM2 promotes the repressive action of LIM homeodomain transcription factor Lhx2 in the expression of porcine pituitary glycoprotein hormone alpha subunit gene

We have cloned a porcine orthologue of cofactor CLIM2 (Ldb1/NLI) from the porcine pituitary cDNA library by protein-protein interaction with the Yeast Two-Hybrid System using porcine Lhx2 as a bait protein. Porcine CLIM2 shows a high identity (99%) in the dimerization domain, nuclear localization signal and LIM binding domain with those of man and mouse. The expression of CLIM2 gene in the anterior pituitary lobe was detected during the porcine fetal and postnatal period by RT-PCR analysis, suggesting that this protein is constitutively expressing and plays a basic role in the anterior pituitary. Transfection assay to the pituitary tumor derived LbetaT2 cells, and the Chinese hamster ovary cells demonstrated that CLIM2 acts as a corepressor of the porcine Lhx2 function. Interestingly, CLIM2 alone apparently repressed the high level of alphaGSU gene expression in LbetaT2 cells. These data suggest that CLIM2 is a basic factor in the pituitary development and function, and plays the role of repressor to modify the function of Lhx2 on the alphaGSU gene expression.     

Cofactor CLIM2 promotes the repressive action of LIM homeodomain transcription factor Lhx2 in the expression of porcine pituitary glycoprotein hormone α subunit gene

We have cloned a porcine orthologue of cofactor CLIM2 (Ldb1/NLI) from the porcine pituitary cDNA library by protein–protein interaction with the Yeast Two-Hybrid System using porcine Lhx2 as a bait protein. Porcine CLIM2 shows a high identity (99%) in the dimerization domain, nuclear localization signal and LIM binding domain with those of man and mouse. The expression of CLIM2 gene in the anterior pituitary lobe was detected during the porcine fetal and postnatal period by RT-PCR analysis, suggesting that this protein is constitutively expressing and plays a basic role in the anterior pituitary. Transfection assay to the pituitary tumor derived LβT2 cells, and the Chinese hamster ovary cells demonstrated that CLIM2 acts as a corepressor of the porcine Lhx2 function. Interestingly, CLIM2 alone apparently repressed the high level of αGSU gene expression in LβT2 cells. These data suggest that CLIM2 is a basic factor in the pituitary development and function, and plays the role of repressor to modify the function of Lhx2 on the αGSU gene expression.     

Cre-mediated recombination in the pituitary gland

Organ-specific expression of a cre recombinase transgene allows for the analysis of gene function in a particular tissue or cell type. Using a 4.6 kb promoter from the mouse glycoprotein hormone alpha-subunit (alphaGSU or Cga) gene, we have generated and characterized a line of transgenic mice that express cre recombinase in the anterior and intermediate lobes of the pituitary gland. Utilizing a cre-responsive reporter transgene, alphaGSU-cre transgene expression was detected in the pituitary primordium and in all five cell types of the adult anterior pituitary. alphaGSU-cre transgene activity was also detected in the cardiac and skeletal muscle. Little or no activity was evident in the gonads, adrenal glands, brain, ventromedial hypothalamus, or kidneys. The alphaGSU-cre transgenic mice characterized here will be a valuable tool for examining gene function in the pituitary gland.     

Expression screening for Lhx3 downstream genes identifies Thg-1pit as a novel mouse gene involved in pituitary development.

Thg-1pit, a novel mouse gene, was detected in a screen for genes that are differentially expressed in the developing pituitary of wild-type and Lhx3 null mutant embryos. The predicted translation product of the Thg-1pit gene contains a C-terminal TSC-box adjacent to a leucine zipper motif. These features are characteristic for the TSC-22/DIP/bun family of proteins. The onset of prominent Thg-1pit expression coincides with Lhx3 activation at early stages of pituitary development. Expression is further enhanced as cells begin to differentiate within the developing pituitary gland. No expression is observed in the pituitary rudiment of mutants that lack Lhx3 function. A possible role is thus suggested for Lhx3 activities in the regulation of Thg-1pit function during early steps of pituitary organogenesis.     

Antagonism of the testis- and ovary-determining pathways during ovotestis development in mice

Ovotestis development in B6-XY^POS mice provides a rare opportunity to study the interaction of the testis- and ovary-determining pathways in the same tissue. We studied expression of several markers of mouse fetal testis (SRY, SOX9) or ovary (FOXL2, Rspo1) development in B6-XY^POS ovotestes by immunofluorescence, using normal testes and ovaries as controls. In ovotestes, SOX9 was expressed only in the central region where SRY is expressed earliest, resulting in testis cord formation. Surprisingly, FOXL2-expressing cells also were found in this region, but individual cells expressed either FOXL2 or SOX9, not both. At the poles, even though SOX9 was not up-regulated, SRY expression was down-regulated normally as in XY testes, and FOXL2 was expressed from an early stage, demonstrating ovarian differentiation in these areas. Our data (1) show that SRY must act within a specific developmental window to activate Sox9; (2) challenge the established view that SOX9 is responsible for down-regulating Sry expression; (3) disprove the concept that testicular and ovarian cells occupy discrete domains in ovotestes; and (4) suggest that FOXL2 is actively suppressed in Sertoli cell precursors by the action of SOX9. Together these findings provide important new insights into the molecular regulation of testis and ovary development.     

The developmental expression of foxl2 in the dogfish Scyliorhinus canicula

The FoxL2 genes are a subfamily of the Fox (forkhead box) gene family. FOXL2 is mutated in the disorder Blepharophimosis, Ptosis, and Epicanthus Inversus Syndrome (BPES), which is characterized by eyelid malformations, and Premature Ovarian Failure (POF). In the mouse expression is seen in the perioptic mesenchyme, developing eyelids, ovary and pituitary. We have isolated a foxl2 cDNA from the dogfish Scyliorhinus canicula (also known as the lesser spotted catshark), allowing the characterisation of this gene's sequence and expression from a lineage that diverged early in the evolution of gnathostomes. Molecular phylogenetic analysis strongly grouped this sequence with the gnathostomes within the FoxL2 subfamily. We demonstrate the early expression of Scyliorhinus canicula foxl2 in the mandibular head mesoderm and later in continuous populations of mandibular arch cells and mandibular head mesenchyme cells around the developing pituitary. As development proceeds expression decreases in the mesenchyme of the head but is seen in the mesenchyme around the eye and later in the developing eyelids. Additionally expression is seen in regions of pharyngeal arch mesoderm and in ectoderm from which gill buds will form. This expression is maintained in the developing and elongating gill buds. Thus, S. canicula foxl2 is a marker for the mandibular mesoderm and gill buds and its expression is conserved in the perioptic mesenchyme, developing eyelids and pituitary.