Spatial and temporal mechanisms of cell fate determination in the developing CNS

Abstract

The generation of neural cell diversity in the developing central nervous system relies on mechanisms that provide spatial and temporal information to neural progenitor cells. The deployment of morphogen gradients is an important strategy to impart spatial information to the field of responding cells. In this process, cells translate different concentrations of signal into the expression of distinct sets of cell fate-determining transcription factors, which determine cell fate as progenitors leave the cell cycle and differentiate into neurons. However, the mechanisms by which time regulates cell fate determination are poorly understood. The aim of this thesis is to better understand the mechanisms of spatial and temporal patterning in the specification of neural cell types.

In the ventral half of the neural tube, the graded activity of Sonic hedgehog (Shh) has been proposed to specify the patterned generation of distinct neuronal subtypes. It remains unclear, however, whether non-graded mechanisms of Shh signaling also contribute to this process. We show that Shh-induced Nkx2 proteins intrinsically amplify Shh responses and that this activity is important to specify floor plate (FP) and V3 fates in the ventral spinal cord. Conversely, Pax6 antagonizes Shh signaling and constrains its inductive activity over time. Furthermore, our data suggest that the spatial patterning of FP and V3 cells reflects a switch of neuronal potential in neural progenitors and not a requirement for different concentrations of Shh. Together, this study indicates that the output of graded Shh signaling depends on dynamic and non-graded changes of competence in responding cells.

At the hindbrain level, the progenitor domain dorsally abutting the FP generates visceral motor neurons (vMN) at early stages of development. To better understand the genetic program of vMN specification, we studied the role of proteins expressed in vMN progenitors during this process. We show that Nkx2.2 is sufficient to activate the expression of Phox2b, an important determinant of vMN fate. Moreover, the redundant activities of Nkx6.1 and Nkx6.2 proteins are not required for the generation of vMNs, but are important to prevent the parallel activation of dorsal cell fate differentiation programs and to ensure proper migration and axonal projection of vMNs. Thus, our data establish complementary roles for Nkx2.2 and Nkx6 proteins in the establishment of vMN identity.

In contrast to spatial patterning, the mechanisms that regulate the sequential generation of distinct cell types from a common pool of progenitors remain poorly resolved. To better understand these mechanisms we analyzed the sequential generation of vMN and serotonergic neurons (5HTN) from a common pool of Nkx2.2+ progenitors in the ventral hindbrain, and found that the temporal specification of these cell types depends on the integrated activities of Nkx and Hox proteins to regulate the temporal expression of Phox2b. In turn, Phox2b functions as a cell fate selector promoting vMN and repressing 5HTN fate. To further understand the vMN-to-5HTN switch, we screened for factors that could regulate this process, and identified Tgfβ2 as a signal that executes the switch through a temporal cross-repressive interaction with Phox2b. Moreover, we show that prolonged Shh activity establishes the initial period of vMN fate and induces Tgfβ2 expression with a temporal delay. Together, our studies reveal that a Shh-Tgfβ signaling relay mechanism regulates the sequential generation of vMNs and 5HTNs in a dynamic process that can be modulated by determinants controlling spatial patterning.