Molecular genetic analysis of hindlimb development in zebrafish
Evolutionary Cell and Developmental Biology (Zoology)
Final Report Abstract
In zebrafish, the molecular mechanisms underlying pectoral fin initiation and development are well characterized. In contrast, the formation of the pelvic fins (homologous to tetrapod hindlimbs) is only poorly understood and comparably few genes are known that act in the initiation process. In the prevailing model of proximal-to-distal (PD) specification of the vertebrate limb, derived from experiments performed in the chick model, antagonising gradients of proximal (retinoic acid, RA) and distal (fibroblast growth factors, Fgfs) signals control limb bud regionalization. However, evidence from the mouse suggests that endogenous RA plays no role in establishing or maintaining cell fates in the hindlimbs. It is thus possible that RA- independence of hindlimb development is specific to mammals. To provide support for this hypothesis, we examined whether zebrafish exhibit a specific requirement for RA in hindlimb initiation and development. To systematically examine the contribution of the retinoic acid (RA) signaling pathway to hindlimb/pelvic fin development, we first developed a staging series of zebrafish larvae that covers pelvic development (between 3-5 weeks post fertilization) and is based on fluorescent reporter genes that mark mesenchymal cartilage precursor cells, endothelial cells and cartilage. This staging series (stages S0-S14) allowed us to apply a pharmacological antagonist of RA synthesis throughout defined stages of pelvic fin development and generated RA loss-offunction phenotypes similar in effect to a complete allelic series: Without full RA signaling, starting several hours prior to the emergence of pelvic fin buds (S0), the pelvic girdle and subsequently the fin buds fail to form. Dependence on RA extends further until S4, when fin buds form a small lobe, resulting in skeletal defects that include a smaller fin base and reduced outgrowth of anterior processes as well as fewer fin rays. We conclude that RA is required for pelvic fin induction and serves subsequent roles in patterning processes of the pelvic skeleton. These findings suggest that, similar to the findings in vertebrate forelimbs and hindlimbs in birds, RA is essential for hindlimb formation. We then established appropriate lines using the Gal4/UAS system to be able to study the underlying molecular mechanisms and to have a more accurate control of RA signaling. We generated an effector line that efficiently blocks RA signaling (UAS:dnrara2a) as well as several lines that drive expression of an inducible form of Gal4 (ERT2-Gal4-VP16) under the transcriptional control of paired fin-specific enhancers for pitx1 and prrx1a/-1b1. We are currently in the process of optimizing the conditions to inducibly inhibit RA signaling during critical phases of pelvic fin development. In order to identify a new gene with essential roles in pelvic fin development, we have mapped and characterized the NZ045 mutant, which exhibits reduced pelvic fins and shortened dorsal and anal fins, by sequencing the single candidate gene located between the closest linked markers. We could confirm that mutants do not possess point mutations that would lead to a non-synonymous amino acid change or disrupt the protein in any other way. We could identify, however, two new coding exons at the 5’-end of the gene, which include a start codon homologous to the sequence in other vertebrates. Experiments are under way to determine whether candidate gene expression is altered in mutants. Together, our findings implicate RA in zebrafish hindlimb development and support the validity of the PD-model of limb development in a fish model. New lines utilizing the Gal4/UAS system will allow us to uncover the underlying mechanisms.
