Gata6 Promotes GLI3 Repressor Activities in the Limb

Gli3 is a major regulator of Hedgehog signaling during limb development. In the anterior mesenchyme, GLI3 is proteolytically processed into GLI3R, a truncated repressor form that inhibits Hedgehog signaling. Although numerous studies have identified mechanisms that regulate Gli3 function in vitro, it is not completely understood how Gli3 function is regulated in vivo. In this study, we show a novel mechanism of regulation of GLI3R activities in limb buds by Gata6, a member of the GATA transcription factor family. We show that conditional inactivation of Gata6 prior to limb outgrowth by the Tcre deleter causes preaxial polydactyly, the formation of an anterior extra digit, in hindlimbs. A recent study suggested that Gata6 represses Shh transcription in hindlimb buds. However, we found that ectopic Hedgehog signaling precedes ectopic Shh expression. In conjunction, we observed Gata6 and Gli3 genetically interact, and compound heterozygous mutants develop preaxial polydactyly without ectopic Shh expression, indicating an additional prior mechanism to prevent polydactyly. These results support the idea that Gata6 possesses dual roles during limb development: enhancement of Gli3 repressor function to repress Hedgehog signaling in the anterior limb bud, and negative regulation of Shh expression. Our in vitro and in vivo studies identified that GATA6 physically interacts with GLI3R to facilitate nuclear localization of GLI3R and repressor activities of GLI3R. Both the genetic and biochemical data elucidates a novel mechanism by Gata6 to regulate GLI3R activities in the anterior limb progenitor cells to prevent polydactyly and attain proper development of the mammalian autopod

Visualizing Trimming Dependence of Biodistribution and Kinetics with Homo- and Heterogeneous N-Glycoclusters on Fluorescent Albumin

A series of N-glycans, each sequentially trimmed from biantennary sialoglycans, were homo- or heterogeneously clustered efficiently on fluorescent albumin using a method that combined strain-promoted alkyne-azide cyclization and 6π-azaelectrocyclization. Noninvasive in vivo kinetics and dissection analysis revealed, for the first time, a glycan-dependent shift from urinary to gall bladder excretion mediated by sequential trimming of non-reducing end sialic acids. N-glycoalbumins that were trimmed further, in particular, GlcNAc- and hybrid biantennary-terminated congeners, were selectively taken up by sinusoidal endothelial and stellate cells in the liver, which are critical for diagnosis and treatment of liver fibrillation. Our glycocluster strategy can not only reveal the previously unexplored extracellular functions of N-glycan trimming, but will be classified as the newly emerging glycoprobes for diagnostic and therapeutic applications

Celf1 Is Required for Formation of Endoderm-Derived Organs in Zebrafish

We recently reported that an RNA binding protein called Cugbp Elav-like family member 1 (Celf1) regulates somite symmetry and left-right patterning in zebrafish. In this report, we show additional roles of Celf1 in zebrafish organogenesis. When celf1 is knocked down by using an antisense morpholino oligonucleotides (MO), liver buds fail to form, and pancreas buds do not form a cluster, suggesting earlier defects in endoderm organogenesis. As expected, we found failures in endoderm cell growth and migration during gastrulation in embryos injected with celf1-MOs. RNA immunoprecipitation revealed that Celf1 binds to gata5 and cdc42 mRNAs which are known to be involved in cell growth and migration, respectively. Our results therefore suggest that Celf1 regulates proper organogenesis of endoderm-derived tissues by regulating the expression of such targets

Celf1 is required for formation of endoderm-derived organs in zebrafish

We recently reported that an RNA binding protein called Cugbp Elav-like family member 1 (Celf1) regulates somite symmetry and left-right patterning in zebrafish. In this report, we show additional roles of Celf1 in zebrafish organogenesis. When celf1 is knocked down by using an antisense morpholino oligonucleotides (MO), liver buds fail to form, and pancreas buds do not form a cluster, suggesting earlier defects in endoderm organogenesis. As expected, we found failures in endoderm cell growth and migration during gastrulation in embryos injected with celf1-MOs. RNA immunoprecipitation revealed that Celf1 binds to gata5 and cdc42 mRNAs which are known to be involved in cell growth and migration, respectively. Our results therefore suggest that Celf1 regulates proper organogenesis of endoderm-derived tissues by regulating the expression of such targets

Temporal changes of Sall4 lineage contribution in developing embryos and the contribution of Sall4-lineages to postnatal germ cells in mice

Abstract Mutations in the SALL4 gene cause human syndromes with defects in multiple organs. Sall4 expression declines rapidly in post-gastrulation mouse embryos, and our understanding of the requirement of Sall4 in animal development is still limited. To assess the contributions of Sall4 expressing cells to developing mouse embryos, we monitored temporal changes of the contribution of Sall4 lineages using a Sall4 GFP-CreER T2 knock-in mouse line and recombination-dependent reporter lines. By administering tamoxifen at various time points we observed that the contributions of Sall4 lineages to the axial level were rapidly restricted from the entire body to the posterior part of the body. The contribution to forelimbs, hindlimbs, craniofacial structures and external genitalia also declined after gastrulation with different temporal dynamics. We also detected Sall4 lineage contributions to the extra-embryonic tissues, such as the yolk sac and umbilical cord, in a temporal manner. These Sall4 lineage contributions provide insights into potential roles of Sall4 during mammalian embryonic development. In postnatal males, long-term lineage tracing detected Sall4 lineage contributions to the spermatogonial stem cell pool during spermatogenesis. The Sall4 GFP-CreER T2 line can serve as a tool to monitor spatial-temporal contributions of Sall4 lineages as well as to perform gene manipulations in Sall4-expressing lineages

GATA6 regulates subcellular localization of GLI3R.

<p><b>A</b>: Representative in vitro images of nuclear GATA6+nuclear GLI3R (upper), nuclear GATA6+cytosolic GLI3R (middle) and cytosolic GATA6+cytosolic GLI3R (bottom). <b>B</b>: Quantitation of subcellular localization of GATA6 and GLI3R. NC: predominantly nuclear localized. GATA6 mutants, indicated at the bottom, are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006138#pgen.1006138.g005" target="_blank">Fig 5E</a>. The number of cells examined for each set of transfection is indicated in the panel. <b>C-H</b>: Representative images of the anterior-proximal mesenchyme of hindlimb buds at E10.25. <b>C, E, G</b>: wild type, <b>D, F, H</b>: <i>Gata6</i> cKO. <b>I</b>: Quantitation of subcellular localization of GLI3R in the anterior-proximal mesenchyme of hindlimb buds at E10.25. Gray and black bars represent wild-type and <i>Gata6</i> cKO samples, respectively. The graph shows percentage of GLI3R localization patterns, such as predominantly nuclear (N>C), similarly in the nucleus and cytoplasm (N = C), or predominantly cytoplasmic (NGata6 cKO embryos were examined. * indicates P<0.05. <b>J</b>: Western blot of nuclear fractions from anterior part of wild-type and <i>Gata6</i> cKO hindlimb buds at E10.25–10.5. Histone H3 (H3) is included as a loading control.</p

Genetic interaction between <i>Gata6</i> and <i>Gli3</i> in preaxial polydactyly development.

<p><b>A-J</b>: Alcian blue-stained autopod of indicated genotypes at E15.5. A-E: forelimbs, F-J: hindlimbs. Thin red arrows point to bifurcated d1 (<b>C</b>) and small projection (<b>H</b>) in fore- and hind-limbs, respectively in <i>Gli3</i>^<i>+/-</i> mutants. Thick red arrows in <b>D</b> and <b>I</b> point to anterior ectopic digits. Asterisks in E and J indicate digit tips of <i>Gli3</i>^<i>-/-</i> autopod. <b>K-O</b>: Expression pattern of <i>Shh</i> in hindlimb buds of indicated genotypes at E11.5. Black and red arrows point to normal and ectopic signals, respectively. <b>P-V</b>: <i>Sox9</i> in situ hybridization in hindlimbs of indicated genotypes at E12.5. Red arrows in <b>S</b> and <b>T</b> point to anterior ectopic digit condensation. Asterisks in <b>U</b> indicate distal tips of digit condensation. Red arrowheads in <b>V</b> point to distally-fused condensation.</p

Expression pattern of SHH targets and digit condensation in <i>Gata6</i> cKO; <i>Shh</i> allelic series.

<p>Expression pattern of <i>Gli1</i> (<b>A-D</b>), <i>Ptch1</i> (<b>E-H</b>) and <i>Sox9</i> (<b>I-L</b>) of wild type (<b>A, E, I</b>), <i>Gata6</i> cKO (<b>B, F, J</b>), <i>Gata6</i> cKO; <i>Shh</i>^<i>+/-</i> (<b>C, G, K</b>) and <i>Gata6</i> cKO; <i>Shh</i>^<i>-/-</i> (<b>D, H, L</b>) hindlimb buds. <b>A-H</b>: E11.5, <b>I-L</b>: E12.5. In <b>A</b>-<b>H</b>, black arrows and red arrows point to normal and ectopic signals, respectively. Blue arrowheads indicate loss of expression in <b>D</b> and <b>H</b>. In <b>I-L</b>, digit condensations are labeled as 1–5, and ectopic condensation is marked with red arrows.</p

Expression pattern of <i>Gli1</i>, <i>Ptch1</i> and <i>Pax9</i> in <i>Gata6</i>; <i>Gli3</i> allelic series.

<p>In situ hybridization of <i>Gli1</i> (<b>A-G</b>), <i>Ptch1</i> (<b>H-N</b>) and <i>Pax9</i> (<b>O-U</b>) of hindlimb buds of indicated genotypes at E11.5. Black and red arrows point to normal and ectopic signals, respectively. Blue arrows and arrowheads indicate reduced and loss of <i>Pax9</i> signals, respectively.</p