Charecterization of Mesodermal Percursor Expression in Zebrafish Integrin Mutants 

Congenital heart defects (CHD) are developmental and functional abnormalities of an infant's heart at birth. CHD are the most common type of birth defect affecting approximately 1 in every 115 babies. Of all CHD, only about 13% have known causes. Zebrafish are used as a model organism to study congenital heart defects. Zebrafish serve as an excellent model to study CHD as they have the ability to survive without a functioning cardiovascular system for up to 4 days due to their thin skin which allows for oxygen diffusion. Zerbrafish have high conservation of signaling pathways and mechanisms allowing for translation into other models. Interaction between mesodermal precursors and their extracellular environment is critical to proper development. The mesoderm is a germ layer in the embryo where many organs derive from. Integrins are transmembrane receptor proteins that bind extracellular matrix proteins which leads to signal transduction of migration and proliferation. Integrins are made up of a combination of non-covalently bound alpha and beta subunits. Integrin ɑ4 and ɑ5 knockout has previously shown to lead to mesodermal derived tissue malformation. Presented here is a further understanding of how ɑ4 and ɑ5 knockout affect characterization of mesodermal percursor populations. Comparison of siblings and mutants was used to determine that there was no statistically significant differences in any precorusor populations other than the “left area” population. This work will bring a understanding of the cellular mechanisms that lead to heart defects during development. 

Integrins are cellular receptors formed by two linked protein chains that act like tiny anchors. These anchors connect cells to a surrounding network of molecules called the extracellular matrix, which provides support and structure. When Integrins latch onto a specific protein in the extracellular matrix called Fibronectin, they trigger a series of cellular signals that influence critical processes like cell movement, specialization, and survival. Integrin double mutants present with smaller heads, cardiac edema, shortened body axis. Previous research has shown that analysis of siblings and mutant ɑ4 and ɑ5 knockout leads to a large number of fully separated heart cell clusters as well as smaller hearts on average. Heart tissue is derived from the mesoderm. 

This research began by completing In Situ Hybridization (ISH) for integrin double mutants. ISH is a biological technique used to locate specific DNA sequences, which in this case was two prior discovered sequences of DNA confirmed to be present in mesodermal precursor populations. ISH was performed on both siblings and mutants for both protein markers (HAND2 and Nxk2.5). This allows for visualization of the gene target wherever it is expressed leading to expression of mesodermal DNA sequences only in mesodermal tissue. After ISH, a polymerase chain reaction (PCR) was performed. PCR is a technique that repeatedly duplicates the DNA sequence found previously through heating and cooling nucleic acid strands until millions of copies of the original sequence are present. This allowed for these millions of copies to be analyzed through gel electorphoresis. Gel electrophoresis is the process of separating molecules by charge and size. Because siblings and mutants have different sequence sizes due to the knockout of  ɑ4 and ɑ5 this separation leads to identification of individual zebrafish. After expression patterns and identity for each fish were obtained the samples were analyzed with the Fiji imaging software. This allowed for distance and area measurements to be obtained in pixels. This data was transformed into microns and statistically analyzed in the Prism GraphPad. 

Analysis of siblings and mutants showed that ɑ4 and ɑ5 knockout has no affect on HAND2 expressed precursor populations in the top, middle, and bottom areas as well as both gap distance measurements  and all three width measurements at the 10 somite stage. Nkx2.5 expression in the yolk sac showed a statistically significant larger area in mutants than siblings. All other area and distance measurements had no statistically significant difference. Differences in integrin knockout expression patterns previously seen at 52 hpf in heart cells not seen in 13 of 14 measurements at the 10 somite stage.   

Analyze other mesodermal derived organ morphology such as kidney, interrenal gland, and thyroid gland. Assess expression of precursor populations at cell stages between 10 somite and 52 hpf. Assess effects ɑ4 and ɑ5 knockout has on the bulbous arteriosus. Statistically compare complete areas for HAND2 and Nkx2.5 measurements. Determine specific death rates and times in mutant fish.