| Popis: |
Cell type specification in the ventral spinal cord is controlled by a suite of transcription factors (TFs) whose expression is induced by opposing gradients of retinoic acid (RA) and sonic hedgehog (SHH). The action of these TFs results in the creation of five distinct progenitor domains, four of which produce interneurons and one that produces motor neurons (MNs). Though the transcription factors (Pax6, Sp8, Nkx2.2, Olig2) involved in progenitor domain boundary setting and in the specification of adult cell types from these domains are known, major questions remain as to how these factors are able to specify specific fates. Questions also remain about the function of the TFs involved in boundary specification. Do any of these factors have additional functions? Are they necessary for adult cell type specification? In this thesis I investigate how the putative domain boundary TF Sp8 affects adult fate choice, and how TF specificity might be achieved in nascent MNs born in the spinal cord. In particular, I examine the role of Sp8, in motor neuron cell type choice. I also investigate candidate co TFs that could generate cell type enhancer specificity. In Chapter 2 I describe a novel function for a domain boundary setting TF, Sp8, in specifying adult MN type. In this chapter I leverage in vitro MN differentiation models to examine KO and overexpression lines to define the action of this TF. In addition to its ability to repress dorsal P3 domain this TF also represses the formation of cranial MNs in favor of skeletal MNs and is able to induce skeletal MN TFs. In Chapter 3 I describe a series of experiments that dissect enhancer TF motif content in a search for a TF that, along with known MN TFs, differentiates between active and inactive enhancers bound by the MN TFs Isl1 and Lhx3. Though neither Klf investigated plays this role, their true role in gene regulation in MNs remains obscure. Though the data are preliminary, the motifs investigated in this chapter seem to have some measure of control over enhancer activity. Overall this thesis paints a picture of novel activity for Sp8, separate from its defined minor function in the developing ventral spinal cord, opening new ways to understand cell fate regulation in the ventral spinal cord. It also rules out two candidate MN master TF co-regulators (Klf6 and Klf7), while suggesting the importance of their binding motifs for enhancer function. I propose that the Sp/Klf family of TFs have novel roles in MN gene regulation. |
