Vitamin A is an essential nutrient; it is not synthesized by mammals and therefore must be obtained through the diet. During times of fasting or dietary vitamin A insufficiency, retinol, the alcohol form of the vitamin is released from the liver, its main storage tissue, for circulation in complex with retinol-binding protein 4 (RBP) to provide an adequate supply to peripheral tissues. Stimulated by Retinoic Acid 6 (STRA6), the transmembrane RBP receptor, mediates retinol uptake across blood-tissue barriers such as the retinal pigment epithelium of the eye, the placenta and the choroid plexus of the brain. Our understanding as to how this protein functions has been greatly enhanced by the high-resolution 3D structure of zebrafish STRA6 in complex with calmodulin (CaM) solved by single-particle cryogenic-electron microscopy. However, the nature of the interaction of STRA6 with retinol remains unclear. Here, I present the high-resolution structures of zebrafish and sheep STRA6 reconstituted in nanodisc lipid bilayers in the presence and absence of retinol. The nanodisc reconstitution system has allowed us to study this protein in a close to physiological environment and examine its interaction with the cell membrane and relationship with its ligand, retinol. We also present the structure of sheep STRA6 in complex with human RBP. The structure of the STRA6-RBP complex confirms predictions in the literature as to the nature of the protein-protein interaction needed for retinol transport. Calcium-bound CaM is bound to STRA6 in the RBP-STRA6 structure, consistent with a regulatory role of this calcium binding protein in STRA6-RBP interaction. The analysis of the three states of STRA6 – pre, post and during interaction with retinol – provide unique insights into the mechanism of STRA6-mediated cellular retinol uptake.

Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were both cumbersome or required breeding and production of fish where thrombocytes are GFP labeled. Therefore, I established a flow cytometry based method of counting the number of thrombocytes. I used mepacrine to fluorescently label the blood cells and used the white cell fraction. Standard antisense oligonucleotide designed to the central portion of each of the target hox mRNAs, was piggybacked by a control morpholino and intravenously injected into the adult zebrafish. The thrombocyte count was measured 48 hours post injection. In this study, I found that the knockdown of hoxc11b resulted in increased number of thrombocytes and knockdown of hoxa10b, hoxb2a, hoxc5a, and hoxd3a showed reduction in the thrombocyte counts. I then screened the other 47 hox genes in the zebrafish genome using flow sorting method and found that knockdown of hoxa9a and hoxb1a also resulted in decreased thrombocyte number. Further, I used the dye DiI, which labels only young thrombocytes at specific concentrations and observed that the knockdown of hoxa10b, hoxb2a, hoxc5a, hoxd3a, hoxa9a and hoxb1a, lead to a decrease in young thrombocytes; whereas hoxc11b knockdown lead to increase in number of young thrombocytes. Using bromodeoxyuridine, I also showed that there is increase in release of young thrombocytes into peripheral circulation in hoxc11b knockdown fish which suggests that hoxc11b significantly promotes cell proliferation rather effecting apoptosis. In conclusion, I found six hox genes that are positive regulators and one hox gene is a negative regulator for thrombocyte development.

GPR17, a uracil nucleotide cysteinyl leukotriene receptor, belongs to the GPCR (G protein coupled receptor) family. It has been shown recently that inhibiting this protein in the nervous system in mice can lead to blockage of oligodendrocyte maturation, which supports myelin repair. Interestingly, our laboratory found GPR17 in thrombocytes. However, we do not know whether it has any function in thrombocyte aggregation or the nature of the ligand. In this paper, we studied the role of GPR17 in hemostasis, which is a fundamental defense mechanism in the event of injury. Using zebrafish as a model system, our laboratory has studied specifically thrombocytes, which play a significant role in hemostasis. The major reasons to use zebrafish as a model system are that their thrombocytes are functionally equivalent to human platelets, the adult fish are amenable to knockdown experiments, and they are readily available in the market. This study was performed by using a piggy back knockdown method where we used a chemical hybrid of control morpholino and an antisense oligonucleotide sequence leads to the degradation the mRNA for GPR17. After knockdown GPR17 in thrombocytes, the percent difference of the thrombocytes aggregation between the control and knockdown blood samples was measured by flow cytometry. We used various thrombocyte agonists to study differences in aggregation between the control and knockdown blood samples. The study showed that knockdown of GPR17 resulted in no significant differences in percent thrombocyte aggregation between control and agonist treated samples except for a slight increase in collagen-treated samples. Thus, it appears that GPR17 has no significant role in hemostasis.

No description available.

No description available.

Transmembrane protein 59 (tmem59) is a gene that encodes a protein involved in autophagy and apoptosis in human. A previous study in zebrafish showed that tmem59 mRNA was several folds higher in thrombocytes than those found in red blood cells (RBCs). Therefore, we hypothesized that tmem59 has a role in thrombocytes function. We injected a hybrid of control vivo-morpholino (cVMO) and tmem59 specific antisense standard oligonucleotide (tmem59SO) into adult zebrafish to knockdown tmem59.This piggyback knockdown approach resulted in fish that had more bleeding in gill bleeding assay than the control fish. The thrombocytes fromtmem59 knockdown zebrafish aggregated faster with ADP and collagen agonists. Also, the number of blood cells was reduced after the knockdown of tmem59. We also found the effects of MS-222 anesthesia on hemostasis and found that the bleeding was reduced yielding less blood and the blood cell counts increased probably due to vasoconstriction of the blood vessels. In summary, we found tmem59 is a negative regulator of hemostasis and inferred that anesthesia should be avoided in hemostasis studies.

Previous studies have shown that fish release alarming substances into the water to alert their kin to escape from danger. In our laboratory, we found that zebrafish produce trypsin and release it from their gills into the environment when they are under stress. By placing the zebrafish larvae in the middle of a small tank and then placing trypsin at one end of the tank, we observed that the larvae moved away from the trypsin zone and almost to the opposite end of the tank. This escape response was significant and did not occur in response to the control substances, bovine serum albumin (BSA), Russell's viper venom (RVV), and collagen. Also, previously, we had shown that the trypsin could act via a protease-activated receptor-2 (PAR2) on the surface of the cells. Therefore, we hypothesized that trypsin would induce a change in neuronal activity in the brain via PAR2-mediated signaling in cells on the surface of the fish body. To investigate whether the trypsin-responsive cells were surface cells, we generated a primary cell culture of zebrafish keratinocytes, confirmed these cells' identity by specific marker expression, and then incubated these cells with the calcium indicator Fluo-4 and exposed them to trypsin. By using calcium flux assay in a flow-cytometer, we found that trypsin-treated keratinocytes showed an increase in intracellular calcium release. To test whether PAR2 mediates the escape response to trypsin, we treated larvae with a PAR2 antagonist and showed that the trypsin-initiated escape response was abrogated. Furthermore, par2a mutants with knockdown of par2a by the piggyback knockdown method failed to respond to trypsin. Trypsin treatment of adult fish led to an approximately 2-fold increase in brain c-fos mRNA levels 45 mins after trypsin treatment, suggesting that trypsin signals may have reached the brain, probably via a spinothalamic pathway. Taken together, our results reveal a novel trypsin-initiated escape response in fish. These studies should enhance our understanding of fish communication in general and alarm behavior in particular. Furthermore, since pain receptors in other animals are also PAR2, our finding may be useful in exploring pathways of pain reception.

von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.

The development of the cardiovascular system, including the heart and circulating blood within a vascular network, relies on mesoderm-derived cells to contribute to the development of both cardiac and hematopoietic tissues. This dissertation focuses on exploring the roles of crip2, downstream of the transcription factor Nkx2.5 established from an RNA sequencing dataset, in cardiac and hematopoietic development using the zebrafish model. In Chapter 2, we investigate the influence of Crip proteins on the development of the zebrafish heart. Congenital heart defects (CHDs), affecting approximately 1% of live births, arise from structural anomalies during heart development primarily caused by genetic mutations. While there isn’t just one driver of CHDs, transcription factors such as Nkx2.5, play a pivotal role in guiding cardiac morphogenesis. NKX2-5-associated CHDs often involve outflow tract (OFT) malformations. The development of the heart involves two progenitor cell populations, the first heart field (FHF) and second heart field (SHF), contributing to the linear heart tube and subsequent growth. Despite understanding the role of Nkx2.5, the spatiotemporal mechanisms directed by Nkx factors in SHF progenitor specification, proliferation, and identity maintenance remain elusive. This study aims to uncover novel effectors of Nkx transcriptional regulation, using RNA sequencing on dissected wild-type and nkx2.5-/- zebrafish hearts at 26 hours post fertilization (hpf). This work focuses on a LIM domain protein, cysteine rich intestinal protein 2 (crip2), identified as a mis-regulated gene in nkx2.5-deficient embryos, and we explore its role downstream of nkx genes in SHF-derived arterial pole formation. While crip2 is abundantly expressed in the developing heart, the family member crip3 also shows a mild expression pattern. Loss-of-function mutations in crip2 and crip3 (referred to as cripDM) reveal normal cardiac chamber specification. Atrioventricular canal morphology remains unaffected in cripDM embryos. The OFT in cripDM embryos displays a significant dilation, accompanied by increased ltbp3 expression. Surprisingly, the smooth muscle cell population of the OFT does not explain the size increase. This research expands our understanding of OFT development, highlighting the multi-layered contributions of various cell types and factors. In Chapter 3, we further examine the role of crip2 in the development of hematopoietic stem cells given its endothelial expression pattern. Hematopoietic stem and progenitor cells (HSPCs) have multilineage potential and can sustain long-term self-renewal. The ability to derive patient-specific HSCs in culture has immense therapeutic potential to overcome the shortage of compatible donors for HSC transplantations. However, differentiation protocols largely fail to produce long-lived HSCs from human pluripotent stem cells. Understanding the complex genetic networks and signaling pathways required to generate HSCs will facilitate clinical applications in patients. The hemogenic endothelium (HE) is a specialized niche of endothelial cells within the ventral portion of the dorsal aorta that gives rise to HSPCs during the definitive wave of hematopoiesis in the zebrafish embryo. Our data reveal that crip2 has a previously unrecognized function in establishing the proper endothelial cell environment for HSPC specification. CripDM embryos exhibit decreased emergence of HSCs by 26 hpf. Loss of HSPCs in the cripDM results in decreased erythroid, myeloid, and lymphoid lineage production between 30 -72 hpf; these perturbations in the hematopoietic lineages recover by 96 hpf. To decipher the spatiotemporal mechanisms underlying the cripDM phenotype, we performed single cell RNA (scRNA) sequencing of sorted, Kdrl:mCherry+ cells at 30 hpf. Our analysis reveals upregulation of genes essential for vascular development and mis-regulation of Notch signaling pathways in the cripDM embryos. Building on these data, our ongoing studies aim to investigate how crip2 regulates the endothelial niche of the ventral aorta to produce HSCs early in definitive hematopoiesis. We anticipate that our insights will inform potential therapeutic interventions for improvements of human HSC production in vitro.

Craniofacial malformations arise from developmental defects in the head, face, and neck and account for one third of congenital defects at birth. Clinical phenotypes such as DiGeorge Syndrome, the most common microdeletion condition, illustrate a developmental link between cardiovascular and craniofacial morphogenesis. Moreover, recent fate mapping studies in mice and zebrafish support this notion through identification of a multipotent progenitor in the cardiopharyngeal field that gives rise to the heart, branchiomeric muscles, and pharyngeal arch (PA) arteries. NKX2-5 is a key cardiac transcription factor associated with human congenital heart disease and mouse models of Nkx2-5 deficiency highlight critical roles in cardiac development. In zebrafish, nkx2.5 and nkx2.7 are paralogous genes in the NK4 family expressed in cardiomyocytes and PAs. Despite the shared cellular origins of cardiac and craniofacial tissues, the function of NK4 factors in head and neck patterning has not been elucidated. Here, we demonstrate that Nkx2.7 serves as a previously unappreciated, crucial regulator of craniofacial muscle and cartilage formation. Our studies reveal a unique requirement for nkx2.7 in PA1- and PA2-derived branchiomeric muscle and cartilage elements for which nkx2.5 cannot compensate. Moreover, molecular evolutionary analysis of NK4 genes reveals that nkx2.5 and nkx2.7 are ohnologs resulting from two rounds of vertebrate whole genome duplications with an early split between them, underscoring the concept that these genes play independent roles during development. The distinct mechanistic function of nkx2.7 is elucidated by cell counting experiments that uncover the requirement of nkx2.7 in specification of PA1 and PA2 branchiomeric muscle progenitors. Furthermore, single cell RNA-sequencing performed on microdissected PA tissues from wild-type and nkx2.7-/- embryos identifies decreased expression of the ventral neural crest gene signature essential for cartilage and jaw joint morphogenesis. Together, our studies shed light on an evolutionarily conserved, unique function of Nkx2.7 in vertebrate craniofacial development and have the potential to advance our understanding of the etiologies and therapeutic interventions for patients with congenital deformities of the head and neck.