REGULATION OF A NODAL INHIBITOR DAND5 VIA FLOW SENSATION AND MIRNAS

Left-right (LR) development of internal organs is highly conserved among vertebrates. Deregulation of factors controlling this process contributes to developmental defects, such as those leading to congenital heart defects. LR patterning in zebrafish is regulated early in development by leftward fluid flow generated by cilia of cells lining Kupffer’s Vesicle (KV). This is proposed to lead to increased intracellular Ca²⁺ concentrations which eventually down-regulate the nodal inhibitor, dand5, on the left of KV. A cation channel, Pkd2, is believed to be responsible for transporting Ca²⁺ into cells via the cilium. Recent studies argue against this mechanosensory mechanism and support the hypothesis that Pkd2 localized to the endoplasmic reticulum (ER) is responsible for increases in Ca²⁺. We were also interested in identifying the effector responsible for dand5 regulation further downstream of flow sensing. We hypothesized that 1) dand5 expression in KV is regulated by Pkd2 activity in the ER and 2) dand5 asymmetry is regulated by miRNAs that target the 3’ UTR of dand5 mRNA. To confirm our first hypothesis, we blocked Pkd2 protein expression in embryos using morpholinos (MO). In-situ hybridizations were then used to visualize dand5 expression in KV. Embryos treated with pkd2 MO (n=33) exhibited randomized heart jogging, with 45.46% having jogs to the left, 21.21% centered, and 33.33% to the right. In-situ hybridizations of these embryos (n=12) showed that 16.67% had right biased expression within KV and 83.33% had bilaterally symmetric expression of dand5, compared to uninjected embryos (n=12) which had 91.67% right biased expression and 8.33% bilaterally symmetric expression. We also examined the level of phenotypic rescue in pkd2 mutants injected with hPKD2 RNAs that produced proteins with different cell localization behaviors. Embryos rescued through RNA injections were imaged 72 hours post fertilization (hpf). RNA injections were administered during the single cell stage. pkd2 mutant zebrafish exhibiting curly tails and heart jogging defects were partially rescued by wildtype PKD2 RNA and by PKD2 S812D RNA, a variant who’s protein primarily localizes to the ER. To address our second hypothesis, we used a MO that blocked predicted dand5 miRNA target sites (dand5- TP). dand5- TP and control MO were injected into the yolk syncytial layer (YSL) at ~1K cell stage and embryos were imaged at 30% epiboly and 14 somite stage. Heart jogging was observed ~24 hpf. Using MO target protectors (TP), we showed that dand5 in KV is downregulated by miRNAs. Embryos injected with dand5- TP into YSL (n=39) exhibited left (69.23%), centered (10.26%), or right (20.51%) heart jogging phenotypes. Further investigations are needed to determine the localization of Pkd2 needed for proper development, as well as determining the connection between flow sensation, Pkd2 activity, and asymmetric expression of dand5 in KV.