Specification of an anterior neuroectoderm patterning by Frizzled8a-mediated Wnt8b signalling during late gastrulation in zebrafish

Wnts have been shown to provide a posteriorizing signal that has to be repressed in the anterior neuroectoderm for normal anteroposterior (AP) patterning. We have previously identified a zebrafish frizzled8a (fz8a) gene expressed in the presumptive anterior neuroectoderm as well as prechordal plate at the late gastrula stage. We have investigated the role of Fz8a-mediated Wnt8b signalling in anterior brain patterning in zebrafish. We show that in zebrafish embryos: (1) Wnt signalling has at least two different stage-specific posteriorizing activities in the anterior neuroectoderm, one before mid-gastrulation and the other at late gastrulation; (2) Fz8a plays an important role in mediating anterior brain patterning; (3) Wnt8b and Fz8a can functionally interact to transmit posteriorizing signals that determine the fate of the posterior diencephalon and midbrain in late gastrula embryos; and (4) Wnt8b can suppress fz8a expression in the anterior neuroectoderm and potentially affect the level and/or range of Wnt signalling. In conclusion, we suggest that a gradient of Fz8a-mediated Wnt8b signalling may play crucial role in patterning the posterior diencephalon and midbrain regions in the late gastrula.     

Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish

The mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to result from changes in gene expression via the aryl hydrocarbon receptor (AHR). The induction of cytochrome P450 1A (CYP1A) in various organs is a cardinal effect of TCDD. However, whether CYP1A is involved in endpoints of TCDD toxicity is controversial. We investigated the role of CYP1A in TCDD-induced developmental toxicities using gene knock-down with morpholino antisense oligos. Exposure of zebrafish embryos to TCDD, at concentrations eliciting the hallmark endpoints of developmental toxicity, induced CYP1A in the heart and vascular endothelium throughout the body. This induction by TCDD was markedly inhibited by morpholinos to zebrafish arylhydrocarbon receptor 2 (zfAHR2-MO) and to zebrafish CYP1A (zfCYP1A-MO). The zfAHR2-MO but not the zfCYP1A-MO inhibited zfCYP1A mRNA expression, indicating the specificities of these morpholinos. Injection of either zfAHR2-MO or zfCYP1A-MO blocked the representative signs of TCDD developmental toxicity in zebrafish, pericardial edema and trunk circulation failure. The morpholinos appeared do not affect normal development in TCDD-untreated embryos. These results suggest a mediatory role of zfCYP1A induction through zfAHR2 activation in causing circulation failure by TCDD in zebrafish. This is the first molecular evidence demonstrating an essential requirement for CYP1A induction in TCDD-evoked developmental toxicities in any vertebrate species.     

Evidence for Involvement of Wnt Signalling in Body Polarities,Cell Proliferation,and the Neuro-Sensory System in an Adult Ctenophore

Signalling through the Wnt family of secreted proteins originated in a common metazoan ancestor and greatly influenced the evolution of animal body plans. In bilaterians, Wnt signalling plays multiple fundamental roles during embryonic development and in adult tissues, notably in axial patterning, neural development and stem cell regulation. Studies in various cnidarian species have particularly highlighted the evolutionarily conserved role of the Wnt/β-catenin pathway in specification and patterning of the primary embryonic axis. However in another key non-bilaterian phylum, Ctenophora, Wnts are not involved in early establishment of the body axis during embryogenesis. We analysed the expression in the adult of the ctenophore Pleurobrachia pileus of 11 orthologues of Wnt signalling genes including all ctenophore Wnt ligands and Fz receptors and several members of the intracellular β-catenin pathway machinery. All genes are strongly expressed around the mouth margin at the oral pole, evoking the Wnt oral centre of cnidarians. This observation is consistent with primary axis polarisation by the Wnts being a universal metazoan feature, secondarily lost in ctenophores during early development but retained in the adult. In addition, local expression of Wnt signalling genes was seen in various anatomical structures of the body including in the locomotory comb rows, where their complex deployment suggests control by the Wnts of local comb polarity. Other important contexts of Wnt involvement which probably evolved before the ctenophore/cnidarian/bilaterian split include proliferating stem cells and progenitors irrespective of cell types, and developing as well as differentiated neuro-sensory structures.     

Expression of wnt and frizzled genes during early sea star development

The Wnt signaling pathway is highly conserved across metazoa and has pleiotropic functions in the development of many animals. Binding of a secreted Wnt ligand to its Frizzled (Fz) receptor activates Dishevelled, which then drives one of three major signaling cascades, canonical (β-catenin), calcium, or planar cell polarity signaling. These pathways have distinct developmental effects and function in different processes in different organisms. Here we report the expression of six wnt and three fz genes during embryogenesis of the sea star, Patiria miniata, as a first step in uncovering the roles of Wnt signaling in the development of this organism. wnt3, wnt4, wnt8, and wnt16 are expressed in nested domains in the endoderm and lateral ectoderm from blastula through late gastrula stages; wnt2 and wnt5 are expressed in the mesoderm and anterior endoderm. Expression of different fz paralogs is detected in the mesoderm; posterior endoderm and ectoderm; and anterior ectoderm. Taken together, this suggests that Wnt signaling can occur throughout most of the embryo and may therefore play multiple roles during sea star development.     

Expression of wnt and frizzled genes during early sea star development

The Wnt signaling pathway is highly conserved across metazoa and has pleiotropic functions in the development of many animals. Binding of a secreted Wnt ligand to its Frizzled (Fz) receptor activates Dishevelled, which then drives one of three major signaling cascades, canonical (β-catenin), calcium, or planar cell polarity signaling. These pathways have distinct developmental effects and function in different processes in different organisms. Here we report the expression of six wnt and three fz genes during embryogenesis of the sea star, Patiria miniata, as a first step in uncovering the roles of Wnt signaling in the development of this organism. wnt3, wnt4, wnt8, and wnt16 are expressed in nested domains in the endoderm and lateral ectoderm from blastula through late gastrula stages; wnt2 and wnt5 are expressed in the mesoderm and anterior endoderm. Expression of different fz paralogs is detected in the mesoderm; posterior endoderm and ectoderm; and anterior ectoderm. Taken together, this suggests that Wnt signaling can occur throughout most of the embryo and may therefore play multiple roles during sea star development.     

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to “off-target” effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.     

Distinct roles for Fgf,Wnt and retinoic acid in posteriorizing the neural ectoderm

Early neural patterning in vertebrates involves signals that inhibit anterior (A) and promote posterior (P) positional values within the nascent neural plate. In this study, we have investigated the contributions of, and interactions between, retinoic acid (RA), Fgf and Wnt signals in the promotion of posterior fates in the ectoderm. We analyze expression and function of cyp26/P450RAI, a gene that encodes retinoic acid 4-hydroxylase, as a tool for investigating these events. Cyp26 is first expressed in the presumptive anterior neural ectoderm and the blastoderm margin at the late blastula. When the posterior neural gene hoxb1b is expressed during gastrulation, it shows a strikingly complementary pattern to cyp26. Using these two genes, as well as otx2 and meis3 as anterior and posterior markers, we show that Fgf and Wnt signals suppress expression of anterior genes, including cyp26. Overexpression of cyp26 suppresses posterior genes, suggesting that the anterior expression of cyp26 is important for restricting the expression of posterior genes. Consistent with this, knock-down of cyp26 by morpholino oligonucleotides leads to the anterior expansion of posterior genes. We further show that Fgf- and Wnt-dependent activation of posterior genes is mediated by RA, whereas suppression of anterior genes does not depend on RA signaling. Fgf and Wnt signals suppress cyp26 expression, while Cyp26 suppresses the RA signal. Thus, cyp26 has an important role in linking the Fgf, Wnt and RA signals to regulate AP patterning of the neural ectoderm in the late blastula to gastrula embryo in zebrafish.     

Left‐right asymmetry in the chick embryo requires core planar cell polarity protein Vangl2

Consistent left‐right patterning is a fascinating and biomedically important problem. In the chick embryo, it is not known how cells determine their position (left or right) relative to the primitive streak, which is required for subsequent asymmetric gene expression cascades. We show that the subcellular localization of Vangl2, a core planar cell polarity (PCP) protein, is consistently polarized, giving cells in the blastoderm a vector pointing toward the primitive streak. Moreover, morpholino‐mediated loss‐of‐function of Vangl2 by electroporation into chicks at very early stages randomizes the normally left‐sided expression of Sonic hedgehog. Strikingly, Vangl2 morpholinos also induce a desynchronization of asymmetric gene expression within the left and right domains of Hensen's node. These data reveal the existence of polarized planar cell polarity protein localization in gastrulating chick and demonstrate that the PCP pathway is functionally required for normal asymmetry in the chick upstream of Sonic hedgehog. These data suggest a new and widely applicable class of models for the spread and coordination of left‐right patterning information in the embryonic blastoderm. genesis 47:719–728, 2009. © 2009 Wiley‐Liss, Inc.     

Combinatorial activity of Flamingo proteins directs convergence and extension within the early zebrafish embryo via the planar cell polarity pathway

The seven-transmembrane protocadherin, Flamingo, functions in a number of processes during Drosophila development, including planar cell polarity (PCP). To assess the role(s) of Flamingo1/Celsr1 (Fmi1) during vertebrate embryogenesis we have exploited the zebrafish system, identifying two Fmi1 orthologues (zFmi1a and zFmi1b) and employing morpholinos to induce mis-splicing of zebrafish fmi1 mRNAs, to both imitate mutations identified in Drosophila flamingo and generate novel aberrant Flamingo proteins. We demonstrate that in the zebrafish gastrula, Fmi1 proteins function in concert with each other and with the vertebrate PCP proteins, Wnt11 and Strabismus, to mediate convergence and extension during gastrulation, without altering early dorso-ventral patterning. We show that zebrafish Fmi1a promotes extension of the entire antero-posterior axis of the zebrafish gastrula including prechordal plate and ventral diencephalic precursors. However, while we show that control over axial extension is autonomous, we find that Fmi1a is not required within lateral cells undergoing dorsal convergence.     

Cloning and developmental expression of a novel, secreted frizzled-related protein from the sea urchin, Strongylocentrotus purpuratus

Wnt proteins and their receptors, members of the frizzled protein family, play a key role in regulating a wide range of developmental processes. Recently, putative regulators of Wnt signaling known as secreted frizzled-related proteins (SFRPs) have been identified in several vertebrates. Here, we describe the cloning of a novel SFRP (suSFRP1) from the sea urchin, Strongylocentrotus purpuratus. SuSFRP1 contains a putative signal sequence, four cysteine-rich domains and a single Ig domain. The developmental expression of suSFRP1 mRNA is highly dynamic and can be separated into three phases: (1) abrupt accumulation in most or all cells of the embryo at the early blastula stage; (2) restriction of expression to the prospective endoderm and animal pole region of the gastrula; and (3) expression in prospective muscle cells of the coelomic pouches during late embryogenesis.     

TALE-family homeodomain proteins regulate endodermal sonic hedgehog expression and pattern the anterior endoderm

sonic hedgehog (shh) is expressed in anterior endoderm, where it is required to repress pancreas gene expression and to pattern the endoderm, but the pathway controlling endodermal shh expression is unclear. We find that expression of meis3, a TALE class homeodomain gene, coincides with shh expression in the endoderm of zebrafish embryos. Using a dominant negative construct or anti-sense morpholino oligos (MOs) to disrupt meis3 function, we observe ectopic insulin expression in anterior endoderm. This phenotype is also observed when meis3 MOs are targeted to the endoderm, suggesting that meis3 acts within the endoderm to restrict insulin expression. We also find that meis3 is required for endodermal shh expression, indicating that meis3 acts upstream of shh to restrict insulin expression. Loss of pbx4, a TALE gene encoding a Meis cofactor, produces the same phenotype as loss of meis3, consistent with Meis3 acting in a complex with Pbx4 as reported in other systems. Lastly, we observe a progressive anterior displacement of endoderm-derived organs upon disruption of meis3 or pbx4, apparently as a result of underdevelopment of the pharyngeal region. Our data indicate that meis3 and pbx4 regulate shh expression in anterior endoderm, thereby influencing patterning and growth of the foregut.     

Sequential antagonism of early and late Wnt-signaling by zebrafish colgate promotes dorsal and anterior fates

The establishment of the vertebrate body plan involves patterning of the ectoderm, mesoderm, and endoderm along the dorsoventral and antero-posterior axes. Interactions among numerous signaling molecules from several multigene families, including Wnts, have been implicated in regulating these processes. Here we provide evidence that the zebrafish colgate(b382) (col) mutation results in increased Wnt signaling that leads to defects in dorsal and anterior development. col mutants display early defects in dorsoventral patterning manifested by a decrease in the expression of dorsal shield-specific markers and ectopic expression of ventrolaterally expressed genes during gastrulation. In addition to these early patterning defects, col mutants display a striking regional posteriorization within the neuroectoderm, resulting in a reduction in anterior fates and an expansion of posterior fates within the forebrain and midbrain-hindbrain regions. We are able to correlate these phenotypes to the overactivation of Wnt signaling in col mutants. The early dorsal and anterior patterning phenotypes of the col mutant embryos are selectively rescued by inactivation of Wnt8 function by morpholino translational interference. In contrast, the regionalized neuroectoderm posterioriorization phenotype is selectively rescued by morpholino-mediated inactivation of Wnt8b. These results suggest that col-mediated antagonism of early and late Wnt-signaling activity during gastrulation is normally required sequentially for both early dorsoventral patterning and the specification and patterning of regional fates within the anterior neuroectoderm.