Regulation of Zebrafish Hindbrain Development by Fibroblast Growth Factor and Retinoic Acid: A Dissertation

Abstract

Fibroblast growth factor (Fgf) and Retinoic acid (RA) are known to be involved in patterning the posterior embryo. Work has shown that Fgf can convert anterior tissue into posterior fates and that embryos deficient in Fgf signaling lack posterior trunk and tail structures. Likewise, studies performed on RA have shown that overexpression of RA posteriorizes anterior tissue, while disrupting RA signaling yields a loss of posterior fates. While it appears these signals are necessary for posterior development, the role Fgf and RA play in development of the hindbrain is still enigmatic. A detailed study of the requirements for Fgf and RA in the early vertebrate hindbrain are lacking, namely due to a deficiency in gene markers for the presumptive hindbrain at early developmental stages. In this study, we make use of recently isolated genes, which are expressed in the presumptive hindbrain region at early developmental stages, to explore Fgf and RA regulation of the early vertebrate hindprain. We employed both overexpression and loss of function approaches to explore the role of Fgf in early vertebrate development with an emphasis on the presumptive hindbrain region in zebrafish embryos. By loss of function analysis, we show that Fgf regulates genes expressed exclusively in the hindbrain region (meis3 and hoxbla) as well as genes whose expression domains encompass both the hindbrain and more caudal regions (nlz and hoxb1b), thus demonstrating a requirement for Fgf signaling throughout the anteroposterior axis of the hindbrain (rostral to caudal hindbrain) by mid-gastrula stages. To further characterize early gene regulation by Fgf, we utilized an in vitro system and found that Fgf is sufficient to induce nlz directly and hoxb1b indirectly, while it does not induce meis3 or hoxb1a. Furthermore, in vivo work demonstrates that Fgf soaked beads can induce nlz and hoxb1b adjacent to the bead and meis3at a distance. Given the regulation of these genes in vitro and in vivo by Fgf and their position along the rostrocaudal axis of the embryo, our results suggest an early acting Fgf resides in the caudal end of the embryo and signals at a distance to the hindbrain. We detect a similar regulation of hindbrain genes by RA at gastrula stages as well, suggesting that both factors are essential for early hindbrain development. Interestingly however, we find that the relationship between Fgf and RA is dynamic throughout development. Both signals are required at gastrula stages as disruption of either pathway alone disrupts hindbrain gene expression, but a simultaneous disruption of both pathways at later stages is required to disrupt the hindbrain. We suggest that Fgf and RA are present in limiting concentrations at gastrula stages, such that both factors are required for gene expression or that one factor is necessary for activation of the other. Our results also reveal a changing and dynamic relationship between Fgf and RA in the regulation of the zebrafish hindbrain, suggesting that at segmentation stages, Fgf and RA may no longer be limiting or that they are no longer interdependent. As we have demonstrated that an early Fgf signal is required for gastrula stage hindbrain development, we next questioned which Fgf performed this function. We have demonstrated that the early Fgf signal required for hindbrain development is not Fgf3 or Fgf8, two Fgfs known to be involved in signaling centers at the mid-hindbrain boundary (MHB) and rhombomere (r) 4. We further show that two recently identified Fgfs, Fgf4 and Fgf24 are also insufficient alone or in combination with other known Fgfs to regulate hindbrain gene expression. However, as Fgfs may act combinatorially, we do not rule out the possibility of their involvement in early hindbrain gene regulation. However, as time passes and additional Fgfs are isolated and cloned, the elusive Fgf signal required for early hindbrain development will likely be identified. Taken together, we propose that an early acting Fgf residing in the caudal end of the embryo regulates hindbrain genes together with RA at gastrula stages. We suggest that both Fgf and RA are required for gene expression at gastrula stages, but this requirements changes over time as Fgf and RA become redundant. We also demonstrate that the Fgf required for gastrula stage hindbrain development has yet to be identified.