Progenitor cell maturation and initiation of neurogenesis in the developing vertebrate neural retina.

Abstract

The mature vertebrate central nervous system is composed of an enormous number of neuronal and glial cells. A relatively small number of progenitor cells generate these cells during a finite period of time of development. Progenitor cells during early stages of central nervous system development divide so that each division produces two progeny that divide again. This `preneurogenic' mode of division is essential for the exponential increase of number of progenitor cells. Later, progenitor cells change their mode of division to `neurogenic', where one or both daughter cells produced by a division withdraw from the mitotic cycle and differentiate. This more mature, neurogenic division is critical for generation of a functional nervous system. The aim of the project described in this thesis was to understand: 1) the molecular differences that dictate the two modes of progenitor cell division, namely preneurogenic and neurogenic, 2) the mechanism that regulates the switch in the mode of division, and 3) the molecular trigger that initiates differentiation. Molecular differences between preneurogenic and neurogenic progenitor cells were identified, and are described in more detail in chapter II. The early, preneurogenic progenitor cells express the transcription factor, Sox2, and a ligand for the Notch receptor, Delta1. The more mature, neurogenic progenitor cells express Sox2 and the bHLH transcription factor, E2A, and do not express Delta1. Perturbation of Notch signaling resulted in conversion of progenitor cells from preneurogenic to neurogenic and in premature neurogenesis. Furthermore, Sonic hedgehog was found to be expressed by a subset of newly differentiating cells. Misexpression of Sonic hedgehog led to premature maturation of preneurogenic progenitor cells and neurogenesis. These results suggest that Notch signaling maintains progenitor cells in the preneurogenic state and that Sonic hedgehog initiates progenitor cell maturation. Certain proneural bHLH transcription factors were found to initiate neurogenesis, and are described in more detail in chapter III. Expression of a number of proneural bHLH factors comes up in a stereotypic temporal sequence prior to the onset of ganglion cell differentiation. Ascl1 and Neurog2 were expressed first, which was followed by expression of Neurod1 and Neurod4. Finally, Atoh7 was expressed, which preceded the appearance of ganglion cells. Individual progenitor cells expressed heterogeneous combinations of proneural genes prior to ganglion cell genesis. Misexpression of Ascl1 or Neurog2 in preneurogenic retina was sufficient to initiate ganglion cell genesis. Misexpression of Neurog2 initiated the stereotypic sequence of proneural gene expression that normally preceded ganglion cell genesis. Ascl1 expression was also sufficient to initiate ganglion cell genesis. However, it functioned by a mechanism distinct from that of Neurog2. These results suggest that ganglion cell genesis may be initiated by two different mechanisms.