Two Distinct Modes of Signaling by Vascular Endothelial Growth Factor C Guide Blood and Lymphatic Vessel Patterning in Zebrafish: A Dissertation

Vascular Endothelial Growth Factor Receptor-3 (VEGFR3/Flt4) and its ligand Vegfc are necessary for development of both blood and lymphatic vasculature in vertebrates. In zebrafish, Vegfc/Flt4 signaling is essential for formation of arteries, veins, and lymphatic vessels. Interestingly, Flt4 appears to utilize distinct signaling pathways during the development of each of these vessels. To identify components of this pathway, we performed a transgenic haploid genetic screen in zebrafish that express EGFP under the control of a blood vessel specific promoter. As a result, we indentified a mutant allele of vascular endothelial growth factor c (vegfc), vegfcum18. vegfcum18 mutants display defects in vein and lymphatic vessel development but normal segmental artery (SeA) formation. Characterization of this allele led to the finding that the primary defect in vegfcum18 mutants was a general failure in vein and lymphatic vessel sprouting. Further genetic and biochemical analysis of this mutant revealed profound paracrine defects, which likely result in the observed loss of lymphatic and venous structures. Furthermore, double mutant analysis demonstrated that defects during SeA formation in vegfcum18 mutants were masked by inputs from the Vegfa signaling pathway. Endothelial cell autonomous expression of vegfcum18 induced angiogenic effects on blood vessels while endothelial cells lacking vegfc displayed defects in tip cell occupancy, suggesting a cell autonomous-autocrine role for Vegfc during developmental angiogenesis. Finally, we present genetic evidence that links processing of Vegfc by Furin during the formation of lymphatics in zebrafish. Together the data presented here suggest two discrete modes of signaling during blood and lymphatic vessel development, thus implying that regulation of Vegfc secretion and processing may play a pivotal role in the formation of these distinct vessel types in zebrafish

A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfc(um18) could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis

Inhibition of FGF receptor blocks adaptive resistance to RET inhibition in CCDC6-RET-rearranged thyroid cancer

Genetic alterations in RET lead to activation of ERK and AKT signaling and are associated with hereditary and sporadic thyroid cancer and lung cancer. Highly selective RET inhibitors have recently entered clinical use after demonstrating efficacy in treating patients with diverse tumor types harboring RET gene rearrangements or activating mutations. In order to understand resistance mechanisms arising after treatment with RET inhibitors, we performed a comprehensive molecular and genomic analysis of a patient with RET-rearranged thyroid cancer. Using a combination of drug screening and proteomic and biochemical profiling, we identified an adaptive resistance to RET inhibitors that reactivates ERK signaling within hours of drug exposure. We found that activation of FGFR signaling is a mechanism of adaptive resistance to RET inhibitors that activates ERK signaling. Combined inhibition of FGFR and RET prevented the development of adaptive resistance to RET inhibitors, reduced cell viability, and decreased tumor growth in cellular and animal models of CCDC6-RET-rearranged thyroid cancer

Hedgehog signaling via a calcitonin receptor-like receptor can induce arterial differentiation independently of VEGF signaling in zebrafish

Multiple signaling pathways control the specification of endothelial cells (ECs) to become arteries or veins during vertebrate embryogenesis. Current models propose that a cascade of Hedgehog (Hh), vascular endothelial growth factor (VEGF), and Notch signaling acts instructively on ECs to control the choice between arterial or venous fate. Differences in the phenotypes induced by Hh, VEGF, or Notch inhibition suggest that not all of the effects of Hh on arteriovenous specification are mediated by VEGF. We establish that full derepression of the Hh pathway in ptc1;ptc2 mutants converts the posterior cardinal vein into a second arterial vessel that manifests intact arterial gene expression, intersegmental vessel sprouting, and HSC gene expression. Importantly, although VEGF was thought to be absolutely essential for arterial fates, we find that normal and ectopic arterial differentiation can occur without VEGF signaling in ptc1;ptc2 mutants. Furthermore, Hh is able to bypass VEGF to induce arterial differentiation in ECs via the calcitonin receptor-like receptor, thus revealing a surprising complexity in the interplay between Hh and VEGF signaling during arteriovenous specification. Finally, our experiments establish a dual function of Hh during induction of runx1+ HSCs

Inhibition of FGF receptor blocks adaptive resistance to RET inhibition in CCDC6-RET-rearranged thyroid cancer.

Genetic alterations in RET lead to activation of ERK and AKT signaling and are associated with hereditary and sporadic thyroid cancer and lung cancer. Highly selective RET inhibitors have recently entered clinical use after demonstrating efficacy in treating patients with diverse tumor types harboring RET gene rearrangements or activating mutations. In order to understand resistance mechanisms arising after treatment with RET inhibitors, we performed a comprehensive molecular and genomic analysis of a patient with RET-rearranged thyroid cancer. Using a combination of drug screening and proteomic and biochemical profiling, we identified an adaptive resistance to RET inhibitors that reactivates ERK signaling within hours of drug exposure. We found that activation of FGFR signaling is a mechanism of adaptive resistance to RET inhibitors that activates ERK signaling. Combined inhibition of FGFR and RET prevented the development of adaptive resistance to RET inhibitors, reduced cell viability, and decreased tumor growth in cellular and animal models of CCDC6-RET-rearranged thyroid cancer

Stable Vascular Connections and Remodeling Require Full Expression of VE-Cadherin in Zebrafish Embryos

BACKGROUND: VE-cadherin is an endothelial specific, transmembrane protein, that clusters at adherens junctions where it promotes homotypic cell-cell adhesion. VE-cadherin null mutation in the mouse results in early fetal lethality due to altered vascular development. However, the mechanism of action of VE-cadherin is complex and, in the mouse embryo, it is difficult to define the specific steps of vascular development in which this protein is involved. METHODOLOGY AND PRINCIPAL FINDINGS: In order to study the role VE-cadherin in the development of the vascular system in a more suitable model, we knocked down the expression of the coding gene in zebrafish. The novel findings reported here are: 1) partial reduction of VE-cadherin expression using low doses of morpholinos causes vascular fragility, head hemorrhages and increase in permeability; this has not been described before and suggests that the total amount of the protein expressed is an important determinant of vascular stability; 2) concentrations of morpholinos which abrogate VE-cadherin expression prevent vessels to establish successful reciprocal contacts and, as a consequence, vascular sprouting activity is not inhibited. This likely explains the observed vascular hyper-sprouting and the presence of several small, collapsing vessels; 3) the common cardinal vein lacks a correct connection with the endocardium leaving the heart separated from the rest of the circulatory system. The lack of closure of the circulatory loop has never been described before and may explain some downstream defects of the phenotype such as the lack of a correct vascular remodeling. CONCLUSIONS AND SIGNIFICANCE: Our observations identify several steps of vascular development in which VE-cadherin plays an essential role. While it does not appear to regulate vascular patterning it is implicated in vascular connection and inhibition of sprouting activity. These processes require stable cell-cell junctions which are defective in absence of VE-cadherin. Notably, also partial modifications in VE-cadherin expression prevent the formation of a stable vasculature. This suggests that partial internalization or change of function of this protein may strongly affect vascular stability and organization

The ciliopathy gene cc2d2a controls zebrafish photoreceptor outer segment development through a role in Rab8-dependent vesicle trafficking

Ciliopathies are a genetically and phenotypically heterogeneous group of human developmental disorders whose root cause is the absence or dysfunction of primary cilia. Joubert syndrome is characterized by a distinctive hindbrain malformation variably associated with retinal dystrophy and cystic kidney disease. Mutations in CC2D2A are found in ∼10% of patients with Joubert syndrome. Here we describe the retinal phenotype of cc2d2a mutant zebrafish consisting of disorganized rod and cone photoreceptor outer segments resulting in abnormal visual function as measured by electroretinogram. Our analysis reveals trafficking defects in mutant photoreceptors affecting transmembrane outer segment proteins (opsins) and striking accumulation of vesicles, suggesting a role for Cc2d2a in vesicle trafficking and fusion. This is further supported by mislocalization of Rab8, a key regulator of opsin carrier vesicle trafficking, in cc2d2a mutant photoreceptors and by enhancement of the cc2d2a retinal and kidney phenotypes with partial knockdown of rab8. We demonstrate that Cc2d2a localizes to the connecting cilium in photoreceptors and to the transition zone in other ciliated cell types and that cilia are present in these cells in cc2d2a mutants, arguing against a primary function for Cc2d2a in ciliogenesis. Our data support a model where Cc2d2a, localized at the photoreceptor connecting cilium/transition zone, facilitates protein transport through a role in Rab8-dependent vesicle trafficking and fusion

HHEX is a transcriptional regulator of the VEGFC/FLT4/PROX1 signaling axis during vascular development.

Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis

Establishment of a Transgenic Zebrafish Line for Superficial Skin Ablation and Functional Validation of Apoptosis Modulators In Vivo

BACKGROUND: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)(cy17) (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR(+) fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR(+) signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR(+) fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+) fluorescent signaling. CONCLUSION/SIGNIFICANCE: The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo