My laboratory continues to investigate the role of Sonic hedgehog (Shh) and its downstream signaling components in developing and mature nervous system. The role of Gli3 in neurogenesis: Gli3, one of three vertebrate Gli transcription factors in Hedgehog (Hh) pathway, is processed into a repressor form (Gli3R) in the absence of Hh signal and acts as the major negative transducer of the pathway. Although the role of Gli3 in embryonic patterning has been extensively studied, its role in cortical neurogenesis, especially in the regulation of neural progenitors in proliferation and cell fate specification, is largely unknown. In order to bypass the patterning defects caused by loss of Gli3, we conditionally deleted Gli3 after patterning is complete in mouse. Our results from birthdating and in utero electroporation experiments demonstrate that the Gli3, specifically Gli3R, is critical for specifying the fate of cortical neurons that are generated according to a stereotypical temporal order. Moreover, Gli3 is required for maintaining the cortical progenitors in active cell cycle, suggesting that cells may acquire differentiated status in response to the loss of Gli3 during neurogenesis. Around birth, the embryonic neural stem cells (NSCs) undergo a transition to form the postnatal neurogenic niche in the subvenricular zone (SVZ) of the lateral ventricle. The cytoarchitecture of the SVZ is arranged in a pinwheel structure where multi-ciliated ependymal cells surround a cluster of postnatal NSCs in the center. Since our previous results showed that Gli3, a negative transducer of Sonic hedgehog (Shh) pathway, is required for maintenance of cortical progenitors, we investigated whether Gli3 plays a similar role in the postnatal NSCs. We found that the establishment of pinwheel structures in the SVZ was disrupted in Gli3cko mice where Gli3 is conditionally deleted with Nestin-Cre mice. Specifically, the cells in the SVZ of Gli3cko mice displayed characteristics of both ependymal cells and NSCs: multi-cilia and GFAP expression. Since both ependymal cells and NSCs originate from embryonic NSCs, our findings suggest that Gli3 is critical for their fate specification and establishment of postnatal cytoarchitecture. In addition, ependymal cell-specific deletion of Gli3 using FoxJ1-Cre also resulted in upregulation of NSC markers, suggesting that Gli3 is also required for maintenance of ependymal cell characteristics and suppress NSC features. The genetic lineage of midbrain dopaminergic neurons: Dopamine neurons (DNs) in the ventral midbrain (vMb) play diverse roles in cognitive function, emotion, addiction and motor control. However, the origin of such diversity has not been extensively studied. Here, we show the evidence for the importance of the early genetic lineages of DNs that may determine one aspect of such diversity, the spatial distribution within the vMb. Specifically, we investigated the contribution of cells derived from progenitors expressing Sonic hedgehog (Shh) and Gli1, the downstream target gene of Shh signaling. Using Genetic Inducible Fate Mapping (GIFM) approaches, we found that Shh and Gli1 lineages contribute to spatially overlapping population of DNs with little distinction. However, the temporal contribution of Gli1 genetic lineage preceded the Shh lineage by one day in development, suggesting that the Shh-response may pre-set the future Shh-expression domain within vMb. In addition, the spatial contribution of Gli1 lineages changed from medial to lateral DNs reflecting its dynamic expressions between E8.5-E11.5. In contrast, Shh lineages contributed to a broader domain encompassing both medial and lateral DNs. Interestingly, we found that there is a progression from anterior to posterior contribution of both lineages suggesting that there may exist the important differences in anterior vs. posterior DNs. In addition, we we found that Shh is expressed approximately a day later in Gli1-lineage cells which had turned off Gli1 expression. Thus, there is a wave of medial to lateral shift in Gli1 expression domain that is taken over by Shh expression in vMb. In order to understand whether the inhibition of autocrine Shh signaling is a prerequisite for induction and maintenance of Shh expression within vMb and proper DN development, we are performing gain-of-function and loss-of-function of Shh signaling in Shh- or Gli1-expression domain. Taken together, our results will enhance understanding of temporal sequence of events necessary for proper development of DNs of vMb. Hippocampal neurogenesis: Temporal and spatial control of marking cells of specific genetic lineages provides valuable insights into the gene function during development and maturation processes. However, the current genetic inducible fate mapping (GIFM) approaches utilize only one recombination event that results in an irreversible marking. We developed a dual GIFM system that allow us to control recombination events at two distinct time points with CreER(T2) and FlpE:PR, which are tightly regulated with Tamoxifen and RU486, respectively. In addition, we have developed two universal reporter mouse lines that conditionally express fluorescent protein-tagged wheat germ agglutinin (tWGA:FP), which is transferred across synapses during neural activities. Combinatorial use of our dual GIFM with the novel trans-synaptic reporter system enables us to trace the neural circuits that are formed between adult born granule neurons from Shh-responding neural stem cells with pyramidal neurons of CA3 and subsequently of CA1. Gene expression profiling of adult neural stem cells and their niche: Stem cells reside in the niche, a sheltering microenvironment that is thought to coordinate normal homeostatic production of functional mature cells. We have investigated the nature of the niche signals responsible for the unique properties of neural stem cells (NSCs) in the subventricular zone (SVZ) of the lateral ventricle. While several studies have linked known signaling molecules including Sonic hedgehog (Shh) and Wnts to adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of genetic inducible fate mapping system and transgenic mice to isolate distinct cell types of neurogenic SVZ including NSCs, ependymal cells, astrocytes and transit-amplifying cells. We are currently obtaining the secretory molecule expression profiling (SMEP) of each cell type using Signal Sequence Trap method. We will validate the specific expression of identified genes and assess the physiological function of isolated signaling components in vitro and in vivo.
