SUMMARYA central challenge of developmental and evolutionary biology is to understand how anatomy is encoded in the genome. Elucidating the genetic mechanisms that control the development of specific anatomical features will require the analysis of model morphogenetic processes and an integration of biological information at genomic, cellular and tissue levels. The formation of the endoskeleton of the sea urchin embryo is a powerful experimental system for developing such an integrated view of the genomic regulatory control of morphogenesis. The dynamic cellular behaviors that underlie skeletogenesis are well understood and a complex transcriptional gene regulatory network (GRN) that underlies the specification of embryonic skeletogenic cells (primary mesenchyme cells, PMCs) has recently been elucidated. Here, we link the PMC specification GRN to genes that directly control skeletal morphogenesis. We identify new gene products that play a proximate role in skeletal morphogenesis and uncover transcriptional regulatory inputs into many of these genes. Our work extends the importance of the PMC GRN as a model developmental GRN and establishes a unique picture of the genomic regulatory control of a major morphogenetic process. Furthermore, because echinoderms exhibit diverse programs of skeletal development, the newly expanded sea urchin skeletogenic GRN will provide a foundation for comparative studies that explore the relationship between GRN evolution and morphological evolution. KEY WORDS: Gene regulatory network, Primary mesenchyme, Sea urchin, Skeletal morphogenesis, SkeletonThe genomic regulatory control of skeletal morphogenesis in the sea urchin Kiran Rafiq, Melani S. Cheers and Charles A. Ettensohn* DEVELOPMENT 580 this library allowed us to identify several components of the PMC GRN, including delta (Sweet et al., 2002), the transcription factors alx1 and erg (Zhu et al., 2001; Ettensohn et al., 2003), and several biomineralization-related genes (Illies et al., 2002; Cheers and Ettensohn, 2005;Livingston et al., 2006). In the present study, we greatly expand the current PMC GRN model by identifying many additional morphoregulatory genes and by uncovering regulatory inputs into these genes. Our work extends the value of the PMC GRN as a model developmental GRN and establishes a framework for understanding the genomic circuitry that encodes a major anatomical feature. MATERIALS AND METHODS Embryo cultureStrongylocentrotus purpuratus embryos were obtained and cultured at 15°C as described previously (Zhu et al., 2001). The PMC cDNA library and expressed sequence tag (EST) collectionThe construction and arraying of the PMC cDNA library has been described (Zhu et al., 2001;Livingston et al., 2006). Briefly, the library was generated from polyA(+) RNA that was isolated from micromeres (presumptive PMCs) that were cultured until sibling control embryos reached the mid-gastrula stage, ~36 hours post-fertilization. The cDNA library was not normalized or subtracted in any way. Reverse transcription was primed usin...

The failure to expand functional pancreatic ␤-cell mass in response to increased metabolic demand is a hallmark of type 2 diabetes. Lineage tracing studies indicate that replication of existing ␤-cells is the principle mechanism for ␤-cell expansion in adult mice. Here we demonstrate that the proliferative response of ␤-cells is dependent on the orphan nuclear receptor hepatocyte nuclear factor-4␣ (HNF-4␣), the gene that is mutated in Maturity-Onset Diabetes of the Young 1 (MODY1). Computational analysis of microarray expression profiles from isolated islets of mice lacking HNF-4␣ in pancreatic ␤-cells reveals that HNF-4␣ regulates selected genes in the ␤-cell, many of which are involved in proliferation. Using a physiological model of ␤-cell expansion, we show that HNF-4␣ is required for ␤-cell replication and the activation of the Ras/ERK signaling cascade in islets. This phenotype correlates with the down-regulation of suppression of tumorigenicity 5 (ST5) in HNF-4␣ mutants, which we identify as a novel regulator of ERK phosphorylation in ␤-cells and a direct transcriptional target of HNF-4␣ in vivo. Together, these results indicate that HNF-4␣ is essential for the physiological expansion of adult ␤-cell mass in response to increased metabolic demand.[Keywords: Ras; extracellular regulated kinase; mitogen activated protein kinase; ␤-cells; HNF-4␣; type 2 diabetes; gestational diabetes] Supplemental material is available at http://www.genesdev.org.

There was an error published in Development 141, 950-961.Column M of supplementary Table S1 contained an error and has been replaced. The authors apologise to readers for this mistake. ABSTRACT A central challenge of developmental and evolutionary biology is to understand the transformation of genetic information into morphology. Elucidating the connections between genes and anatomy will require model morphogenetic processes that are amenable to detailed analysis of cell/tissue behaviors and to systems-level approaches to gene regulation. The formation of the calcified endoskeleton of the sea urchin embryo is a valuable experimental system for developing such an integrated view of the genomic regulatory control of morphogenesis. A transcriptional gene regulatory network (GRN) that underlies the specification of skeletogenic cells (primary mesenchyme cells, or PMCs) has recently been elucidated. In this study, we carried out a genome-wide analysis of mRNAs encoded by effector genes in the network and uncovered transcriptional inputs into many of these genes. We used RNA-seq to identify >400 transcripts differentially expressed by PMCs during gastrulation, when these cells undergo a striking sequence of behaviors that drives skeletal morphogenesis. Our analysis expanded by almost an order of magnitude the number of known (and candidate) downstream effectors that directly mediate skeletal morphogenesis. We carried out genome-wide analysis of (1) functional targets of Ets1 and Alx1, two pivotal, early transcription factors in the PMC GRN, and (2) functional targets of MAPK signaling, a pathway that plays an essential role in PMC specification. These studies identified transcriptional inputs into >200 PMC effector genes. Our work establishes a framework for understanding the genomic regulatory control of a major morphogenetic process and has important implications for reconstructing the evolution of biomineralization in metazoans.

ObjectiveGlucagon-like peptide-1 (GLP-1) plays a major role in pancreatic β-cell function and survival by increasing cytoplasmic cAMP levels, which are thought to affect transcription through activation of the basic leucine zipper (bZIP) transcription factor CREB. Here, we test CREB function in the adult β-cell through inducible gene deletion.MethodsWe employed cell type-specific and inducible gene ablation to determine CREB function in pancreatic β-cells in mice.ResultsBy ablating CREB acutely in mature β-cells in tamoxifen-treated CrebloxP/loxP;Pdx1-CreERT2 mice, we show that CREB has little impact on β-cell turnover, in contrast to what had been postulated previously. Rather, CREB is required for GLP-1 to elicit its full effects on stimulating glucose-induced insulin secretion and protection from cytokine-induced apoptosis. Mechanistically, we find that CREB regulates expression of the pro-apoptotic gene p21 (Cdkn1a) in β-cells, thus demonstrating that CREB is essential to mediating this critical aspect of GLP-1 receptor signaling.ConclusionsIn sum, our studies using conditional gene deletion put into question current notions about the importance of CREB in regulating β-cell function and mass. However, we reveal an important role for CREB in the β-cell response to GLP-1 receptor signaling, further validating CREB as a therapeutic target for diabetes.

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