From Zebrafish to Humans: How TGF-B Signaling Impacts Great Vessel Morphogenesis

Congenital heart defects (CHD) are among the most common birth defects, affecting about 1% of live births. Outflow tract (OFT) defects are the most populace of CHD cases annually. However, the etiology of OFT defects remains puzzling due to the complex cell and molecular interactions that guide heart development. Here, we report a novel zebrafish mutant we named pendulum (pen) with a malformed OFT and a defective adjacent bulboventricular valve. The pen mutant shows a positionally displaced ventral aorta (VA), the single vessel that connects the OFT to the pharyngeal arch arteries (PAA). Additionally, we found via RNA-Seq analysis that pen mutants have enhanced TGF-β signaling activity at 72 hours post fertilization (hpf) compared to its counterpart wild-type control siblings. The uniqueness of pen mutant makes it a suitable model to study the patterning of the great vessels (OFT/VA/PAA). As such, we aim to define the effect of hyperactivation of TGF-β signaling on the overall great vessel architecture. To tackle this goal, we devise a 2-step approach: (1) analyze the ventral aorta patterning, and (2) define the OFT/VA junction position relative to the PAA in pen mutants. Findings from this project will provide exciting insights into understanding how enhanced TGF-β signaling directs zebrafish OFT/VA/PAA patterning. This research will contribute to the broader discipline of developmental biology by revealing how TGF-β signaling impacts cardiac development, a critical step in understanding CHD and allowing for potential prevention or treatment. Understanding these mechanisms has direct implications for improving potential outcomes for individuals with CHD– a global plague. For my own personal development, this project will provide me with hands-on experience in developmental biology and scientific experimentation, and will deepen my understanding of the developmental mechanisms in human disease. As a first-generation student and aspiring physician, this experience will strengthen my ability to understand the clinical relevance behind research, which will allow me to foster the scientific inquiry necessary to be a successful physician. 

This study uses a zebrafish mutant model, pendulum (pen), to investigate how elevated TGF-β signaling affects great vessel development. Embryos from pen mutants and wild-type siblings are collected at 48, 60, and 72 hours post-fertilization to capture key stages of outflow tract (OFT) and ventral aorta (VA) patterning. To visualize specific cell populations, we perform immunofluorescence staining using endothelial, myocardial, and smooth muscle markers, combined with a transgenic fluorescent reporter line. Samples are imaged using confocal microscopy, and images are analyzed with ImageJ to quantify vessel morphology, including VA length, position, and endothelial cell number. Additionally, we measure the spatial relationship between the OFT–VA junction and the pharyngeal arch arteries (PAA) to assess structural organization. Statistical analyses are performed to compare mutant and control groups, allowing us to determine how TGF-β hyperactivation influences great vessel architecture.

1. Elevated TGF-β signaling disrupts normal great vessel patterning in pen mutants

2. OFT–VA connectivity defect confirmed (fails to shift from PAA3 to PAA4)

3. VA patterning partially affected, with dynamic anterior–posterior changes over time

4. Highlights importance of precise TGF-β regulation during cardiac development

Future Directions:

1. Analyze earlier timepoint (48 hpf) to identify when defects first arise

2. Further investigate how TGF-β signaling influences early vessel patterning

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IACUC protocol 1022_1028_Aug