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

Abstract

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.

Introduction

The pituitary gland represents an excellent model system to study inductive signals and molecular mechanisms that mediate organ fate commitment and cell specialization. Pituitary development from a rudiment to a functional gland occurs in distinct successive steps that are mediated by defined molecular regulatory events (reviewed by Treier et al., 1998, Sheng and Westphal, 1999). Several inductive signals, including members of BMP and WNT protein families as well as transcription factors, notably members of the LIM-homeobox gene family (reviewed by Hobert and Westphal, 2000), play key roles in pituitary formation. Initially, at day E8.5 of embryonic development, the formation of a pituitary placode is induced by the release of BMP4 from the ventral diencephalon. Subsequent differentiation of the placode into Rathke's pouch is induced by the LIM-homeobox gene Isl1 (Ericson et al., 1998). At E9.25, a second diencephalon-derived molecule, FGF8, triggers the pouch to express, among others, the LIM-homeobox genes Lhx3 and Lhx4. Opposing gradients of FGF8 and BMP2, dorsal-to-ventral and ventral-to-dorsal, respectively, are responsible for the determination within the developing pouch of the six hormone-producing cell types (Treier et al., 1998). The graded expression of these molecules, between E9.25 and E12, in turn activates a cascade of events that eventually leads to the formation of a definitive pouch at E12.5 (Takuma et al., 1998).

Interestingly, a definitive pouch is formed in both Lhx3^−/− and Lhx4 ^−/− single null mutant embryos but not in Lhx3/4 double knockout mutants (Sheng et al., 1996, Sheng et al., 1997), indicating redundant function of the two genes at this stage of pituitary development. There is an absolute requirement for Lhx3 function at subsequent stages of pituitary development since, in Lhx3 null mutants, a defect in the differentiation of hormone producing pituitary cells is not rescued by Lhx4.

While our knowledge concerning factors whose temporal appearance and spatial distribution correlate well with defined steps of pituitary formation has significantly been advanced, the genes that are activated by these factors are still largely unknown. Of special interest in this context are the target genes of Lhx3. This is because the Lhx3 gene plays a crucial role in controlling the development of Rathke's pouch and in triggering the appearance of specialized hormone-producing cells (Sheng et al., 1997). Identification of genes that act downstream of Lhx3 is therefore an important task towards understanding key molecular mechanisms that underlie pituitary development.

In the present work, we have addressed this issue by searching for genes that are differentially expressed in the developing pituitary gland of wild type and Lhx3 null mutant embryos. To identify genes involved in the initiation of pituitary cell differentiation, we performed a differential screening of cDNA populations isolated from pituitaries of wild type and null mutant E12.5 embryos. One of the genes identified by this procedure is related to TSC-22, first isolated from mouse osteoblastic cells as an early TGF-β responsive gene (Shibanuma et al., 1992). The presence of a TSC-box adjacent to a leucine zipper motif in the C-terminal region of the encoded amino acid sequence identified the product of our gene as a relative of the TSC-22/DIP/bun family of proteins.

Owing to the 79% amino acids identity between this mouse protein and human THG-1, we termed it Thg-1pit to signify both its nature and its expression in the developing pituitary gland. We show that the temporal expression of this gene correlates well with the onset of pituitary cell differentiation. Furthermore, lack of Thg-1pit expression in pituitary primordia of Lhx3 ^−/− null mutant embryos is consistent with the possibility that our gene is a component of an Lhx3-dependent pathway of gene activation.