P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1.

Abstract

A pituitary LIM homeodomain factor, PLim, is expressed as Rathke's pouch forms and as specific pituitary cell phenotypes are established, suggesting functional roles throughout pituitary development. While selectively expressed in Pituitary organ commitment appears to occur shortly after a region of the somatic ectoderm makes direct contact with neuroectodermal cells in an area of mesenchymal incompetence (1, 2). Subsequently, five distinct cell types, each characterized by the expression of a unique hormone, appear in a spatially and temporally specific fashion (reviewed in refs. 3 and 4). The pituitary-specific POU-domain transcription factor Pit-1 (3, 4) is selectively activated in the caudomedial part of the nascent gland at embryonic day 15.5 (e15.5) in the mouse and is required for activation of the prolactin (Prl), growth hormone (GH), and thyroid-stimulating hormone }3-subunit (TSHI3) genes in this region, as well as for somatotrope, lactotrope, and thyrotrope cell proliferation (5-7). Additional activating factors work with Pit-1 to achieve cellspecific gene activation (8)(9)(10)(11)(12)(13)(14)(15).A second family of homeodomain transcription factors, initially defined by RNA. These RT-PCR-generated DNA fragments were used to screen a mouse pituitary cDNA library and a mouse genomic library. RNase protection assays were performed with an intron 3-containing cDNA clone (no. 11) as template for T7RNA polymerase-directed synthesis of a radiolabeled antisense probe. In situ hybridization of 20-,um sagittal sections of mouse embryos and organs (e9, e9.5, elO.5, and e15.5) fixed in buffered 10% formalin was performed as described (1) by using T7 RNA polymerase to generate two separate 35S-labeled cRNA probes (486 and 550 nt) corresponding to fragments of N-terminal or C-terminal coding sequences of P-Lim.In Vitro Protein-Protein Interaction, DNA Binding, and Transfection Assays. Restriction fragments of P-Lim cDNA were ligated in frame into pGEX-KG (39) to yield glutathione S-transferase (GST) fusion proteins, and PCR was used to generate deletions of each LIM region separately (ALIM-1, A1-87; ALIM-2, A1-29 and A86-154) and of the entire LIM region (P-ALim = A1-151).[35S]Methionine-labeled in vitro translated protein was incubated for 20 min at 37°C with 2-3 jig of GST fusion protein bound to 25 p,l of glutathioneagarose beads in a total volume of 100 ,ul of 20 mM Hepes, pH 7.9/100 mM NaCl/1 mM EDTA/4 mM MgCl2/1 mM dithiothreitol, 0.02% (vol/vol) Nonidet P-40/10% (vol/vol) glycerol/0.5% (wt/vol) nonfat dry milk, with ethidium bromide at 50 ,ug/ml to eliminate potential protein-DNA interactions (32).Protein-mediated gel shift assays, in vitro culture, and transient transfection of cells were performed as described (9,14). For the mouse a-glycoprotein subunit (aGSU) reporter gene, a BamHI-Pst I fragment containing the promoter region of the aGSU starting from bp -440 was ligated to the luciferase gene in the pGL2 basic vector (Promega). Mouse TSH,B (from kb -1.2), rat Prl promoter/enhancer, and mous...

Roles of the LHX3 and LHX4 LIM-homeodomain factors in pituitary development

“…Mouse Lhx3a and Lhx3b mRNAs have distinct temporal expression profiles during development and are differentially expressed in cell lines that model the specialized pituitary cell types (Zhadanov et al, 1995a;Sloop et al, 1999;Sloop et al, 2001a;West et al, 2004). During rodent embryogenesis, Lhx3 mRNAs can be detected in the ventral spinal cord, the pons, the medulla oblongata, the pineal gland, the lungs, and in the developing and established anterior/intermediate pituitary gland (Seidah et al, 1994;Bach et al, 1995;Zhadanov et al, 1995a;Meier et al, 1999;Weng et al, 2006). Similar expression patterns are seen during human development (Sloop et al, 1999;Schmitt et al, 2000;Sobrier et al, 2004).…”

“…Bach et al, 1995;Sloop et al, 1999;West et al, 2004;McGillivray et al, 2005;Granger et al, 2006). Recently, LHX3 has been found to be an upstream activator of FOXL2, a transcription factor with predicted roles in the differentiation of cells expressing αGSU (Ellsworth et al, 2006).…”

“…Recently, LHX3 has been found to be an upstream activator of FOXL2, a transcription factor with predicted roles in the differentiation of cells expressing αGSU (Ellsworth et al, 2006). For activation of some promoters in the pituitary and nervous system, LHX3 can participate in complex transcriptional interactions with other factors such as PIT1 and ISL1 (Bach et al, 1995;Thaler et al, 2002;Granger et al, 2006).…”

Specificity of single LIM motifs in targeting and LIM/LIM interactions in situ.

“…LIM-binding proteins identified so far are transcription factors of the POU (Bach et al 1995) and bHLH (German et al 1992;Valge-Archer et al 1994;Wadman et al 1994) types, but further interacting species are likely to exist (see, e.g., Wu and Gill 1994}. The nature of the site responsible for association of MLP to actin-based filaments is not known.…”

“…Thus, LIMonly proteins can bind specifically to certain transcription factors (German et al 1992;Valge-Archer et al 1994;Wadman et al 1994;Bach et al 1995). In addition, these proteins can bind one another directly through their LIM motifs.…”