Insights from a novel zebrafish mutant on cardiovascular development

Alcian Blue staining and In Situ results suggest a potential connection between pendulum mutant craniofacial defects and an upregulation of TGF-β

Abstract

Vertebrate heart formation depends on coordinated signaling among the first heart field, the second heart field (SHF), and neural crest cells (NCC). The cardiac outflow tract (OFT) and the adjacent bulboventricular (BV) valve are formed by both SHF and NCC lineages. Approximately 30% of human congenital heart malformations affect these and other OFT structures. Though this subject has been extensively studied, the cellular and molecular basis of OFT development remains poorly understood. Here, we report a novel zebrafish mutant, pendulum (pen), which enables us to explore these mechanisms. In pen mutants, we observe endocardial expansion and reduced smooth muscle cells in the OFT, indicating a defective SHF contribution. Transcriptome profiling indicates TGF-β signaling upregulation, which is crucial in SHF and NCC lineage. Extending our analysis to NCC derivatives, we employed two-color acid-free cartilage and bone staining at 4 days post fertilization (4 dpf) and observed disrupted craniofacial cartilage organization, which points towards defective NCC migration and differentiation. These findings establish pen as a model of value for studying SHF–NCC interaction and provide a system for research into therapeutic strategies against OFT malformations.

Background: Zebrafish Outflow Tract

The zebrafish OFT, located at the arterial pole of the two-chambered heart (ventricle and atrium), comprises the smooth muscle component known as bulbous arteriosus (BA) critical for blood exiting the heart.

Background: The Pendulum Mutant

The novel zebrafish mutant, pen, is phenotypically characterized by pericardial edema (B, arrow), blood pooling (D, arrow), slower heart rate, and jaw defects at 72 hpf. The pen phenotype acts as a recessive allele, occurring in ~25% of the progeny of crosses between two carriers.

Background: Outflow Tract Analysis in Pen Mutant

OFT BA analysis via DAF-2DA2 immunofluorescence reveals intact smooth muscle (green) in WT (A, white arrow) and absent (B) and strongly reduced (C) in pen at the arterial pole of the ventricle (red) at 72 hpf.

Background: Gene Analysis of Pen Mutant

A transcriptomic analysis of pen mutants reveals downregulation of key cardiac pathways (red box, D) and upregulation of TGF-Beta signaling (red box, E), indicating disrupted cardiac and neural crest patterning

Results

In situ Dlx 5

Dlx5a is involved in neural crest cell migration and organization during zebrafish craniofacial development. In our assay, we showcased the presence of mRNA expression of dlx5a across different timepoints in the pharyngeal arches (black arrow). It is evident here that dlx5a expression is unaffected, indicative of the presence of NCC. Scale bar: 20µm.

Alcian Blue: Craniofacial assessment

The pen mutants exhibit severe disorganization of facial cartilage compared to siblings (p < 0.05, χ² test). Scale bar: 20 µm

Craniofacial Quantification

Quantification of craniofacial cartilage in pen mutants reveals shorter cartilage lengths and a wider angle between Meckel’s cartilage and the palatoquadrate—indicative of microcephaly and micrognathia.These features support a defect in NCC migration and differentiation (p<0.05, Mann–Whitney U test).

Otilith Assessment

A clear indication of the defective chondrocyte differentiation is translated into the varying defects in the ossification process in otoliths of pen mutants. (p < 0.05, χ² test).

Conclusions and Future research

pen mutants develop early valve and smooth muscle abnormalities, consistent with SHF pathology.

These correlate with abnormal cardiomyocyte contraction and enhanced TGF-β signaling.

Though NCC development is normal, pen disrupts NCC migration and differentiation.

This occurs in the form of craniofacial deformities caused by the enhanced TGF-β signaling.

Overall, we have established a model of value to study the SHF-NCC interaction to better understand CHD OFT malformations.

Future experimentation: Exploration of whether TGF-β signaling inhibition is able to rescue pen mutant phenotype.

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