During vertebrate development, early patterning of numerous tissues and cell types occurs consistently with great precision and accuracy. Tissues are patterned by morphogens, one major one being a derivative of dietary Vitamin A called retinoic acid (RA). Intriguingly, patterning in response to RA is both robust (i.e. less variable) and accurate, with tissues consistently forming in the same manner from embryo to embryo with sharp boundaries, despite variations in genetic background, environmental factors, and noise inherent in biological processes. Despite their importance in development, the mechanisms that regulate this robustness and accuracy are still not well understood. To unravel these regulatory mechanisms, we utilized various methods from computational modeling to dissect patterning events in hindbrain segmentation to gene knockouts in animal models to study individual roles of specific binding proteins in regulating responses to RA. To understand the mechanisms that promote robust responses to RA, I first examine the process of hindbrain segmentation by the RA morphogen gradient. During vertebrate hindbrain development, the neural tube is subdivided along the anterior-posterior (A-P) axis into seven distinct segments called rhombomeres (r). Boundaries between rhombomeres are initially rough and jagged, with cells expressing different segment identity genes intermingling at transition zones spanning a few cell diameters wide at prospective boundaries. Mechanisms responsible for boundary sharpening have been investigated for the boundaries between r3/4 and r4/5 in the hindbrain revealing insights into interactions between RA and segment defining genes hoxb1a and krox20 in determining segmental patterning. However, these previous studies were limited in that they focused solely on those boundaries, a single morphogen gradient, and did not model the correct tissue dynamics that occurred such as convergent extension. To expand upon these studies, together with my mathematical collaborator Yuchi Qiu, we computationally modeled these processes using new parameters that included both changing tissue dimensions and more complex gene regulatory networks. Through our new model, we revealed a novel role for rapid tissue elongation in boundary sharpening and maintenance of small segment size. We also explained how two different morphogens, fibroblast growth factor (FGF) and RA which vary in range of signaling activity, synergize to specify multiple boundaries (r2-6) and consistent segment size robustly and accurately.
Then to understand at the cellular level what mechanisms regulate robust responses to RA, I investigated the roles of cellular retinoic acid binding proteins (Crabps). Vertebrates have two highly conserved Crabps, Crabp1 and Crabp2, which transport RA to its receptors in the nucleus or to RA degradation enzymes in the cytoplasm. These dual roles make Crabps excellent candidates for regulating intracellular RA levels and promoting robust cellular responses to RA. To investigate this, I generated mutants for all Crabp1s and Crabp2s in zebrafish. I discovered stark contrasts in sex ratios between Crabp null mutants, with males being overly represented in Crabp2 null mutants. Upon closer examination of mutant gonads and through exogenous RA treatments, I showed Crabp2s mediate RA signaling to promote germ cell proliferation and male sex determination as opposed to Crabp1s, which inhibit germ cell proliferation promoting female sex determination. This revealed a new role of Crabps in mediating RA responses during gonad development and maintaining appropriate sex ratios in a population. In my thesis, I provide insights into the mechanisms in development used to achieve both accurate and robust tissue patterning and cellular responses to RA.
