Expression of Novel Genes During Zebrafish Development

Abstract

Congenital heart defects (CHDs) affect 1% of births, often resulting from dysregulated cardiac development and genetic mutations [1]. We use the transparent zebrafish (Danio rerio) model to analyze orthologus cardiovascular genes that may be associated with CHDs. To accomplish this, we employed co-expression analysis to identify novel regulatory genes, and then analyzed where these genes are expressed. pabpc4 and shdb were identified as candidates due to either co-expression with the ventricular marker vmhc or relation to the she gene family, respiectively. We hypothesized that pabpc4 functions within specific cardiac or striated muscle domains, and that shdb will be expressed as a vascular adaptor protein [3]. 

Sequences were identified via ZFIN, and primers were designed using Primer3 and ApE. Following PCR amplification of 96 hpf cDNA and subsequent gel purification, purified DNA templates were used to synthesize antisense RNA probes. Gene expression patterns were localized using in situ hybridization (ISH), imaged via Zeiss V8 microscopy, and analyzed qualitatively based on observed staining patterns. pabpc4 was found to be expressed in the fins at 48 hpf, in the yolk at 72 hpf, in the ventrical and the swim bladder at 96 hpf. Expression was found in the somites, facial-cranial muscles and in a peculiar spot on the lateral head of all stages, hypothesized to be a gland. shdb was found to be expressed mainly in the nervous tissue of the zebrafish embryo and particularly in the pectoral fins. 

Future research will include investigating the peculiar staining patterns of pabpc4, including more stages in the shdb ISH analysis, and troubleshooting the Proteinase K protocol. 

Background

Congenital heart defects (CHD) are the most common type of birth defect, affecting nearly 1% of live births each year. CHD is caused by abnormal cardiac formation within the womb, often due to genetic mutations or altered gene regulation [1]. 

The zebrafish (Danio rerio) serves as a powerful cardiovascular model due to its rapid morphogenesis, embryonic transparency, and high degree of orthology to human cardiovascular genes. 

Researchers can employ several strategies to identify novel genes, including co-expression analysis or targeting gene families known to be active within areas of interest. Our target genes pabpc4 and shdb were identified using these techniques. 

pabpc4 (poly (A) protein binding cytoplasmic 4) is co-expressed with vmhc (ventricular myosin heavy chain), which is expressed in ventricular cardiomyocyte precursors [4]. Because vmhc co-expressed genes may be expressed in only the heart (ventricle-only or both ventricle and atrium), expressed in all striated muscles, or expressed ubiquitously, we hypothesize that pabpc4 will manifest in one of these distinct domains.

Given that shdb (Src homology 2 domain containing transforming protein D, b) is a member of the same gene family as she, an adaptor protein involved in vascular development and dorsal aorta diameter, we hypothesized that shdb expression would similarly localize to the developing embryo's vasculature [3].

To begin this process, we started by designing our primers using primer3 and ApE. We found the desired gene using the ZFIN online database and transferred it to the other software to design the primers. After receiving our primers, we resuspended them and ran a PCR on 96 hpf complementary DNA using a forward primer and a reverse primer with the T7 RNA polymerase binding site. Once we had the PCR product, we ran the product using gel electrophoresis to check that the band size matched up with the predicted product size. Once this was verified, we proceeded with a gel purification in which we purified the DNA from the gel that was run. After purifying our DNA from the gel, we made our RNA antisense probes to be used in further in situ hybridization (ISH)  to show where our genes were expressed. In situ hybridizations were imaged using Zeiss V8 microscope. Based on staining sites, expression patterns were determined qualitatively.  

Upon analyzing our results, we have decided that the results can only be confirmed through further testing. This further testing would include more in situ hybridization experiments, specifically with embryos of different developmental stages (24 hpf, 48 hpf, 72 hpf, 96 hpf), with additional treatments to help fully develop the probe. We also noticed during our in situ hybridization experiments that many embryos were becoming very fragile and falling apart. We hypothesize that this may have been caused by an issue with our Proteinase K treatment. In the future, we would like to revisit this and analyze what could be going wrong with the Proteinase K. 

1. Centers for Disease Control and Prevention. (2024, May 15). About congenital heart defects. https://www.cdc.gov/heart-defects/about/index.html

2. Kotiya, D., Leibold, N., Verma, N., Jicha, G. A., Goldstein, L. B., & Despa, F. (2023). Rapid, scalable assay of amylin-β amyloid co-aggregation in brain tissue and blood. Journal of Biological Chemistry, 299(5), 104682. https://doi.org/10.1016/j.jbc.2023.104682

3. Schumacher, J. A., Wright, Z. A., Rufin Florat, D., Anand, S. K., Dasyani, M., Batta, S. P. R., Laverde, V., Ferrari, K., Klimkaite, L., Bredemeier, N. O., Gurung, S., Koller, G. M., Aguera, K. N., Chadwick, G. P., Johnson, R. D., Davis, G. E., & Sumanas, S. (2024). SH2 domain protein E and ABL signaling regulate blood vessel size. PLoS Genetics, 20(1), e1010851. 

4. Yoshida, M., Yoshida, K., Kozlov, G., Lim, N. S., De Crescenzo, G., Pang, Z., Berlanga, J. J., Kahvejian, A., Gehring, K., Wing, S. S., & Sonenberg, N. (2006). Poly(A) binding protein (PABP) homeostasis is mediated by the stability of its inhibitor, Paip2. The EMBO Journal, 25(9), 1934–1944. https://doi.org/10.1038/sj.emboj.7601079

5. Xue, Z., Hennig, P., Hurd, E. A., Moore, J. W., Keren-Paz, A., Kriventseva, E. V., ... & Guttman, M. (2018). The lncRNA Braveheart is required for cardiovascular lineage commitment. Molecular Cell, 72(2), 335–347. https://doi.org/10.1016/j.molcel.2018.08.032

IACUC 1022

IBC Miami0063_Schumacher_2025