Embryos of oviparous reptiles develop on the surface of a large mass of yolk, which they metabolize to become relatively large hatchlings. Access to the yolk is provided by tissues growing outward from the embryo to cover the surface of the yolk. A key feature of yolk sac development is a dedicated blood vascular system to communicate with the embryo. The best known model for yolk sac development and function of oviparous amniotes is based on numerous studies of birds, primarily domestic chickens. In this model, the vascular yolk sac forms the perimeter of the large yolk mass and is lined by a specialized epithelium, which takes up, processes and transports yolk nutrients to the yolk sac blood vessels. Studies of lizard yolk sac development, dating to more than 100 years ago, report characteristics inconsistent with this model. We compared development of the yolk sac from oviposition to near hatching in embryonic series of three species of oviparous scincid lizards to consider congruence with the pattern described for birds. Our findings reinforce results of prior studies indicating that squamate reptiles mobilize and metabolize the large yolk reserves in their eggs through a process unknown in other amniotes. Development of the yolk sac of lizards differs from birds in four primary characteristics, migration of mesoderm, proliferation of endoderm, vascular development and cellular diversity within the yolk sac cavity. Notably, all of the yolk is incorporated into cells relatively early in development and endodermal cells within the yolk sac cavity align along blood vessels which course throughout the yolk sac cavity. The pattern of uptake of yolk by endodermal cells indicates that the mechanism of yolk metabolism differs between lizards and birds and that the evolution of a fundamental characteristic of embryonic nutrition diverged in these two lineages. Attributes of the yolk sac of squamates reveal the existence of phylogenetic diversity among amniote lineages and raise new questions concerning the evolution of the amniotic egg. J. Morphol. 278:574–591, 2017. © 2017 Wiley Periodicals, Inc.

Increasing evidence suggests that normal pulmonary vascular morphogenesis is critical for the formation and maintenance of the lung parenchyma, both pre- and postnatally. Indeed, the disruption of angiogenic pathways, whether through inherent genetic predisposition or as a consequence of life-saving interventions, may underlie many pulmonary diseases of infancy, including alveolar capillary dysplasia (ACD) and bronchopulmonary dysplasia (BPD). To understand the etiology of – and advance treatments for – such diseases, we must first identify the fundamental genetic regulators that orchestrate normal parenchymal development. Neuropilin-1 (NRP1) is a transmembrane receptor that plays essential roles in normal and pathological vascular development, and binds two distinct ligand families: Vascular endothelial growth factor (VEGF) and Class 3 Semaphorins (SEMA3). Although VEGF-NRP1 interactions in systemic vascular development have been described, the importance of SEMA3-NRP1 signalling in systemic or pulmonary vascular morphogenesis is uncertain. We hypothesized that SEMA3-NRP1 and VEGF-NRP1 interactions are fundamental pathways in the orchestration of pulmonary vascular development in both the embryo and neonate. Disruption of these pathways would therefore lead to significant interruption of normal angiogenic and vascular maturation processes that are relevant to the pathogenesis of pulmonary diseases. Using extensive histopathological analyses of NRP1 loss-of-function mice, we show evidence of a significant role for SEMA3-NRP1 signalling in fetal microvascular development: congenital loss of SEMA3-NRP1 signalling resulted in severely attenuated development of the distal vasculature and alveolar-capillary interface, leading to fatal respiratory distress at birth that is reminiscent of clinical ACD. By contrast, VEGF-NRP1 and SEMA3-NRP1 signalling appear unessential for normal alveolar and vascular development in the postnatal period, per se, despite increased mortality. Our results demonstrate the critical involvement of SEMA3-NRP1 signalling in endothelial development and substantiate the idea that NRP1 mediates opposing and cooperative functions between SEMA3 and VEGF ligands.

To maintain human health, the lymphatic system requires a structurally and functionally sound network of lymph vessels to absorb lipid-based nutrients, preserve extracellular fluid homeostasis, and mediate immune responses. Aside from lymphedema, investigations in the past few decades have found that impairment of the lymphatic vasculature is also involved in processes such as inflammation, tumor metastasis, fat metabolism, and obesity. However, despite a long history of study and rekindled vigor in the field of lymphatic vascular research, our knowledge of lymph vessel development and physiology is still quite limited. Recently, mutations in a protein family known as connexins (Cxs) were identified as the cause of lymphatic dysfunction in some cases of inherited lymphedema. This dissertation explores the role of primarily two specific connexins, Cx37 and Cx43, and the transcription factor Foxc2 in the morphogenesis and function of the lymphatic vasculature in mice. To accomplish this, phenotypic characterization of mice with genetic deficiencies (knockout mice) in Cx37, Cx43, and/or Foxc2 was performed principally via necropsy, histological techniques (immuno-fluorescence microscopy and H&E staining), and Evans blue dye (EBD) injections. Developmental abnormalities were found in lymphatic vascular growth, patterning, and remodeling in mice lacking Cx37, Cx43, Foxc2 or a combined deficiency of these proteins. Reductions or complete loss of lymphatic valves were a common finding in mice lacking one or more of these proteins. These valve deficits underlay lymphatic insufficiencies that resulted in lymphedema and chylothorax in some genotypes. Foxc2 was found to be a regulator of Cx37 expression. Moreover, Foxc2 was also dependent on Cx37 function for proper morphogenesis of lymph vessels. These findings pertaining to the expression of connexins in the lymphatic vasculature, their role in lymphatic valvulogenesis, and the interdependence of Cx37 and Foxc2 during lymph-vascular development represent my original contributions to human knowledge.

Arabidopsis thaliana contains two genes annotated as methylthioadenosine nucleosidases (MTN): MTN1, At4g38800 and MTN2, At4g34840. This enzyme activity hydrolyzes the methylthioadenosine (MTA) produced by nicotianamine (NA), polyamine (PA), and ethylene biosynthesis to methylthioribose (MTR) within the Yang cycle. Comprehensive analysis of the mtn1-1mtn2-1 mutant line with 14 % residual MTN activity revealed a complex phenotype that includes male and female infertility and abnormal vascular development. Based on metabolite profiling, mtn1-1mtn2-1 has a reduced NA content, altered PA profiles with higher putrescine (Put) and lower spermidine (Spd) and spermine (Spm) levels, disrupted metal ion profiles, and abnormal auxin distribution. The modeling of Arabidopsis PA synthases developed by comparison with the crystal structures of human Spd and spermine synthases complexed with MTA suggests that Arabidopsis PA synthases are product inhibited by MTA. Thus, these pleiotropic mutant phenotypes possibly are the result of one metabolite directly inhibiting numerous pathways. By creating and analyzing a series of mutants and transgenic lines with moderate levels of MTN activity the complex phenotype of mtn1-1mtn2-1 was dissected in order to determine the fundamental trait associated with MTN deficiency. Two double mutants were identified by crossing single T-DNA mutants, and an artificial micro RNA (amiRNA) line was generated by transforming mtn1-1 with amiRNA specific to MTN2. The T-DNA double mutants, mtn1 4mtn2-1, and mtn1-1mtn2-5 had 98 % and 28 % MTN activity, respectively, whereas the amiRNA line has 16 % MTN activity. The growth, development, and metabolite analysis of these mutants revealed that their delayed bolting, correlated with an increased number of leaves, was the common trait observed across all lines. Xylem proliferation defects and increased number of vascular bundles per unit area were shared in all lines except mtn1 4mtn2-1. Based on these results, auxin distribution is proposed as the key target of the accumulated MTA that results from MTN deficiency. The infertility related to MTN-deficiency was restored by supplying 100 ??M of Spd to the mtn1-1mtn2-1 seedlings over 14 days. The data presented in this thesis reveals two potential links that work synergistically to recover fertility in this mtn1-1mtn2-1 line. Based on a detailed analysis of the female gynoecia morphology, transcript, hormone and metabolite profiles, it is proposed that the Spd partially reverses the mutant phenotypes through the recovery of auxin distribution and /or vascular development. Interestingly, the Spd effect seems to be transgenerational: they give rise to plants that are genotypically mtn1-1mtn2-1 but phenotypically WT over generations. Taken together, all of the results suggest that MTN-deficient mutants provide the potential for unraveling the molecular mechanism associated with nicotianamine, polyamines, auxin, and vascular development with respect to enhancing the efficiency of nutrient use and yields in plants.

Intracerebral hemorrhage (ICH) is a severe form of stroke, with a high mortality rate and often resulting in irreversible neurological deterioration. Although animal studies have provided insight into the etiology of the disease, many of the causative genes and mechanisms implicated in cerebral-vascular malformations are unknown. Treatment options remain ineffective. With the present models, the pathophysiological consequences of ICH can only be assessed in situ and after histological analysis. Furthermore, common deficiencies of the current models include the heterogeneity, low expression and low reproducibility of the desired phenotype. Hence, there is a requirement for novel approaches to model ICH pathogenesis. Zebrafish (Danio rerio) has gained recognition as a vertebrate model for stroke research. Through a combination of pharmacological blockers, metabolite rescue, genetic approaches, and confocal imaging analysis, I demonstrate a requirement for the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway in regulating developmental cerebral-vascular stabilization. A transient loss in HMGCR function induces ICH, characterised by progressive dilation of blood vessels, vascular permeability and vessel rupture. These effects are likely due to reduced prenylation of Rho GTPases, evidenced by morpholino-mediated blocking of the prenylation pathway and in vivo assessment of endothelial-specific localization of cdc42, a Rho GTPase family protein. These results are in conformity with recent clinical and experimental evidence. I have further shown that this model consistently replicates common pathoghysiological processes associated with ICH. The hemorrhages are associated with the disruption of the blood-brain barrier, vessel disintegration, hematoma expansion and edema into the adjacent brain regions. Also, enhanced apoptosis, activation of inflammatory mediators in the periphery of the hematoma, enriched heme oxygenase 1 (HO-1) expression and localised thrombosis were observed in these embryos. I show that the patterning and distribution of catecholaminergic neurons, response to sensory stimulus and swimming speed were impaired as a consequence of ICH. These results suggest that HMGCR contributes to cerebral-vascular stabilisation through Rho GTPase mediated-signalling and that zebrafish can serve as a powerful paradigm for the systemic analysis of the etiological and pathophysiological underpinnings of ICH and can help establish the basis for future studies into screening for putative therapeutics and elucidating mechanisms aiding functional recovery.

Increasing evidence suggests that normal pulmonary vascular morphogenesis is critical for the formation and maintenance of the lung parenchyma, both pre- and postnatally. Indeed, the disruption of angiogenic pathways, whether through inherent genetic predisposition or as a consequence of life-saving interventions, may underlie many pulmonary diseases of infancy, including alveolar capillary dysplasia (ACD) and bronchopulmonary dysplasia (BPD). To understand the etiology of – and advance treatments for – such diseases, we must first identify the fundamental genetic regulators that orchestrate normal parenchymal development. Neuropilin-1 (NRP1) is a transmembrane receptor that plays essential roles in normal and pathological vascular development, and binds two distinct ligand families: Vascular endothelial growth factor (VEGF) and Class 3 Semaphorins (SEMA3). Although VEGF-NRP1 interactions in systemic vascular development have been described, the importance of SEMA3-NRP1 signalling in systemic or pulmonary vascular morphogenesis is uncertain. We hypothesized that SEMA3-NRP1 and VEGF-NRP1 interactions are fundamental pathways in the orchestration of pulmonary vascular development in both the embryo and neonate. Disruption of these pathways would therefore lead to significant interruption of normal angiogenic and vascular maturation processes that are relevant to the pathogenesis of pulmonary diseases. Using extensive histopathological analyses of NRP1 loss-of-function mice, we show evidence of a significant role for SEMA3-NRP1 signalling in fetal microvascular development: congenital loss of SEMA3-NRP1 signalling resulted in severely attenuated development of the distal vasculature and alveolar-capillary interface, leading to fatal respiratory distress at birth that is reminiscent of clinical ACD. By contrast, VEGF-NRP1 and SEMA3-NRP1 signalling appear unessential for normal alveolar and vascular development in the postnatal period, per se, despite increased mortality. Our results demonstrate the critical involvement of SEMA3-NRP1 signalling in endothelial development and substantiate the idea that NRP1 mediates opposing and cooperative functions between SEMA3 and VEGF ligands.

During normal placental development, a primitive vascular network develops through vasculogenesis and angiogenesis, and is then remodelled through maturation and regression. The mechanism behind this regression is unknown, but data from other systems suggests that it could be due to an endothelial-mesenchymal transition (EndMT). If this is the case, then dysregulated EndMT could lead to increased vascular regression, which could result in placental hypovascularisation. As the placental vasculature is the area of exchange between maternal and fetal circulations, a reduction in its surface area could result in fetal growth restriction (FGR). The hypothesis of this thesis is that EndMT occurs during normal placental development, but is increased during FGR and contributes to placental hypovascularisation. A primary cell model consisting of endothelial and mesenchymal cells was isolated from human first trimester placental villous stroma. These cells were shown to lose CD31 mRNA (n = 1-3) and protein (n = 15) over 4 passages, with no loss of cell viability (n = 8). EndMT-associated transcription factors were also present in these cells at all 4 passages (n = 2-4). When cells were isolated from this mixed cell model based on their CD31-positivity and examined immediately after isolation, a small proportion also expressed αSMA (n = 5). Co-expression of endothelial and mesenchymal markers suggests that an EndMT was occurring. After 24 hours in culture, the proportion of these cells expressing αSMA increased (n = 5), and some cells co-expressed vWF and αSMA, while others lost their CD31-positivity, indicating that these cells had undergone EndMT. Cells isolated based on their CD31-positivity were treated with factors shown to inhibit EndMT in other systems. However, culture with 10µM SB431542 (TGFβ receptor inhibitor; n = 6), 10µM Dorsomorphin (BMP receptor inhibitor; n = 3), or 0.1µM PDGFR-β Tyrosine Kinase Inhibitor IV (n = 3) did not inhibit gain of αSMA by these cells. Culture on Matrigel in endothelial growth medium containing VEGF and FGF also failed to stabilise the endothelial phenotype (n = 3). The possibility that EndMT occurs in placenta in vivo was examined; genes associated with EndMT were shown to be present in placenta (n = 5), and there was limited evidence of CD31 or vWF co-expression with αSMA in tissue. Preliminary evidence was obtained to suggest that expression of EndMT-associated genes was altered in FGR placentas compared to normal. In summary, the data presented in this thesis demonstrate that an EndMT occurs in primary placental microvascular endothelial cells in vitro. Furthermore, these studies provide evidence to suggest that this transition also occurs in vivo and could be altered in placentas from pregnancies complicated by FGR.

Intracerebral hemorrhage (ICH) is a severe form of stroke, with a high mortality rate and often resulting in irreversible neurological deterioration. Although animal studies have provided insight into the etiology of the disease, many of the causative genes and mechanisms implicated in cerebral-vascular malformations are unknown. Treatment options remain ineffective. With the present models, the pathophysiological consequences of ICH can only be assessed in situ and after histological analysis. Furthermore, common deficiencies of the current models include the heterogeneity, low expression and low reproducibility of the desired phenotype. Hence, there is a requirement for novel approaches to model ICH pathogenesis. Zebrafish (Danio rerio) has gained recognition as a vertebrate model for stroke research. Through a combination of pharmacological blockers, metabolite rescue, genetic approaches, and confocal imaging analysis, I demonstrate a requirement for the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway in regulating developmental cerebral-vascular stabilization. A transient loss in HMGCR function induces ICH, characterised by progressive dilation of blood vessels, vascular permeability and vessel rupture. These effects are likely due to reduced prenylation of Rho GTPases, evidenced by morpholino-mediated blocking of the prenylation pathway and in vivo assessment of endothelial-specific localization of cdc42, a Rho GTPase family protein. These results are in conformity with recent clinical and experimental evidence. I have further shown that this model consistently replicates common pathoghysiological processes associated with ICH. The hemorrhages are associated with the disruption of the blood-brain barrier, vessel disintegration, hematoma expansion and edema into the adjacent brain regions. Also, enhanced apoptosis, activation of inflammatory mediators in the periphery of the hematoma, enriched heme oxygenase 1 (HO-1) expression and localised thrombosis were observed in these embryos. I show that the patterning and distribution of catecholaminergic neurons, response to sensory stimulus and swimming speed were impaired as a consequence of ICH. These results suggest that HMGCR contributes to cerebral-vascular stabilisation through Rho GTPase mediated-signalling and that zebrafish can serve as a powerful paradigm for the systemic analysis of the etiological and pathophysiological underpinnings of ICH and can help establish the basis for future studies into screening for putative therapeutics and elucidating mechanisms aiding functional recovery.

A zebrafish transcript dubbed GA2692 was initially identified via a whole-mount in situ hybridization screen for vessel specific transcripts. Its mRNA expression during embryonic development was detected in ventral hematopoietic and vasculogenic mesoderm and later throughout the vasculature up to 48 hours post fertilization. Morpholino-mediated knockdown of GA2692 in embryos resulted in multiple defects in vasculature, particularly, at sites undergoing active capillary sprouting: the intersegmental vessels, sub-intestinal vessels and the capillary sprouts of the pectoral fin vessel. During the course of these studies, a homology search indicated that GA2692 is the zebrafish orthologue of mammalian HspA12B, a distant member of the heat shock protein 70 (Hsp70) family. By a combination of northern blot and realtime PCR analysis, we showed that HspA12B is highly expressed in human endothelial cells in vitro. Knockdown of HspA12B by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells blocked wound healing, migration and tube formation, whereas overexpression of HspA12B enhanced migration and accelerated wound healing - data that are consistent with the in vivo fish phenotype obtained in the morpholino-knockdown studies. Phosphorylation of Akt was consistently reduced by siRNAs against HspA12B. Overexpression of a constitutively active form of Akt rescued the inhibitory effects of knockdown of HspA12B on migration of human umbilical vein endothelial cells. Collectively, our data suggests that HspA12B is a highly endothelial-cell-specific distant member of the Hsp70 family and plays a significant role in endothelial cells during development and angiogenesis in vitro, partially attributable to modulation of Akt phosphorylation.

The vasculature is a highly intricate system of "highways" that shuttles blood from the heart to every tissue and organ in the human body. These vessels are responsible for carrying oxygen, trafficking hormones, delivering nutrients, and removing waste products from the body. The formation of a functioning vascular system depends on the close coordination of many cell types and, on the capillary level, specifically endothelial cells and pericytes as well as the surrounding protein microenvironment, known as the extracellular matrix (ECM). Impaired coordination amongst the cellular and protein constituents results in the improper functioning of the vascular network and can eventually contribute to the failure of organ systems. This dissertation research focuses on how improper ECM deposition affects vascular assembly. We utilized several approaches to affect ECM composition, specifically: 1) hypoxia exposure and 2) reducing ECM pharmacologically and utilizing lentiviral-mediated silencing of Type IV Collagen (Col-IV, gene Col4a1) expression. In these experimental settings, we observed downstream changes in the coordination between endothelial cells and pericytes while forming vascular networks. In short, this dissertation work suggests that excess ECM deposition, and particularly that of Col-IV, has unique deleterious effects on the developing vasculature as compared to reduced ECM deposition. The findings from this work suggest mechanisms underlying how the vasculature may be destabilized in hypoxia-associated pathologies, such as preeclampsia. / Doctor of Philosophy / Every tissue and organ in the human body receives blood from the heart via the extremely complex network of "highways" known as the vasculature. These vessels oversee moving nutrients, oxygen, hormones, and waste materials out of the body. At the capillary level, endothelial cells and pericytes, as well as the surrounding protein milieu known as the extracellular matrix (ECM), are required for the development of a functional vascular system. If the vascular network fails to develop and operate properly because of poor protein and cellular coordination, it can eventually lead to the failure of organ systems. The study for this dissertation focuses on how vascular development is impacted by insufficient ECM deposition. We used several strategies to modify the composition of the ECM, including 1) hypoxia exposure, 2) pharmaceutical ECM reduction, and 3) lentiviral-mediated delivery of shRNA to silence Type IV Collagen (Col-IV, gene Col4a1) production. We noticed alterations in the coordination between endothelial cells and pericytes as vascular networks were being formed in these experimental environments. In summary, this dissertation work contends that, in contrast to reduced ECM deposition, excess ECM deposition, and specifically that of Col-IV, has distinct detrimental consequences on the developing vasculature. The results of this study offer methods by which diseases associated with hypoxia, such preeclampsia, may cause the vasculature to become unstable.