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.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/zfnv-1n10 |
| Date | January 2024 |
| Creators | Aleman, Angelika Gabriele |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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