Justin C. Grindley

Disruption of PAX6 function in mice homozygous for the Pax6Sey-1Neu mutation produces abnormalities in the early development and regionalization of the diencephalon

Pax6 expression in the diencephalon of the mouse embryo is restricted both antero-posteriorly and dorso-ventrally, with changes in level occurring at prosomere boundaries. Small eye (Pax6Sey-1Neu) mice homozygous for Pax6 mutations have multiple defects in early forebrain development. In the diencephalon of Pax6Sey-1Neu/Pax6Sey-1Neu mice there is an apparent enlargement of the zona limitans (the boundary region between prosomeres p2 and p3), and a blurring of the p1-p2 boundary. PAX6 function is also required for the normal development of the posterior commissure at the midbrain-p1 boundary. In the posterior diencephalon PAX6 appears to regulate its own transcription, and that of Wnt7b. In p2 and p3, ventral markers are expressed more dorsally than normal, and this is accompanied in p3 by a reduction in the size of the zona incerta. Thus, PAX6 is essential for the normal development and regionalization of the diencephalon.

Characterization of Nfatc1 regulation identifies an enhancer required for gene expression that is specific to pro-valve endocardial cells in the developing heart

Nfatc1 is an endocardial transcription factor required for development of cardiac valves. Herein, we describe identification and characterization of a tissue-specific enhancer in the first intron of murine Nfatc1 that activates a heterogenic promoter and directs gene expression in a subpopulation of endocardial cells of the developing heart: the pro-valve endocardial cells. This enhancer activity begins on embryonic day (E) 8.5 in endocardial cells at the ventricular end of the atrioventricular canal, intensifies and extends from E9.5 to E11.5 in endocardium along the atrioventricular canal and outflow tract. By E12.5, the enhancer activity is accentuated in endocardial cells of forming valves. Sequential deletion analysis identified that a 250 bp DNA fragment at the 3′ end of the intron 1 is required for endocardial-specific activity. This region contains two short conserved sequences hosting a cluster of binding sites for transcription factors, including Nfat and Hox proteins. Electrophoresis mobility shift and chromatin immunoprecipitation assays demonstrated binding of Nfatc1 to the Nfat sites, and inactivation of Nfatc1 downregulated the enhancer activity in pro-valve endocardial cells. By contrast, mutation of the Hox site abolished its specificity, allowing gene expression in non pro-valve endocardium and extracardiac vasculature. Thus, autoregulation of Nfatc1 is required for maintaining high Nfatc1 expression in pro-valve endocardial cells, while suppression through the Hox site prevents its expression outside pro-valve endocardial cells during valve development. Our data demonstrate the first autonomous cell-specific enhancer for pro-valve endocardial cells and delineate a unique transcriptional mechanism that regulates endocardial Nfatc1 expression within developing cardiac valves.

Pax6 is implicated in murine pituitary endocrine function

Pax6, an evolutionarily conserved transcription factor, is expressed in the murine and zebrafish embryonic pituitary, but its role in pituitary development and endocrine function has not been described. To study the role of Pax6 in vivo, we examined Pax6 mutant mouse (SeyNeu) pituitaries. Mice homozygous for the SeyNeu mutation die at birth; therefore, we examined peptide hormone expression by the differentiated pituitary cell types as well as developmental marker expression in the intermediate and anterior lobes of the embryonic pituitary. GH- and PRL-immunopositive cells appear severely decreased in an outbred ICR background at embryonic d 17.5, although mRNA expression of these peptide hormones is present, as is expression of other pituitary markers. This suggests that pituitary cell types are able to differentiate in mutant embryos. To identify the cellular or physiologic mechanism responsible for less GH-and PRL-immunoreactivity in Pax6 mutant mice, we tested serum levels of GH and PRL. Pax6 homozygous mutant mice have GH serum levels one fifth that of controls at embryonic d 17.5, and one-third that of controls at postnatal d 0. PRL serum levels, which are very low during embryonic and neonatal stages, were below assay detection limits in both the wild-type and mutant groups. Taken together, these data suggest that Pax6 is not essential for pituitary differentiation, but rather functions to establish appropriate neonatal homeostatic levels of GH and PRL, possibly through regulation of translational or secretory mechanisms.

Mouse Mesenchyme forkhead 2 (Mf2): expression, DNA binding and induction by sonic hedgehog during somitogenesis

Cloning and sequencing of mouse Mf2 (mesoderm/mesenchyme forkhead 2) cDNAs revealed an open reading frame encoding a putative protein of 492 amino acids which, after in vitro translation, binds to a DNA consensus sequence. Mf2 is expressed at high levels in the ventral region of newly formed somites, in sclerotomal derivatives, in lateral plate and cephalic mesoderm and in the first and second branchial arches. Other regions of mesodermal expression include the developing tongue, meninges, nose, whiskers, kidney, genital tubercule and limb joints. In the nervous system Mf2 is transcribed in restricted regions of the mid- and forebrain. In several tissues, including the early somite, Mf2 is expressed in cell populations adjacent to regions expressing sonic hedgehog (Shh) and in explant cultures of presomitic mesoderm Mf2 is induced by Shh secreted by COS cells. These results suggest that Mf2, like other murine forkhead genes, has multiple roles in embryogenesis, possibly mediating the response of cells to signaling molecules such as SHH.