Research

Cohorts of neurons are organized into anatomically specialized regions that are functionally connected by neural circuits. Allocating distinct types of neurons from uncommitted progenitor cells and the precision of neuronal connectivity requires the coordination of cell fate programming, differentiation, and neural circuit formation (See figure, panel A). I am interested in how genes and signaling pathways function at specific developmental stages to control these processes. My lab studies these mechanisms in the thalamus, striatum, and dopamine system (See figure, panel B) because these regions regulate perception, sensation, sleep, motivation, and movement and are affected in complex brain disorders including autism, epilepsy, and schizophrenia. Using genetic approaches in mice, we ascertain how neuronal subtypes are established and become functionally connected. We also determine how mutations induced at specific embryonic stages affect brain development and cause complex behavioral phenotypes. Our knowledge of developmental mechanisms is being used to advance stem cell and pharmacological therapies to ameliorate brain disease.

RESEARCH.

During development, specific cohorts of neurons are organized into anatomically specialized regions that are functionally connected by neural circuits. The correct allocation of specialized types of neurons from uncommitted progenitor cells and the precision of neuronal connectivity that occurs during brain development requires the coordination of cell fate programming, differentiation, and neural circuit formation. We are testing hypotheses that will uncover how the WNT1 and Tsc1/mTOR signaling pathways regulate neuronal fate and neural circuit formation.

We are interested in mechanisms that regulate the development of subcortical brain regions including the dopamine system, thalamus, and striatum. These subcortical regions are functional nodes that regulate motivation, sleep, movement, perception, and sensation and underpin catastrophic diseases including Parkinson's disease, schizophrenia, sleep disorders, autism, and epilepsy. We are now poised to ascertain how these nodes are established and subsequently how the nodes are connected by specific neural circuits. We also determine how genetic mutations at specific time points affect the development and function of the subcortical nodes. Finally, using our knowledge of developmental mechanisms we aim to advance cell and pharmacological based therapies to ameliorate neurological disease.

The dynamic temporal requirement of Wnt1 in midbrain dopamine neuron development.An overarching question in developmental biology with clinical implications is how specific classes or subtypes of neurons are established from an uncommitted progenitor pool. We are interested in this problem in the context of midbrain dopamine neuron subtypes that are differentially affected in Parkinson's disease and schizophrenia. We focus on the role Wnt1 in dopamine neuron development using a diverse array of genetic resources that we developed. Using Wnt1-YFP transgenic mice, we uncovered that the molecular identity of dopamine neuron progenitors dynamically changes over time. Our short-term lineage tracing and gene expression analysis showed that progenitors that have early and persistent expression of Wnt1 become dopamine neurons. In addition, a cohort of progenitors that expresses a pulse of the transcription factor Gbx2 become dopamine neurons that form the medial forebrain bundle, which is the midbrain dopamine to ventral striatum neural circuit. We then used long-term genetic inducible fate mapping to show that there are two peaks of Wnt1 lineage contribution to dopamine neurons. To determine the spatial and temporal requirement of Wnt1 in dopamine neuron development, we generated a conditional Wnt1fl/fl allele and temporally deleted Wnt1 concomitant with genetic lineage analysis. Our findings show that Wnt1 regulates Lmx1a early in all dopamine neuron progenitors. Subsequently, there are Wnt1-dependent, but Lmx1a-independent progenitors that differentiate into medial-caudal midbrain neurons. Cell cycle analysis revealed that the loss of Wnt1 results in precocious cell cycle exit and ectopic early born dopamine neurons positioned laterally and a depletion of medial dopamine neurons. These studies show the critical nature of timing of gene expression in dopamine neuron differentiation.

Embryonic stem cells (ESCs) and induced pluripotent stem cells present exciting opportunities for cell replacement strategies and to interrogate disease mechanisms and cellular phenotypes. We established ESC technology to evaluate the subtypes of dopamine neurons generated from ESCs in comparison to dopamine neuron progenitors. Interestingly, we showed that ESC-derived neural precursors express WNT1 prior to differentiating into dopamine neurons suggesting that Wnt1 is required to program dopamine neurons from ESCs. We generated novel conditional Wnt1fl/fl and Wnt1del/del ESCs and showed that the absence of Wnt1 resulted in significantly fewer dopamine neurons compared to controls. Interestingly, all calbindin-expressing dopamine neurons were depleted in the absence of Wnt1. These findings suggest both Wnt1-dependent and Wnt1-independent mechanisms control dopamine neuron subtypes. We are testing the hypothesis that the concentration of the signaling molecules SHH and FGF8 cooperate with WNT1 to specify a medial versus lateral dopamine neuron fate. By using control morphogen concentration in ESCs coupled with quantitative assessment of biomarkers and Illumina sequencing, we aim to provide a clear view of the molecular architecture of ESCs programmed to become dopamine neurons. The successful completion of this research will establish mechanisms that shape the distinct subtypes of dopamine neurons to produce specific classes of dopamine neurons.

The dynamic temporal requirement of Tsc1 and mTOR signaling in neural circuit formation. Tuberous Sclerosis (TS) is a developmental genetic disorder that affects 1:6000 live births and causes cognitive deficits in 50% of TS patients, autism in 30-50% of TS patients, and epilepsy in 90% of TS patients. We are studying the thalamus and thalamocortical circuits in TS because human TS patients that perform poorly on cognitive tasks have significant changes in thalamic grey matter volume. Genetically, TS is caused by mutations in Tsc1 following a two-hit model that generates a mosaic tapestry of mutant and unaffected cells. Tsc1 encodes a protein that negatively regulates mammalian target of rapamycin (mTOR) pathway and the deletion of Tsc1 causes mTOR dysregulation culminating in increased S6K1 activity. We are now testing the hypothesis that defects in thalamic circuits disrupt neurological function in TS. We first deleted murine Tsc1 gene at E12.5 when recombination throughout the entire developing thalamus and subsequently analyzed the molecular, cellular, and behavioral consequences in developing and adult mice. Our findings show that the thalamus is sensitive to Tsc1 inactivation as evidenced by mTOR pathway dysregulation within forty-eight hours of Tsc1 deletion. This early deletion resulted in neuronal overgrowth and a defect in thalamic axon organization en route to their cortical targets including layer IV barrels in somatosensory cortex, which we detected using genetic inducible circuit tracing. Interestingly, at their final target site in somatosensory cortex, thalamic axons did not properly delineate the layer IV barrels and appeared to form ectopic connections in the adjacent septal neurons. Importantly, we showed that mutant VB thalamic axons that innervate genetically unaffected somatosensory cortex caused a secondary patterning defect thus exacerbating the primary genetic mutation. Finally, Tsc1 deletion in the thalamus at midgestation caused abnormal compulsive repetitive (grooming) behaviors and frequent spontaneous seizures.

We are now using our model of TS to interrogate when during brain development mTOR inhibitors are most effective at ameliorating neurological deficits without disrupting normal brain development. In addition, we are using mTOR inhibitors to rescue specific developmental defects based on the time and duration of mTOR inhibition. Finally, we are using slice electrophysiology, intracranial multiunit recordings, and high throughput behavioral testing in collaboration with Dr. Barry Connors, Dr. Chris Moore, and Dr. Kevin Bath to further elucidate physiological correlates that link our temporal gene deletion/circuit marking to behavioral abnormalities in TS. Collectively, these approaches will link temporal and spatial Tsc1 deletion to specific physiological and behavioral changes. The clinical relevance of this approach is that we can evaluate how mTOR inhibitors affect specific cellular, physiological and behavioral abnormalities associated with TS. In the broader context, we will understand how thalamic neurons and circuity impact complex developmental brain disorders.

Summary of Research.

My research program uses sophisticated genetics approaches including conditional gene deletion, genetic inducible fate mapping, and genetic circuitry tracing. We are now unraveling the mechanism of Wnt1 regulation of cell cycle exit and dopamine neuron fate decisions. We are further refining how multiple genetic lineages contribute to dopamine neuron subtype identity and are identifying the molecular architecture that shapes dopamine neuron specification and neural circuit formation. Using this knowledge we are programming our ESC lines with a long-term goal of transplanting them into mutant mice that are devoid of dopamine neuron subtypes to test the capacity of ESCs to differentiate, integrate, and rescue circuits associated with the specific loss of dopamine neuron subtypes. We have also developed a powerful genetic system that mimics salient features of human TS and will utilize this system to unravel the mechanism of TSC1/mTOR regulation of neural circuit formation. We are establishing a molecular pathway that links mTOR, pFMRP, and SAPA3 to regulate synapse formation. We are also exploiting our temporal and spatial control of Tsc1 deletion to determine how the timing and duration of mTOR inhibitors affects TS.