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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.

Thesis advisor: Christopher S. Lee / Background: Congenital heart disease (CHD) is the most prevalent birth defect in the United States. Advances in treatment have changed CHD from what once was almost always a life-threatening condition to what is commonly a lifelong chronic condition. Up to 60% of adults with CHD experience large gaps in cardiology care during the transition from pediatric to adult specialty care. Effective CHD management in emerging adulthood maximizes lifelong potential, functioning and quality of life. Past research has failed to consider how emerging adults and their parents work together to manage CHD together as an interdependent team. Thus, there remains a dearth of information on how best to support emerging adults and their parents. Since CHD is a life-long diagnosis there is a critical need to understand the ways in which emerging adults and their parents as primary care partners engage in behavior to manage CHD together. This manuscript dissertation had an overarching goal to develop a deeper understanding of emerging adult and parent contributions to the management of CHD. Methods: First, an integrative review summarized and evaluated the evidence of published and peer-reviewed literature regarding parental perspective of the emerging adult with CHD. Next, a cross-sectional quantitative hypothesis-generating pilot study investigating emerging adult and parent contributions to management of CHD was conducted. And finally, an exploratory qualitative study was completed to describe health care team provider perspectives on the experience of emerging adults and their parents in managing CHD. Results: These three manuscripts have the key following results: 1) parents have concerns about their emerging adult children with CHD related to their future, independence in self-care of CHD, including health care system navigation, 2) there was a positive correlation between emerging adult and parent contributions to self-care (management, monitoring and maintenance) of CHD, and in the domain of navigating the health care system, there was a weak and negative correlation (the more an emerging adult does, the less the parent contributes), and 3) providers in health care teams report differences in both emerging adult and parent factors that impact management, and that self-care in emerging adults with CHD is critical with known health care system barriers that need assessment and improvement to support this population. Conclusion: The constellation of these findings from the dissertation and past work help fill critical knowledge and research gaps in emerging adult and parent/care partner contributions to management of CHD. These findings support the much-needed future work to inform clinical care, research, and policy for emerging adults with CHD and parents to further improve health and quality of life for this population. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Connell School of Nursing. / Discipline: Nursing.