SH2 domain protein E and ABL signaling regulate blood vessel size.

Blood vessels in different vascular beds vary in size, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. Src homology 2 domain containing E (She) protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vessel size in zebrafish embryos and human endothelial cell culture. Zebrafish she mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow, eventually leading to the DA collapse. Vascular endothelial specific overexpression of she resulted in a reduced diameter of the DA, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the she mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA size in she mutants correlated with an increased endothelial expression of claudin 5a (cldn5a), which encodes a protein enriched in tight junctions. Inhibition of cldn5a expression partially rescued the enlarged DA in she mutants, suggesting that She regulates DA size, in part, by promoting cldn5a expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates vessel and lumen size during vascular tubulogenesis.

Requirement of a novel gene, drish, in the zebrafish retinal ganglion cell and primary motor axon development.

BACKGROUND: During neurogenesis, growing axons must navigate through the complex extracellular environment and make correct synaptic connections for the proper functioning of neural circuits. The mechanisms underlying the formation of functional neural networks are still only partially understood. RESULTS: Here we analyzed the role of a novel gene si:ch73-364h19.1/drish in the neural and vascular development of zebrafish embryos. We show that drish mRNA is expressed broadly and dynamically in multiple cell types including neural, glial, retinal progenitor and vascular endothelial cells throughout the early stages of embryonic development. To study Drish function during embryogenesis, we generated drish genetic mutant using CRISPR/Cas9 genome editing. drish loss-of-function mutant larvae displayed defects in early retinal ganglion cell, optic nerve and the retinal inner nuclear layer formation, as well as ectopic motor axon branching. In addition, drish mutant adults exhibited deficient retinal outer nuclear layer and showed defective light response and locomotory behavior. However, vascular patterning and blood circulation were not significantly affected. CONCLUSIONS: Together, these data demonstrate important roles of zebrafish drish in the retinal ganglion cell, optic nerve and interneuron development and in spinal motor axon branching.

Ets1 functions partially redundantly with Etv2 to promote embryonic vasculogenesis and angiogenesis in zebrafish.

ETS transcription factors play an important role in the specification and differentiation of endothelial cells during vascular development. Despite previous studies, the role of the founding member of the ETS family, Ets1, in vascular development in vivo is only partially understood. Here, we generated a zebrafish ets1 mutant by TALEN genome editing and tested functional redundancy between Ets1 and a related ETS factor Etv2/Etsrp/ER71. While zebrafish ets1-/- mutants have a normal functional vascular system, etv2-/-;ets1-/embryos had more severe angiogenic defects and lower expression levels of kdr and kdrl, the two zebrafish homologs of the mammalian Vascular Endothelial Growth Factor Receptor 2 VEGFR2/Flk1, than etv2-/-embryos. Expression of constitutively active Mitogen-Activated Protein Kinase1 (MAP2K1) within endothelial cells partially rescued this angiogenic defect. Interestingly, ets1-/- embryos displayed extensive apoptosis within the trunk vasculature despite exhibiting normal vascular patterning. Loss of Ets1 combined with a partial knockdown of Etv2 function resulted in a decrease in endothelial cell numbers in the axial vasculature, which argues for a role of Ets1 in promoting vasculogenesis. We also demonstrate that although both Ets1 and Etv2 can induce ectopic vascular marker expression in zebrafish embryos, Ets1 activity is dependent on MAPK-mediated phosphorylation of its Thr30 and Ser33 residues, while Etv2 activity is not. Together, our results identify a novel function of Ets1 in regulating endothelial cell survival during vasculogenesis in vivo. Based on these findings, we propose a revised model of how Ets1 and Etv2 play unique and partially redundant roles to promote vascular development.

Integrin α5 and Integrin α4 cooperate to promote endocardial differentiation and heart morphogenesis.

Endocardium is critically important for proper function of the cardiovascular system. Not only does endocardium connect the heart to blood vasculature, it also plays an important role in heart morphogenesis, valve formation, and ventricular trabeculation. The extracellular protein Fibronectin (Fn1) promotes endocardial differentiation, but the signaling pathways downstream of Fn1 that regulate endocardial development are not understood. Here, we analyzed the role of the Fibronectin receptors Integrin alpha5 (Itga5) and Integrin alpha4 (Itga4) in zebrafish heart development. We show that itga5 mRNA is expressed in both endocardium and myocardium during early stages of heart development. Through analysis of both itga5 single mutants and itga4;itga5 double mutants, we show that loss of both itga5 and itga4 results in enhanced defects in endocardial differentiation and morphogenesis compared to loss of itga5 alone. Loss of both itga5 and itga4 results in cardia bifida and severe myocardial morphology defects. Finally, we find that loss of itga5 and itga4 results in abnormally narrow anterior endodermal sheet morphology. Together, our results support a model in which Itga5 and Itga4 cooperate to promote endocardial differentiation, medial migration of endocardial and myocardial cells, and morphogenesis of anterior endoderm.

Zebrafish etv2 knock-in line labels vascular endothelial and blood progenitor cells.

BACKGROUND: ETS transcription factor Etv2/Etsrp is one of the earliest markers for vascular and hematopoietic progenitors and functions as a key regulator of hematovascular development in multiple vertebrates, including zebrafish. Therefore, transgenic etv2 reporter lines provide a valuable tool to study vasculogenesis and hematopoiesis. However, previously generated zebrafish reporter lines do not fully recapitulate the endogenous pattern of etv2 expression. RESULTS: Here we used CRISPR/Cas9-mediated homology-independent DNA repair approach to knock-in a Gal4 transcriptional activator into the zebrafish etv2 genomic locus, thus generating etv2 ci32Gt gene trap line. etv2 ci32Gt ; UAS:GFP embryos show GFP expression in vascular endothelial, myeloid and red blood cells. Because gal4 insertion interrupts the etv2 locus, homozygous etv2 ci32Gt embryos display defects in vasculogenesis and myelopoiesis, and enable visualizing etv2-deficient hematovascular progenitors in live embryos. Furthermore, we performed differential transcriptome analysis of sorted GFP-positive cells from heterozygous and homozygous etv2 ci32Gt embryos. Approximately 500 downregulated genes were identified in etv2 ci32Gt homozygous embryos, which include multiple genes expressed in vascular endothelial and myeloid cells. CONCLUSIONS: The etv2 ci32Gt gene trap line and the data sets of misregulated genes will be valuable resources to study hematopoietic and vascular development.

ETS transcription factor Etsrp / Etv2 is required for lymphangiogenesis and directly regulates vegfr3 / flt4 expression.

The molecular mechanisms initiating the formation of the lymphatic system, lymphangiogenesis, are still poorly understood. Here we have identified a novel role in lymphangiogenesis for an ETS transcription factor, Etv2/Etsrp, a known regulator of embryonic vascular development. Through the use of fully validated photoactivatable morpholinos we show that inducible Etv2 inhibition in zebrafish embryos at 1 day post-fertilization (dpf) results in significant inhibition of lymphangiogenesis, while development of blood vessels is unaffected. In Etv2-inhibited embryos and larvae, the number of lymphatic progenitors is greatly reduced, the major lymphatic vessel, the thoracic duct, is absent or severely fragmented, and lymphangiogenesis-associated marker expression, including lyve1b, prox1a, and vegfr3/flt4, is strongly downregulated. We also demonstrate that lymphatic progenitors in Etv2 deficient embryos fail to respond to Vegfc signaling. Chromatin immunoprecipitation and sequencing (ChIP-Seq) studies using differentiated mouse embryonic stem (ES) cells as well as luciferase reporter studies in the ES cells and in zebrafish embryos argue that Etv2 directly binds the promoter/enhancer regions of Vegfc receptor Vegfr3/Flt4 and lymphatic marker Lyve1, and promotes their expression. Together these data support a model where Etv2 initiates lymphangiogenesis by directly promoting the expression of flt4 within the posterior cardinal vein.

Clec14a genetically interacts with Etv2 and Vegf signaling during vasculogenesis and angiogenesis in zebrafish.

BACKGROUND: C-lectin family 14 Member A (Clec14a) is a transmembrane protein specifically expressed in vascular endothelial cells during embryogenesis. Previous in vitro and in vivo studies have provided conflicting data regarding Clec14a role in promoting or inhibiting angiogenesis, therefore its functional role in vascular development remains poorly understood. RESULTS: Here we have generated a novel clec14a mutant allele in zebrafish embryos using TALEN genome editing. clec14a mutant embryos exhibit partial defects and delay in the sprouting of intersegmental vessels. These defects in angiogenesis are greatly increased upon the knockdown of a structurally related C1qr protein. Furthermore, a partial knockdown of an ETS transcription factor Etv2 results in a synergistic effect with the clec14a mutation and inhibits expression of early vascular markers in endothelial progenitor cells, arguing that clec14a is involved in promoting vasculogenesis. In addition, Clec14a genetically interacts with Vegfa signaling. A partial knockdown of Vegfaa function in the clec14a mutant background resulted in a synergistic inhibition of intersegmental vessel sprouting. CONCLUSIONS: These results argue that clec14a is involved in both vasculogenesis and angiogenesis, and suggest that Clec14a genetically interacts with Etv2 and Vegf signaling during vascular development in zebrafish embryos.

Wnt signaling positively regulates endothelial cell fate specification in the Fli1a-positive progenitor population via Lef1.

During vertebrate embryogenesis, vascular endothelial cells (ECs) and primitive erythrocytes become specified within close proximity in the posterior lateral plate mesoderm (LPM) from a common progenitor. However, the signaling cascades regulating the specification into either lineage remain largely elusive. Here, we analyze the contribution of ß-catenin dependent Wnt signaling to EC and erythrocyte specification during zebrafish embryogenesis. We generated novel ß-catenin dependent Wnt signaling reporters which, by using destabilized fluorophores (Venus-Pest, dGFP), specifically allow us to detect Wnt signaling responses in narrow time windows as well as in spatially restricted domains, defined by Cre recombinase expression (Tg(axin2BAC:Venus-Pest)mu288; Tg(14TCF:loxP-STOP-loxP-dGFP)mu202). We therefore can detect ß-catenin dependent Wnt signaling activity in a subset of the Fli1a-positive progenitor population. Additionally, we show that mesodermal Wnt3a-mediated signaling via the transcription factor Lef1 positively regulates EC specification (defined by kdrl expression) at the expense of primitive erythrocyte specification (defined by gata1 expression) in zebrafish embryos. Using mesoderm derived from human embryonic stem cells, we identified the same principle of Wnt signaling dependent EC specification in conjunction with auto-upregulation of LEF1. Our data indicate a novel role of ß-catenin dependent Wnt signaling in regulating EC specification during vasculogenesis.

Vegf signaling promotes vascular endothelial differentiation by modulating etv2 expression.

Vasculogenesis involves the differentiation of vascular endothelial progenitors de novo from undifferentiated mesoderm, their migration and coalescence to form the major embryonic vessels and the acquisition of arterial or venous identity. Vascular Endothelial Growth Factor (Vegf) signaling plays multiple roles during vascular development. However, its function during embryonic vasculogenesis has been controversial. Previous studies have implicated Vegf signaling in either regulating arteriovenous specification or overall vascular endothelial differentiation. To clarify the role of Vegf in embryonic vasculogenesis and identify its downstream targets, we used chemical inhibitors of Vegf receptor (Vegfr) signaling in zebrafish embryos as well as zebrafish genetic mutants. A high level of chemical inhibition of Vegfr signaling resulted in the reduction of overall vascular endothelial marker gene expression, including downregulation of both arterial and venous markers, ultimately leading to the apoptosis of vascular endothelial cells. In contrast, a low level of Vegfr inhibition specifically blocked arterial specification while the expression of venous markers appeared largely unaffected or increased. Inhibition of Vegfr signaling prior to the initiation of vasculogenesis reduced overall vascular endothelial differentiation, while inhibition of Vegfr signaling starting at mid-somitogenesis stages largely inhibited arterial specification. Conversely, Vegf overexpression resulted in the expansion of both arterial and pan-endothelial markers, while the expression of several venous-specific markers was downregulated. We further show that Vegf signaling affects overall endothelial differentiation by modulating the expression of the ETS transcription factor etv2/ etsrp. etv2 expression was downregulated in Vegfr- inhibited embryos, and expanded in Vegfaa-overexpressing embryos. Furthermore, vascular-specific overexpression of etv2 in Vegfr-inhibited embryos rescued defects in vascular endothelial differentiation. Similarly, vegfaa genetic mutants displayed a combination of the two phenotypes observed with chemical Vegfr inhibition: the expression of arterial and pan-endothelial markers including etv2 was downregulated while the expression of most venous markers was either expanded or unchanged. Based on these results we propose a revised model which explains the different phenotypes observed upon inhibition of Vegf signaling: low levels of Vegf signaling promote overall vascular endothelial differentiation and cell survival by upregulating etv2 expression, while high levels of Vegf signaling promote arterial and inhibit venous specification.

Myocardium and BMP signaling are required for endocardial differentiation.

Endocardial and myocardial progenitors originate in distinct regions of the anterior lateral plate mesoderm and migrate to the midline where they coalesce to form the cardiac tube. Endocardial progenitors acquire a molecular identity distinct from other vascular endothelial cells and initiate expression of specific genes such as nfatc1. Yet the molecular pathways and tissue interactions involved in establishing endocardial identity are poorly understood. The endocardium develops in tight association with cardiomyocytes. To test for a potential role of the myocardium in endocardial morphogenesis, we used two different zebrafish models deficient in cardiomyocytes: the hand2 mutant and a myocardial-specific genetic ablation method. We show that in hand2 mutants endocardial progenitors migrate to the midline but fail to assemble into a cardiac cone and do not express markers of differentiated endocardium. Endocardial differentiation defects were rescued by myocardial but not endocardial-specific expression of hand2. In metronidazole-treated myl7:nitroreductase embryos, myocardial cells were targeted for apoptosis, which resulted in the loss of endocardial nfatc1 expression. However, endocardial cells were present and retained expression of general vascular endothelial markers. We further identified bone morphogenetic protein (BMP) as a candidate myocardium-derived signal required for endocardial differentiation. Chemical and genetic inhibition of BMP signaling at the tailbud stage resulted in severe inhibition of endocardial differentiation while there was little effect on myocardial development. Heat-shock-induced bmp2b expression rescued endocardial nfatc1 expression in hand2 mutants and in myocardium-depleted embryos. Our results indicate that the myocardium is crucial for endocardial morphogenesis and differentiation, and identify BMP as a signal involved in endocardial differentiation.

Vegfa signaling promotes zebrafish intestinal vasculature development through endothelial cell migration from the posterior cardinal vein.

The mechanisms underlying organ vascularization are not well understood. The zebrafish intestinal vasculature forms early, is easily imaged using transgenic lines and in-situ hybridization, and develops in a stereotypical pattern thus making it an excellent model for investigating mechanisms of organ specific vascularization. Here, we demonstrate that the sub-intestinal vein (SIV) and supra-intestinal artery (SIA) form by a novel mechanism from angioblasts that migrate out of the posterior cardinal vein and coalesce to form the intestinal vasculature in an anterior to posterior wave with the SIA forming after the SIV. We show that vascular endothelial growth factor aa (vegfaa) is expressed in the endoderm at the site where intestinal vessels form and therefore likely provides a guidance signal. Vegfa/Vegfr2 signaling is required for early intestinal vasculature development with mutation in vegfaa or loss of Vegfr2 homologs causing nearly complete inhibition of the formation of the intestinal vasculature. Vegfc and Vegfr3 function, however, are dispensable for intestinal vascularization. Interestingly, ubiquitous overexpression of Vegfc resulted in an overgrowth of the SIV, suggesting that Vegfc is sufficient to induce SIV development. These results argue that Vegfa signaling directs endothelial cells to migrate out of existing vasculature and coalesce to form the intestinal vessels. It is likely that a similar mechanism is utilized during vascularization of other organs.

ETS transcription factors Etv2 and Fli1b are required for tumor angiogenesis.

ETS transcription factor ETV2/Etsrp functions as a key regulator of embryonic vascular development in multiple vertebrates. However, its role in pathological vascular development has not been previously investigated. To analyze its role in tumor angiogenesis, we utilized a zebrafish xenotransplantation model. Using a photoconvertible kdrl:NLS-KikGR line, we demonstrated that all tumor vessels originate from the existing embryonic vasculature by the mechanism of angiogenesis. Xenotransplantation of mouse B16 melanoma cells resulted in a significant increase in expression of the ETS transcription factors etv2 and fli1b expression throughout the embryonic vasculature. etv2 null mutants which undergo significant recovery of embryonic angiogenesis during later developmental stages displayed a strong inhibition of tumor angiogenesis. We utilized highly specific and fully validated photoactivatable morpholinos to inhibit Etv2 function after embryonic vasculogenesis has completed. Inducible inhibition of Etv2 function resulted in a significant reduction of tumor angiogenesis and inhibition of tumor growth. Furthermore, inducible inhibition of Etv2 function in fli1b mutant embryos resulted in even stronger reduction in tumor angiogenesis and growth, demonstrating that Etv2 and Fli1b have a partially redundant requirement during tumor angiogenesis. These results demonstrate the requirement for Etv2 and Fli1b in tumor angiogenesis and suggest that inhibition of these ETS factors may present a novel strategy to inhibit tumor angiogenesis and reduce tumor growth.

ETS transcription factors in embryonic vascular development.

At least thirteen ETS-domain transcription factors are expressed during embryonic hematopoietic or vascular development and potentially function in the formation and maintenance of the embryonic vasculature or blood lineages. This review summarizes our current understanding of the specific roles played by ETS factors in vasculogenesis and angiogenesis and the implications of functional redundancies between them.

Etv2 and fli1b function together as key regulators of vasculogenesis and angiogenesis.

OBJECTIVE: The E26 transformation-specific domain transcription factor Etv2/Etsrp/ER71 is a master regulator of vascular endothelial differentiation during vasculogenesis, although its later role in sprouting angiogenesis remains unknown. Here, we investigated in the zebrafish model a role for Etv2 and related E26 transformation-specific factors, Fli1a and Fli1b in developmental angiogenesis. APPROACH AND RESULTS: Zebrafish fli1a and fli1b mutants were obtained using transposon-mediated gene trap approach. Individual fli1a and fli1b homozygous mutant embryos display normal vascular patterning, yet the angiogenic recovery observed in older etv2 mutant embryos does not occur in embryos lacking both etv2 and fli1b. Etv2 and fli1b double-deficient embryos fail to form any angiogenic sprouts and show greatly increased apoptosis throughout the axial vasculature. In contrast, fli1a mutation did not affect the recovery of etv2 mutant phenotype. Overexpression analyses indicate that both etv2 and fli1b, but not fli1a, induce the expression of multiple vascular markers and of each other. Temporal inhibition of Etv2 function using photoactivatable morpholinos indicates that the function of Etv2 and Fli1b during angiogenesis is independent from the early requirement of Etv2 during vasculogenesis. RNA-Seq analysis and chromatin immunoprecipitation suggest that Etv2 and Fli1b share the same transcriptional targets and bind to the same E26 transformation-specific sites. CONCLUSIONS: Our data argue that there are 2 phases of early vascular development with distinct requirements of E26 transformation-specific transcription factors. Etv2 alone is required for early vasculogenesis, whereas Etv2 and Fli1b function redundantly during late vasculogenesis and early embryonic angiogenesis.

Distinct regulation of the anterior and posterior myeloperoxidase expression by Etv2 and Gata1 during primitive Granulopoiesis in zebrafish.

Neutrophilic granulocytes are the most abundant type of myeloid cells and form an essential part of the innate immune system. In vertebrates the first neutrophils are thought to originate during primitive hematopoiesis, which precedes hematopoietic stem cell formation. In zebrafish embryos, it has been suggested that primitive neutrophils may originate in two distinct sites, the anterior (ALPM) and posterior lateral plate mesoderm (PLPM). An ETS-family transcription factor Etsrp/Etv2/ER71 has been implicated in vasculogenesis and hematopoiesis in multiple vertebrates. However, its role during neutrophil development is not well understood. Here we demonstrate using zebrafish embryos that Etv2 has a specific cell-autonomous function during primitive neutropoiesis in the anterior lateral plate mesoderm (ALPM) but has little effect on erythropoiesis or the posterior lateral plate mesoderm (PLPM) expression of neutrophil marker myeloperoxidase mpo/mpx. Our results argue that ALPM-derived neutrophils originate from etv2-expressing cells which downregulate etv2 during neutropoiesis. We further show that Scl functions downstream of Etv2 in anterior neutropoiesis. Additionally, we demonstrate that mpx expression within the PLPM overlaps with gata1 expression, potentially marking the cells with a dual myelo-erythroid potential. Intriguingly, initiation of mpx expression in the PLPM is dependent on gata1 but not etv2 function. Our results demonstrate that mpx expression is controlled differently in the ALPM and PLPM regions and describe novel roles for etv2 and gata1 during primitive neutropoiesis.

Vascular endothelial and endocardial progenitors differentiate as cardiomyocytes in the absence of Etsrp/Etv2 function.

Previous studies have suggested that embryonic vascular endothelial, endocardial and myocardial lineages originate from multipotential cardiovascular progenitors. However, their existence in vivo has been debated and molecular mechanisms that regulate specification of different cardiovascular lineages are poorly understood. An ETS domain transcription factor Etv2/Etsrp/ER71 has been recently established as a crucial regulator of vascular endothelial differentiation in zebrafish and mouse embryos. In this study, we show that etsrp-expressing vascular endothelial/endocardial progenitors differentiate as cardiomyocytes in the absence of Etsrp function during zebrafish embryonic development. Expression of multiple endocardial specific markers is absent or greatly reduced in Etsrp knockdown or mutant embryos. We show that Etsrp regulates endocardial differentiation by directly inducing endocardial nfatc1 expression. In addition, Etsrp function is required to inhibit myocardial differentiation. In the absence of Etsrp function, etsrp-expressing endothelial and endocardial progenitors initiate myocardial marker hand2 and cmlc2 expression. Furthermore, Foxc1a function and interaction between Foxc1a and Etsrp is required to initiate endocardial development, but is dispensable for the inhibition of myocardial differentiation. These results argue that Etsrp initiates endothelial and endocardial, and inhibits myocardial, differentiation by two distinct mechanisms. Our findings are important for the understanding of genetic pathways that control cardiovascular differentiation during normal vertebrate development and will also greatly contribute to the stem cell research aimed at regenerating heart tissues.

Endocardium gives rise to blood cells in zebrafish embryos.

Previous studies have suggested that the endocardium contributes to hematopoiesis in murine embryos, although definitive evidence to demonstrate the hematopoietic potential of the endocardium is still missing. Here, we use a zebrafish embryonic model to test the emergence of hematopoietic progenitors from the endocardium. By using a combination of expression analysis, time-lapse imaging, and lineage-tracing approaches, we demonstrate that myeloid cells emerge from the endocardium in zebrafish embryos. Inhibition of Etv2/Etsrp or Scl/Tal1, two known master regulators of hematopoiesis and vasculogenesis, does not affect the emergence of endocardial-derived myeloid cells, while inhibition of Hedgehog signaling results in their reduction. Single-cell RNA sequencing analysis followed by experimental validation suggests that the endocardium is the major source of neutrophilic granulocytes. These findings will promote our understanding of alternative mechanisms involved in hematopoiesis, which are likely to be conserved between zebrafish and mammalian embryos.

Hedgehog signaling is required for differentiation of endocardial progenitors in zebrafish.

Endocardial cells form the inner endothelial layer of the heart tube, surrounded by the myocardium. Signaling pathways that regulate endocardial cell specification and differentiation are largely unknown and the origin of endocardial progenitors is still being debated. To study pathways that regulate endocardial differentiation in a zebrafish model system, we isolated zebrafish NFATc1 homolog which is expressed in endocardial but not vascular endothelial cells. We further demonstrate that Hedgehog (Hh) but not VegfA or Notch signaling is required for early endocardial morphogenesis. Pharmacological inhibition of Hh signaling with cyclopamine treatment resulted in nearly complete loss of the endocardial marker expression. Simultaneous knockdown of the two zebrafish sonic hedgehog homologs, shh and twhh or Hh co-receptor smoothened (smo) resulted in similar defects in endocardial morphogenesis. Inhibition of Hh signaling resulted in the loss of fibronectin (fn1) expression in the presumptive endocardial progenitors as early as the 10-somite stage which suggests that Hh signaling is required for the earliest stages of endocardial specification. We further show that the endoderm plays a critical role in migration but not specification or differentiation of the endocardial progenitors while notochord-derived Hh is a likely source for the specification and differentiation signal. Mosaic analysis using cell transplantation shows that Smo function is required cell-autonomously within endocardial progenitor cells. Our results argue that Hh provides a critical signal to induce the specification and differentiation of endocardial progenitors.

Lens regeneration in axolotl: new evidence of developmental plasticity.

BACKGROUND: Among vertebrates lens regeneration is most pronounced in newts, which have the ability to regenerate the entire lens throughout their lives. Regeneration occurs from the dorsal iris by transdifferentiation of the pigment epithelial cells. Interestingly, the ventral iris never contributes to regeneration. Frogs have limited lens regeneration capacity elicited from the cornea during pre-metamorphic stages. The axolotl is another salamander which, like the newt, regenerates its limbs or its tail with the spinal cord, but up until now all reports have shown that it does not regenerate the lens. RESULTS: Here we present a detailed analysis during different stages of axolotl development, and we show that despite previous beliefs the axolotl does regenerate the lens, however, only during a limited time after hatching. We have found that starting at stage 44 (forelimb bud stage) lens regeneration is possible for nearly two weeks. Regeneration occurs from the iris but, in contrast to the newt, regeneration can be elicited from either the dorsal or the ventral iris and, occasionally, even from both in the same eye. Similar studies in the zebra fish concluded that lens regeneration is not possible. CONCLUSIONS: Regeneration of the lens is possible in the axolotl, but differs from both frogs and newts. Thus the axolotl iris provides a novel and more plastic strategy for lens regeneration.

SH2 domain protein E (SHE) and ABL signaling regulate blood vessel size.

Blood vessels in different vascular beds vary in lumen diameter, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. Src homology 2 domain containing E (She) protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vascular lumen size in zebrafish embryos and human endothelial cell culture. Zebrafish she mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow. Vascular endothelial specific overexpression of she resulted in a reduced diameter of the DA lumen, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the she mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA lumen in she mutants correlated with an increased endothelial expression of claudin 5a and 5b (cldn5a / cldn5b), which encode proteins enriched in tight junctions. Inhibition of cldn5a expression partially rescued the enlarged DA in she mutants, suggesting that She regulates DA lumen size, in part, by promoting cldn5a expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates lumen size during vascular tubulogenesis.