Search

Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a β-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.

Background: The expression of the Prospero homeodomain transcription factor (Prox1) in a subset of cardinal venous cells specifies the lymphatic lineage in mice. Prox1 is also indispensible for the maintenance of lymphatic cell fate, and is therefore considered a master control gene for lymphangiogenesis in mammals. In zebrafish, there are two prox1 paralogues, the previously described prox1 (also known as prox1a) and the newly identified prox1b. Principal Findings: To investigate the role of the prox1b gene in zebrafish lymphangiogenesis, we knocked-down prox1b and found that depletion of prox1b mRNA did not cause lymphatic defects. We also generated two different prox1b mutant alleles, and maternal-zygotic homozygous mutant embryos were viable and did not show any lymphatic defects. Furthermore, the expression of prox1b was not restricted to lymphatic vessels during zebrafish development. Conclusion: We conclude that Prox1b activity is not essential for embryonic lymphatic development in zebrafish.

More than 100 years ago, Florence Sabin suggested that lymphatic vessels develop by sprouting from preexisting blood vessels, but it is only over the past decade that the molecular mechanisms underpinning lymphatic vascular development have begun to be elucidated. Genetic manipulations in mice have identified a transcriptional hub comprised of Prox1, CoupTFII, and Sox18 that is essential for lymphatic endothelial cell fate specification. Recent work has identified a number of additional transcription factors that regulate later stages of lymphatic vessel differentiation and maturation. This review highlights recent advances in our understanding of the transcriptional control of lymphatic vascular development and reflects on efforts to better understand the activities of transcriptional networks during this discrete developmental process. Finally, we highlight the transcription factors associated with human lymphatic vascular disorders, demonstrating the importance of understanding how the activity of these key molecules is regulated, with a view toward the development of innovative therapeutic avenues. Refereed/Peer-reviewed

Objective— To study whether Notch signaling, which regulates cell fate decisions and vessel morphogenesis, controls lymphatic development. Methods and Results— In zebrafish embryos, sprouts from the axial vein have lymphangiogenic potential because they give rise to the first lymphatics. Knockdown of delta-like-4 (Dll4) or its receptors Notch-1b or Notch-6 in zebrafish impaired lymphangiogenesis. Dll4/Notch silencing reduced the number of sprouts producing the string of parchordal lymphangioblasts; instead, sprouts connecting to the intersomitic vessels were formed. At a later phase, Notch silencing impaired navigation of lymphatic intersomitic vessels along their arterial templates. Conclusion— These studies imply critical roles for Notch signaling in the formation and wiring of the lymphatic network.

Lymphedema, lymphangiectasias, mental retardation and unusual facial characteristics define the autosomal recessive Hennekam syndrome. Homozygosity mapping identified a critical chromosomal region containing CCBE1, the human ortholog of a gene essential for lymphangiogenesis in zebrafish. Homozygous and compound heterozygous mutations in seven subjects paired with functional analysis in a zebrafish model identify CCBE1 as one of few genes causing primary generalized lymph-vessel dysplasia in humans.

In the past, microinjection of plasmid DNA into early embryos represented the state of the art to generate transgenic zebrafish. However, this approach suffers significant drawbacks (mosaic distribution of the injected transgene, late transgene integration at high copy numbers, low transgenesis frequency), making the generation of transgenic lines a laborious task. Coinjection of I-SceI meganuclease with a reporter construct flanked by I-SceI sites overcomes these problems by earlier transgene integration into the host genome. Here, we provide an optimized protocol for I-SceI meganuclease-mediated transgenesis in zebrafish. This simple protocol provides a reliable method to transiently test tissue-specific reporter expression of meganuclease constructs in injected embryos (F0). Furthermore, it substantially facilitates the generation of multiple stable transgenic lines increasing transgenesis frequencies up to 45%, compared with 5% without I-SceI. The reliable reporter activity in F0 and the improved transgenesis frequency make this protocol a powerful tool for use in gain- and loss-of-function, cell tracing, and cell labeling experiments.

In the past, microinjection of plasmid DNA into early embryos represented the state of the art to generate transgenic zebrafish. However, this approach suffers significant drawbacks (mosaic distribution of the injected transgene, late transgene integration at high copy numbers, low transgenesis frequency), making the generation of transgenic lines a laborious task. Coinjection of I-SceI meganuclease with a reporter construct flanked by I-SceI sites overcomes these problems by earlier transgene integration into the host genome. Here, we provide an optimized protocol for I-SceI meganuclease-mediated transgenesis in zebrafish. This simple protocol provides a reliable method to transiently test tissue-specific reporter expression of meganuclease constructs in injected embryos (F0). Furthermore, it substantially facilitates the generation of multiple stable transgenic lines increasing transgenesis frequencies up to 45%, compared with 5% without I-SceI. The reliable reporter activity in F0 and the improved transgenesis frequency make this protocol a powerful tool for use in gain- and loss-of-function, cell tracing, and cell labeling experiments.

The development of arteries, veins and lymphatics from pre-existing vessels are intimately linked processes controlled by a number of well-studied reiteratively acting signalling pathways. To delineate the mechanisms governing vessel formation in vivo, we performed a forward genetic screen in zebrafish and isolated the mutant expando. Molecular characterisation revealed a loss-of-function mutation in the highly conserved kinase insert region of flt4. Consistent with previous reports, flt4 mutants were deficient in lymphatic vascular development. Recent studies have demonstrated a role for Flt4 in blood vessels and showed that Dll4 limits angiogenic potential by limiting Flt4 function in developing blood vessels. We found that arterial angiogenesis proceeded normally, yet the dll4 loss-of-function arterial hyperbranching phenotype was rescued, in flt4 signalling mutants. Furthermore, we found that the Flt4 ligand Vegfc drives arterial hyperbranching in the absence of dll4. Upon knockdown of dll4, intersegmental arteries were sensitised to increased vegfc levels and the overexpression of dll4 inhibited Vegfc/Flt4-dependent angiogenesis events. Taken together, these data demonstrate that dll4 functions to suppress the ability of developing intersegmental arteries to respond to Vegfc-driven Flt4 signalling in zebrafish. We propose that this mechanism contributes to the differential response of developing arteries and veins to a constant source of Vegfc present in the embryo during angiogenesis.

The discovery of new genes, genetic pathways and drug targets involved in the formation of lymphatic vessels and capillaries has long been hampered by the absence of a small model organism amenable to large scale genetic and pharmacological screening. The recent description of functional and conserved lymphatic vascular systems in the two small animal models Xenopus laevis (the African clawed frog) and Danio rerio (the zebrafish) now opens up the possibility to exploit these models for the study of developmental lymphangiogenesis. In this chapter we will describe the discovery and characterisation of the lymphatic vasculature in both frog and fish models. We will also describe and compare the available genetic tools for the study of lymphangiogenesis in these new model systems.

The rapid embryonic development and high fecundity of zebrafish contribute to the great advantages of this model for the study of developmental genetics. Transient disruption of the normal function of a gene during development can be achieved by microinjecting mRNA, DNA or short chemically stabilized anti-sense oligomers, called morpholinos (MOs), into early zebrafish embryos. The ensuing develop ment of the microinjected embryos is observed over the following hours and days to analyze the impact of the microinjected products on embryogenesis. Compared to stable reverse genetic approaches (sta ble transgenesis, targeted mutants recovered by TILLING), these transient reverse genetic approaches are vastly quicker, relatively affordable, and require little animal facility space. Common applications of these methodologies allow analysis of gain-of-function (gene overexpression or dominant active), loss-of-function (gene knock down or dominant negative), mosaic analysis, lineage-restricted studies and cell tracing experiments. The use of these transient approaches for the manipulation of gene expression has improved our understanding of many key developmental pathways including both the Wnt/beta-catenin and Wnt/PCP pathways, as covered in some detail in Chapter 17 of this book. This chapter describes the most common and versatile approaches: gain of function and loss of function using DNA and mRNA injections and loss of function using MOs.

Cerebral cavernous malformations (CCMs) are a prevalent class of vascular anomalies characterized by thin-walled clusters of malformed blood vessels in the brain. Heritable forms are caused by mutations in CCM1, CCM2 and CCM3, but despite the importance of these factors in vascular biology, an understanding of their molecular and cellular functions remains elusive. Here we describe the characterization of a zebrafish embryonic model of CCM. Loss of ccm1 in zebrafish embryos leads to severe and progressive dilation of major vessels, despite normal endothelial cell fate and number. Vascular dilation in ccm1 mutants is accompanied by progressive spreading of endothelial cells and thinning of vessel walls despite ultrastructurally normal cell-cell contacts. Zebrafish ccm2 mutants display comparable vascular defects. Finally, we show that ccm1 function is cell autonomous, suggesting that it is endothelial cellular morphogenesis that is regulated by CCM proteins during development and pathogenesis.

The rapid embryonic development and high fecundity of zebrafish contribute to the great advantages of this model for the study of developmental genetics. Transient disruption of the normal function of a gene during development can be achieved by microinjecting mRNA, DNA or short chemically stabilized anti-sense oligomers, called morpholinos (MOs), into early zebrafish embryos. The ensuing develop ment of the microinjected embryos is observed over the following hours and days to analyze the impact of the microinjected products on embryogenesis. Compared to stable reverse genetic approaches (sta ble transgenesis, targeted mutants recovered by TILLING), these transient reverse genetic approaches are vastly quicker, relatively affordable, and require little animal facility space. Common applications of these methodologies allow analysis of gain-of-function (gene overexpression or dominant active), loss-of-function (gene knock down or dominant negative), mosaic analysis, lineage-restricted studies and cell tracing experiments. The use of these transient approaches for the manipulation of gene expression has improved our understanding of many key developmental pathways including both the Wnt/beta-catenin and Wnt/PCP pathways, as covered in some detail in Chapter 17 of this book. This chapter describes the most common and versatile approaches: gain of function and loss of function using DNA and mRNA injections and loss of function using MOs.

Neural stem/progenitor cells (NPCs) self-renew and differentiate, generating neuronal and non-neuronal (glial) cell lineages. Although a number of factors, including transcription factors, have been shown to be important in the regulation of NPC proliferation and differentiation, the precise molecular networks remain to be identified. The cAMP Response Element-Binding protein (CREB) is a transcription factor important for neuronal survival, differentiation and plasticity. Recent work suggests that CREB activation, via serine phosphorylation in the kinase inducible domain, is important for neurogenesis in the adult rodent brain. We sought to further investigate CREB function in neurogenesis, using the zebrafish (Danio rerio). Structural and functional analysis of the zebrafish CREB orthologue showed high conservation with mammalian CREB. Activated (phosphorylated) CREB (pCREB) was localised to all known proliferation zones in the adult zebrafish brain, including actively cycling cells. Furthermore, we found that modulating CREB activity during early zebrafish development caused significant defects in neural proliferation, midbrain–hindbrain organization and body patterning. These findings reveal broader and stage-specific physiological roles of CREB function during vertebrate neural development and proliferation.

In separate expression pattern and micro-array screens the zinc finger containing factor, znfl2, has been previously implicated in hematopoiesis. Here we analysed znfl2 expression in detail and performed genetic epistatic analysis in a series of hematopoietic mutants and transient gain-of-function models. znfl2 expression in the hematopoietic intermediate mesoderm and derived erythrocytes required early genes cloche and spadetail, but not gata1. Expression was up-regulated in scl gain-of-function embryos, identifying znfl2 as an early erythroid factor that is regulated upstream or independently of gata1. Furthermore, we identified a duplicate znfl2 gene in the genome (znfl2b) which was expressed in early mesendoderm and weakly in the lateral plate mesoderm, overlapping in expression with znfl2. The production of loss-of-function models for znfl2, znfl2b and znfl2/znfl2b together suggested that these erythrocyte specific zinc finger genes are dispensible for erythropoiesis.

PTH plays a critical role in calcium metabolism in tetrapods. The primary site of PTH expression is the parathyroid glands, although it is also detected in the thymus and hypothalamus. Fish lack anatomically distinct parathyroid glands, and the first animals to evolve parathyroid glands were the amphibians. However, fish do have PTH family ligands and receptors, which are functionally similar to their mammalian counterparts. We report the expression patterns of duplicate zebrafish pth genes during embryogenesis. Both zebrafish pth1 and pth2 transcripts are expressed along the lateral line before the migration of the lateral line primordium and later in development Pth protein is detected in lateral line neuromasts by immunohistochemistry. pth1 Transcripts are also detected in the central nervous system in the ventral neural tube. These temporally and anatomically restricted expression patterns imply a novel role for PTH family hormones during embryonic development of the zebrafish and allow for the genetic dissection of PTH function in this model organism.

The formation of the vascular network requires a tightly controlled balance of pro-angiogenic and stabilizing signals. Perturbation of this balance can result in dysregulated blood vessel morphogenesis and drive pathologies including cancer. Here, we have identified a novel gene, ARHGAP18, as an endogenous negative regulator of angiogenesis, limiting pro-angiogenic signaling and promoting vascular stability. Loss of ARHGAP18 promotes EC hypersprouting during zebrafish and murine retinal vessel development and enhances tumor vascularization and growth. Endogenous ARHGAP18 acts specifically on RhoC and relocalizes to the angiogenic and destabilized EC junctions in a ROCK dependent manner, where it is important in reaffirming stable EC junctions and suppressing tip cell behavior, at least partially through regulation of tip cell genes, Dll4, Flk-1 and Flt-4. These findings highlight ARHGAP18 as a specific RhoGAP to fine tune vascular morphogenesis, limiting tip cell formation and promoting junctional integrity to stabilize the angiogenic architecture.