Gadd45α modulates aversive learning through post-transcriptional regulation of memory-related mRNAs.

Learning is essential for survival and is controlled by complex molecular mechanisms including regulation of newly synthesized mRNAs that are required to modify synaptic functions. Despite the well-known role of RNA-binding proteins (RBPs) in mRNA functionality, their detailed regulation during memory consolidation is poorly understood. This study focuses on the brain function of the RBP Gadd45α (growth arrest and DNA damage-inducible protein 45 alpha, encoded by the Gadd45a gene). Here, we find that hippocampal memory and long-term potentiation are strongly impaired in Gadd45a-deficient mice, a phenotype accompanied by reduced levels of memory-related mRNAs. The majority of the Gadd45α-regulated transcripts show unusually long 3' untranslated regions (3'UTRs) that are destabilized in Gadd45a-deficient mice via a transcription-independent mechanism, leading to reduced levels of the corresponding proteins in synaptosomes. Moreover, Gadd45α can bind specifically to these memory-related mRNAs. Our study reveals a new function for extended 3'UTRs in memory consolidation and identifies Gadd45α as a novel regulator of mRNA stability.

GADD45G functions in male sex determination by promoting p38 signaling and Sry expression.

Male sex determination in mammals is induced by Sry, a gene whose regulation is poorly understood. Here we show that mice mutant for the stress-response gene Gadd45g display complete male-to-female sex reversal. Gadd45g and Sry have a strikingly similar expression pattern in the genital ridge, and they are coexpressed in gonadal somatic cells. In Gadd45g mutants, Sry expression is delayed and reduced, and yet Sry seemed to remain poised for expression, because its promoter is demethylated on schedule and is occupied by active histone marks. Instead, p38 MAPK signaling is impaired in Gadd45g mutants. Moreover, the transcription factor GATA4, which is required for Sry expression, binds to the Sry promoter in vivo in a MAPK-dependent manner. The results suggest that a signaling cascade, involving GADD45G → p38 MAPK → GATA4 → SRY, regulates male sex determination.

Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development.

Gadd45 proteins have been implicated in the cellular response to physiological or environmental stress and the accompanying cell cycle arrest, DNA repair, cell survival and senescence or apoptosis. Although their molecular function is well studied, the expression and role of Gadd45 genes during embryonic development in mice is largely unknown. Here we provide a comprehensive comparison of Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development. In situ hybridizations on sectioned and whole mouse embryos show most prominent Gadd45a expression in the tip of the closing neural tube, the cranial and dorsal root ganglia and the somites. Mouse Gadd45b is expressed strongly in the chorion, but only weakly in the embryo proper, including somites and limb buds. Murine Gadd45g expression strongly resembles Xenopus and medaka fish expression in primary neuron precursors and post-mitotic neurons, indicating a conserved role for Gadd45g in vertebrate neurogenesis. Additionally, Gadd45 genes show conserved expression during somitogenesis. In summary, Gadd45 genes are expressed in evolutionary conserved, but also divergent domains, which predominantly encompass areas of cell differentiation, consistent with their established function in growth arrest and DNA demethylation.

Isolation and propagation of enteric neural crest progenitor cells from mouse embryonic stem cells and embryos.

Neural crest is a source of diverse cell types, including the peripheral nervous system. The transcription factor Sox10 is expressed throughout early neural crest. We exploited Sox10 reporter and selection markers created by homologous recombination to investigate the generation, maintenance and expansion of neural crest progenitors. Sox10-GFP-positive cells are produced transiently from mouse embryonic stem (ES) cells by treatment with retinoic acid in combination with Fgf8b and the cytokine leukaemia inhibitory factor (Lif). We found that expression of Sox10 can be maintained using noggin, Wnt3a, Lif and endothelin (NWLE). ES cell-derived Sox10-GFP-positive cells cultured in NWLE exhibit molecular markers of neural crest progenitors. They differentiate into peripheral neurons in vitro and are able to colonise the enteric network in organotypic gut cultures. Neural crest cells purified from embryos using the Sox10 reporter also survive in NWLE, but progressively succumb to differentiation. We therefore applied selection to eliminate differentiating cells. Sox10-selected cells could be clonally expanded, cryopreserved, and multiplied for over 50 days in adherent culture. They remained neurogenic in vitro and in foetal gut grafts. Generation of neural crest from mouse ES cells opens a new route to the identification and validation of determination factors. Furthermore, the ability to propagate undifferentiated progenitors creates an opportunity for experimental dissection of the stimuli and molecular circu that govern neural crest lineage progression. Finally, the demonstration of robust enteric neurogenesis provides a system for investigating and modelling cell therapeutic approaches to neurocristopathies such as Hirschsprung's disease.

Insm1 (IA-1) is a crucial component of the transcriptional network that controls differentiation of the sympatho-adrenal lineage.

Insm1 (IA-1) encodes a Zn-finger factor that is expressed in the developing nervous system. We demonstrate here that the development of the sympatho-adrenal lineage is severely impaired in Insm1 mutant mice. Differentiation of sympatho-adrenal precursors, as assessed by the expression of neuronal subtype-specific genes such as Th and Dbh, is delayed in a pronounced manner, which is accompanied by a reduced proliferation. Sympathetic neurons eventually overcome the differentiation blockade and mature correctly, but sympathetic ganglia remain small. By contrast, terminal differentiation of adrenal chromaffin cells does not occur. The transcription factors Mash1 (Ascl1), Phox2a, Gata3 and Hand2 (previously dHand) control the differentiation of sympatho-adrenal precursor cells, and their deregulated expression in Insm1 mutant mice demonstrates that Insm1 acts in the transcriptional network that controls differentiation of this lineage. Pronounced similarities between Mash1 and Insm1 phenotypes are apparent, which suggests that Insm1 might mediate aspects of Mash1 function in the subtype-specific differentiation of sympatho-adrenal precursors. Noradrenaline is the major catecholamine produced by developing sympatho-adrenal cells and is required for fetal survival. We demonstrate that the fetal lethality of Insm1 mutant mice is caused by catecholamine deficiency, which highlights the importance of Insm1 in the development of the sympatho-adrenal lineage.

The zinc-finger factor Insm1 (IA-1) is essential for the development of pancreatic beta cells and intestinal endocrine cells.

The pancreatic and intestinal primordia contain epithelial progenitor cells that generate many cell types. During development, specific programs of gene expression restrict the developmental potential of such progenitors and promote their differentiation. The Insm1 (insulinoma-associated 1, IA-1) gene encodes a Zinc-finger factor that was discovered in an insulinoma cDNA library. We show that pancreatic and intestinal endocrine cells express Insm1 and require Insm1 for their development. In the pancreas of Insm1 mutant mice, endocrine precursors are formed, but only few insulin-positive beta cells are generated. Instead, endocrine precursor cells accumulate that express none of the pancreatic hormones. A similar change is observed in the development of intestine, where endocrine precursor cells are formed but do not differentiate correctly. A hallmark of endocrine cell differentiation is the accumulation of proteins that participate in secretion and vesicle transport, and we find many of the corresponding genes to be down-regulated in Insm1 mutant mice. Insm1 thus controls a gene expression program that comprises hormones and proteins of the secretory machinery. Our genetic analysis has revealed a key role of Insm1 in differentiation of pancreatic and intestinal endocrine cells.

The Drosophila gene Start1: a putative cholesterol transporter and key regulator of ecdysteroid synthesis.

Human metastatic lymph node 64 (MLN64) is a transmembrane protein that shares homology with the cholesterol-binding vertebrate steroid acute regulatory protein (StAR)-related lipid transfer domain (START) and is involved in cholesterol traffic and steroid synthesis. We identified a Drosophila melanogaster gene whose putative protein product shows extensive homology with MLN64 and that we name Start1 (FlyBase CG3522). The putative Start1 protein, derived from Start1 cDNA sequences, contains an additional 122 aa of unknown function within the StAR-related lipid transfer domain. Similar inserts seem to exist in the Start1 homologues of Drosophila pseudoobscura and Anopheles gambiae, but not in the homologous protein of the urochordate Ciona intestinalis. Immunostaining using an insert-specific antibody confirms the presence of the insert in the cytoplasm. Whereas RT-PCR data indicate that Start1 is expressed ubiquitously, RNA in situ hybridizations demonstrate its overexpression in prothoracic gland cells, where ecdysteroids are synthesized from cholesterol. Transcripts of Start1 are detectable in embryonic ring gland progenitor cells and are abundant in prothoracic glands of larvae showing wave-like expression during larval stages. In adults, Start1 is expressed in nurse cells of the ovary. These observations are consistent with the assumption that Start1 plays a key role in the regulation of ecdysteroid synthesis. Vice versa, the expression of Start1 itself seems to depend on ecdysone, as in the ecdysone-deficient mutant ecd-1, Start1 expression is severely reduced.