Furry is required for cell movements during gastrulation and functionally interacts with NDR1

Gastrulation is a key event in animal embryogenesis during which germ layer precursors are rearranged and the embryonic axes are established. Cell polarization is essential during gastrulation, driving asymmetric cell division, cell movements, and cell shape changes. The furry (fry) gene encodes an evolutionarily conserved protein with a wide variety of cellular functions, including cell polarization and morphogenesis in invertebrates. However, little is known about its function in vertebrate development. Here, we show that in Xenopus, Fry plays a role in morphogenetic processes during gastrulation, in addition to its previously described function in the regulation of dorsal mesoderm gene expression. Using morpholino knock-down, we demonstrate a distinct role for Fry in blastopore closure and dorsal axis elongation. Loss of Fry function drastically affects the movement and morphological polarization of cells during gastrulation and disrupts dorsal mesoderm convergent extension, responsible for head-to-tail elongation. Finally, we evaluate a functional interaction between Fry and NDR1 kinase, providing evidence of an evolutionarily conserved complex required for morphogenesis.Fil: Cervino, Ailen Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Moretti, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Stuckenholz, Carsten. University of Pittsburgh; Estados Unidos. University of Pittsburgh at Johnstown; Estados UnidosFil: Grecco, Hernan Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Davidson, Lance A.. University of Pittsburgh at Johnstown; Estados Unidos. University of Pittsburgh; Estados UnidosFil: Cirio, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Lhx1 Is Required for Specification of the Renal Progenitor Cell Field

In the vertebrate embryo, the kidney is derived from the intermediate mesoderm. The LIM-class homeobox transcription factor lhx1 is expressed early in the intermediate mesoderm and is one of the first genes to be expressed in the nephric mesenchyme. In this study, we investigated the role of Lhx1 in specification of the kidney field by either overexpressing or depleting lhx1 in Xenopus embryos or depleting lhx1 in an explant culture system. By overexpressing a constitutively-active form of Lhx1, we established its capacity to expand the kidney field during the specification stage of kidney organogenesis. In addition, the ability of Lhx1 to expand the kidney field diminishes as kidney organogenesis transitions to the morphogenesis stage. In a complimentary set of experiments, we determined that embryos depleted of lhx1, show an almost complete loss of the kidney field. Using an explant culture system to induce kidney tissue, we confirmed that expression of genes from both proximal and distal kidney structures is affected by the absence of lhx1. Taken together our results demonstrate an essential role for Lhx1 in driving specification of the entire kidney field from the intermediate mesoderm

Sfrp5 Modulates Both Wnt and BMP Signaling and Regulates Gastrointestinal Organogensis in the Zebrafish, Danio rerio

Sfrp5 belongs to the family of secreted frizzled related proteins (Sfrp), secreted inhibitors of Wingless-MMTV Integration Site (Wnt) signaling, which play an important role in cancer and development. We selected sfrp5 because of its compelling expression profile in the developing endoderm in zebrafish, Danio rerio. In this study, overexpression of sfrp5 in embryos results in defects in both convergent extension (CE) by inhibition of non-canonical Wnt signaling and defects in dorsoventral patterning by inhibition of Tolloid-mediated proteolysis of the BMP inhibitor Chordin. From 25 hours post fertilization (hpf) to 3 days post fertilization (dpf), both overexpression and knockdown of Sfrp5 decrease the size of the endoderm, significantly reducing liver cell number. At 3 dpf, insulin-positive endodermal cells fail to coalesce into a single pancreatic islet. We show that Sfrp5 inhibits both canonical and non-canonical Wnt signaling during embryonic and endodermal development, resulting in endodermal abnormalities. © 2013 Stuckenholz et al

DAF-12 Regulates a Connected Network of Genes to Ensure Robust Developmental Decisions

The nuclear receptor DAF-12 has roles in normal development, the decision to pursue dauer development in unfavorable conditions, and the modulation of adult aging. Despite the biologic importance of DAF-12, target genes for this receptor are largely unknown. To identify DAF-12 targets, we performed chromatin immunoprecipitation followed by hybridization to whole-genome tiling arrays. We identified 1,175 genomic regions to be bound in vivo by DAF-12, and these regions are enriched in known DAF-12 binding motifs and act as DAF-12 response elements in transfected cells and in transgenic worms. The DAF-12 target genes near these binding sites include an extensive network of interconnected heterochronic and microRNA genes. We also identify the genes encoding components of the miRISC, which is required for the control of target genes by microRNA, as a target of DAF-12 regulation. During reproductive development, many of these target genes are misregulated in daf-12(0) mutants, but this only infrequently results in developmental phenotypes. In contrast, we and others have found that null daf-12 mutations enhance the phenotypes of many miRISC and heterochronic target genes. We also find that environmental fluctuations significantly strengthen the weak heterochronic phenotypes of null daf-12 alleles. During diapause, DAF-12 represses the expression of many heterochronic and miRISC target genes, and prior work has demonstrated that dauer formation can suppress the heterochronic phenotypes of many of these target genes in post-dauer development. Together these data are consistent with daf-12 acting to ensure developmental robustness by committing the animal to adult or dauer developmental programs despite variable internal or external conditions

Dysregulated phosphatidylinositol signaling promotes endoplasmic-reticulum-stress-mediated intestinal mucosal injury and inflammation in zebrafish

Dysregulated phosphatidylinositol (PI) signaling has been implicated in human gastrointestinal (GI) malignancies and inflammatory states, underlining the need to study pathophysiological roles of PI in an in vivo genetic model. Here, we study the significance of PI in GI pathophysiology using the zebrafish mutant cdipthi559, which lacks PI synthesis, and unravel a crucial role of PI in intestinal mucosal integrity and inflammation. The cdipthi559 mutants exhibit abnormal villous architecture and disorganized proliferation of intestinal epithelial cells (IECs), with pathologies reminiscent of inflammatory bowel disease (IBD), including apoptosis of goblet cells, abnormal mucosecretion, bacterial overgrowth and leukocyte infiltration. The mutant IECs exhibit vacuolation, microvillus atrophy and impaired proliferation. The cdipthi559 gene expression profile shows enrichment of acute phase response signaling, and the endoplasmic reticulum (ER) stress factors hspa5 and xbp1 are robustly activated in the mutant GI tissue. Temporal electron micrographic analyses reveal that PI-deficient IECs undergo sequential ER-Golgi disruption, mitochondrial depletion, macroautophagy and cell death, consistent with chronic ER-stress-mediated cytopathology. Furthermore, pharmacological induction of ER stress by inhibiting protein glycosylation or PI synthase inhibition in leukocyte-specific reporter lines replicates the cdipthi559 inflammatory phenotype, suggesting a fundamental role of PI metabolism and ER stress in mucosal inflammation. Antibiotics and anti-inflammatory drugs resolved the inflammation, but not the autophagic necroapoptosis of IECs, suggesting that bacterial overgrowth can exacerbate ER stress pathology, whereas persistent ER stress is sufficient to trigger inflammation. Interestingly, the intestinal phenotype was partially alleviated by chemical chaperones, suggesting their therapeutic potential. Using zebrafish genetic and pharmacological models, this study demonstrates a newly identified link between intracellular PI signaling and ER-stress-mediated mucosal inflammation. The zebrafish cdipt mutants provide a powerful tool for dissecting the fundamental mechanisms of ER-stress-mediated human GI diseases and a platform to develop molecularly targeted therapies

Expression profile of <i>sfrp5</i>.

<p><b>A)</b> Expression level of <i>sfrp5</i> as measured by probeset Dr.21012.1.S1 in GI tissue (dark green squares) and non-GI tissue (light green triangles) from 2 through 6 dpf (for details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062470#pone.0062470-Stuckenholz1" target="_blank">[39]</a>). <b>B)</b> Expression of <i>sfrp5</i> and β-actin by RT-PCR of total RNA isolated at indicated time points. <b>C–O)</b> Whole-mount <i>in situ</i> hybridization showing <i>sfrp5</i> expression in zebrafish embryos at 3 hpf (<b>C</b>), shield stage (<b>D</b>), 8 hpf (<b>E</b>), bud stage (<b>F</b>), early (<b>G</b>), mid (<b>H</b>), and late somitogenesis (<b>I</b>), 24 hpf (<b>J</b>), 32 hpf (<b>K</b>), 2 dpf (<b>L</b>), 3 dpf (<b>M</b>), 4 dpf (<b>N</b>), and 6 dpf (<b>O</b>). Lateral views with animal pole to the top (<b>C</b>–<b>F</b>) or with anterior to the left (<b>G</b>–<b>I</b>, <b>M</b>–<b>O</b>). Dorsal view with anterior to the left (<b>J</b>–<b>L</b>).</p