Actin depolymerizing factors cofilin1 and destrin are required for ureteric bud branching morphogenesis.

The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis. While the molecular control of ureteric bud (UB) branching during kidney development has been extensively studied, the detailed cellular events underlying this process remain poorly understood. To gain insight into the role of actin cytoskeletal dynamics during renal branching morphogenesis, we studied the functional requirements for the closely related ADFs cofilin1 (Cfl1) and destrin (Dstn) during mouse development. Either deletion of Cfl1 in UB epithelium or an inactivating mutation in Dstn has no effect on renal morphogenesis, but simultaneous lack of both genes arrests branching morphogenesis at an early stage, revealing considerable functional overlap between cofilin1 and destrin. Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration. Animals with less severe combinations of mutant Cfl1 and Dstn alleles, which retain one wild-type Cfl1 or Dstn allele, display abnormalities including ureter duplication, renal hypoplasia, and abnormal kidney shape. The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching.

Core fucosylation is required for midline patterning during zebrafish development.

Complex carbohydrates represent one of the most polymorphic classes of macromolecules, but their functions during embryonic development remain poorly defined. Herein, we show that knockdown of FucT8, the fucosyltransferase responsible for adding an α1,6 fucosyl residue to the core region of N-linked oligosaccharides, results in defective midline patterning during zebrafish development. Reduced FucT8 expression leads to mild cyclopia, small forebrains, U-shaped somites, among other midline patterning defects. One of the principal FucT8 substrates was identified as Apolipoprotein B (ApoB), the major scaffold protein that is responsible for assembly and secretion of lipoprotein particles in vertebrates. In Drosophila, lipoprotein particles are thought to facilitate cell signaling by serving as a transport vehicle for lipid-modified cell signaling proteins, such as hedgehog. In this regard, knockdown of ApoB expression in zebrafish embryos leads to similar midline patterning defects as those seen in FucT8 morphant embryos. Furthermore, preliminary studies suggest that ApoB facilitates Sonic hedgehog signaling during zebrafish development, analogous to the function of lipoprotein particles during hedgehog signaling in Drosophila.

A beta1,4-galactosyltransferase is required for convergent extension movements in zebrafish.

Our understanding of how complex carbohydrates function during embryonic development is still very limited, primarily due to the large number of glycosyltransferases now known to be involved in their synthesis. To overcome these limitations, we have taken advantage of the zebrafish system to analyze the function of complex carbohydrates during development by down-regulating the expression of specific glycosyltransferases. Herein, we report the identification of the zebrafish ortholog of mammalian beta1,4-galactosyltransferase I, beta4GalT1, and its requirement for proper convergent extension movements during gastrulation. beta4GalT1 is expressed in the oocyte and throughout the embryo during the first 24 h of development. Knockdown of zebrafish beta4GalT1 by two independent morpholino oligonucleotides results in embryos with a truncated anterior-posterior axis, as well as elongated somites and moderate defects in the patterning of the head mesenchyme. Co-injection of zebrafish beta4GalT1 mRNA returns galactosyltransferase activity to control levels and rescues the defects produced by morpholino oligonucleotides. In situ hybridizations of various molecular markers reveal that the axial mesoderm of epiboly stage embryos is abnormally widened in beta4GalT1 morphants, indicative of abnormal convergent extension. Consistent with this, the rate of anterior-posterior axis elongation is reduced relative to control-injected embryos, similar to that seen in known convergent extension mutants. Among the many potential substrates for beta4GalT1 is laminin, a principle component of the extracellular matrix that supports cell movements such as those that occur during convergent extension. Previous in vitro studies have shown that the galactosylation status of laminin directly influences its ability to support cell spreading and migration. In this regard, laminin isolated from beta4GalT1 morphant embryos is poorly galactosylated, which may contribute to defective cell migration during convergent extension movements. This work demonstrates that zebrafish can be used to identify critical developmental roles for specific glycosyltransferases that would not be obvious otherwise, such as an absolute requirement for beta4GalT1 during convergent extension movements.

A beta1,4-galactosyltransferase is required for Bmp2-dependent patterning of the dorsoventral axis during zebrafish embryogenesis.

Complex carbohydrates are highly polymorphic macromolecules that are involved in diverse biological processes; however, a detailed understanding of their function remains obscure. To better define the roles of complex carbohydrates during vertebrate embryogenesis, we have initiated an analysis of glycosyltransferase function using the zebrafish system. In this study, we report the characterization of a zebrafish beta1,4-galactosyltransferase (GalT), which has substantial homology with mammalian beta4GalT5 and is expressed zygotically throughout the zebrafish embryo. Downregulating the expression of beta4GalT5 by injection of specific morpholino oligonucleotides results in dorsalized zebrafish embryos, suggesting a role of beta4GalT5 in Bmp2-mediated specification of the dorsoventral axis. Consistent with this, morpholino-injected embryos have ventrally expanded chordin expression and reduced activation of the Bmp-dependent transcription factors Smad1/5/8. Because other growth factors, such as Egf and Fgf, require binding to extracellular proteoglycans for delivery and/or binding to their cognate receptors, we examined whether proteoglycans isolated from control and morpholino-injected embryos show differential binding affinities for Bmp2. In this regard, proteoglycans isolated from beta4GalT5 morphant embryos are underglycosylated and are unable to bind recombinant Bmp2 as efficiently as proteoglycans from control-injected embryos, whereas the binding of Bmp7 is relatively unaffected. These results suggest that beta4GalT5 is a previously unidentified zebrafish galactosyltransferase that is essential for proper patterning of the dorsoventral axis by regulating Bmp2 signaling. Furthermore, this work demonstrates that a relatively simple carbohydrate modification to endogenous proteoglycans can modulate the specificity of cytokine signaling.