Allocating specialized types of neurons and establishing their functional connections requires cell fate programming, differentiation, and neural circuit formation. We interrogate these coordinated mechanisms in midbrain dopamine neurons and thalamus relay neurons. We study these cells because they control movement and cognition, and are affected in Parkinson's disease, autism, and epilepsy. We also use knowledge of development to advance stem cell and pharmacological therapies in brain disease.

Overview of Research Interests
My lab has two primary research tracts that investigate how signaling pathways shape neuronal fate and neural circuit formation during development. We also established a mouse model of a complex developmental brain disorder that is leading to a deeper understanding of how specific cohorts of neurons and neural circuits contribute to brain disease. Finally, we are exploiting our knowledge of development and disease mechanisms to design cell-based and pharmacological therapies.

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 affective 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.

Active Research Support

2014-2017 DOD-CDMRP Idea Development Award

Title: Timing of mosaic gene deletion in mouse blastocysts and multi-tissue disease development in Tuberous Sclerosis.

Role: Principal Investigator.

 

2013-2015 Simons Foundation Autism Research Initiative

Title: Linking genetic mosaicism, neural circuit abnormalities and behavior.

Role: Principal Investigator.

2012-2015 DOD-CDMRP Idea Development Award (TS110083; $450,000)
Title: Temporal loss of Tsc1: Neural development and brain disease in Tuberous Sclerosis.
Role: Principal Investigator.
The major goals of this project are to identify critical windows of brain development that are affected by the loss of Tsc1 and mTOR dysregulation during embryonic development and to ascertain the impact of mTOR inhibitionon developing neurons during normal development and in Tuberous Sclerosis.

2011-Present COBRE Pilot Project sub-award; $12,500)
Title: Determining the role of mTOR signaling in dopamine neuron cell fate decisions.
Role: M. Zervas Sub-award Project leader (W. Atwood Principal Investigator, NIH 5-27961).
The major goal of this award is to develop stem cell based research projects designed to advance our understanding of programming embryonic stem cells and induced pluripotent stem cells. In addition, these funds are intended to foster a multi-disciplinary collaboration in stem cell biology (Brown Stem Cell Group).

2011-2013 DOD-CDMRP Exploration Hypothesis Development Award (TS100067; $100,000)
Title: Determining Changes in Neural Circuits in Tuberous Sclerosis.
Role: Principal Investigator.
The major goals of this project are to conditionally delete Tsc1 in the thalamus during embryonic development and ascertain the impact on thalamocortical circuitry formation and on thalamic neuron physiology.

2010-2013 NIH/NCRR RI Hospital COBRE Center for Stem Cell Biology
Competitive Proposal Submission for Full Project (701-1960; $450,000)
Title: Determining the Transcriptional Regulation and Cell Signaling Events that Shape the Molecular Identity of Dopamine Neuron Progenitors and Specify Subtypes of Midbrain Dopamine Neurons.
Role: M. Zervas Project Leader (P. Quesenberry Principal Investigator, NIGMS 8P20GM103468-04)
The major goals of this project are to: 1. Elucidate the molecular identity of dopamine neuron progenitors; 2. Determine the genetic basis of dopamine neuron heterogeneity; 3. Investigate the role of WNT, SHH, and FGF8 signaling in establishing the molecular idenity cell and fate specification of dopamine neuron progenitors.

2010-Present Richard B. Salomon Faculty Research Award (2-34310; $15,000)
Title: Genetic Dissection of Midbrain Dopamine Neuron Diversity.
Role: Principal Investigator
The goal of this project is to identify novel transcriptional regulators of dopamine neuron diversity using mouse genetic mutants, fluorescent activated cell sorting and microarray.

Hagan N, Guarente J, Ellisor D and Zervas M (2017). The temporal contribution of the Gbx2 lineage to cerebellar neurons. Front Neuroanat 11:50. doi: 10.3389/fnana.2017.00050 (PMID: 28785208).

Brown S and Zervas M (2017). Temporal expression of Wnt1 defines the competency state and terminal identity of progenitors in the developing cochlear nucleus and inferior colliculus. Front Neuroanat 11:67. doi: 10.3389/fnana.2017.00067 (PMID: 28878630).

Dingle YL, Xiong KB, Machan JT, Seymour KA, Ellisor D, Hoffman-Kim D, Zervas M (2016). Quantitative analysis of dopamine neuron subtypes generated from mouse embryonic stem cells. bioRxiv doi:http://dx.doi.org/10.1101/093419.

Normand EA, Crandall SR, Thorn CA, Murphy EM, Voelcker B, Browning C, Machan JT, Moore CI, Connors BW, Zervas M (2013) Temporal and mosaic Tsc1 deletion in the developing thalamus disrupts thalamocortical circuitry, neural function, and behavior. Neuron 78(5):895-909 (PMID: 23664552).

Yang J, Brown A, Ellisor D, Paul E, Hagan N, Zervas M (2013) Dynamic temporal requirement of Wnt1 in midbrain dopamine neuron development. Development 140(6):1342-1352 (PMID: 23444360).

Ellisor D, Rieser C, Voelcker B, Zervas M (2012) Genetic Dissection of Midbrain Dopamine Neuron Development in vivo. Dev. Biol. 372:249-262 (PMID:23041116).

Hagan N, Zervas M (2012) Wnt1 expression temporally allocates upper rhombic lip progenitors and defines their terminal cell fate in the cerebellum. Mol. Cell Neurosci.49:217-229 (PMCID: PMC3351839).

Hayes L, Zhang Z, Albert P, Zervas M*, Ahn S* (2011) The timing of Sonic Hedgehog and Gli1 expression segregates midbrain dopamine neurons. J. Comp. Neurol. 519:3001-3018 (PMCID: PMC3154975). *Co-corresponding authors.

Brown A, Machan JT, Zervas M (2011) Molecular organization and timing of Wnt1 expression define cohorts of midbrain dopamine neuron progenitors in vivo. J. Comp. Neurol. 519:2978-3000 (PMCID: PMC3359795).

Luu B, Ellisor D, Zervas M (2011) The Lineage Contribution and Role of Gbx2 in Spinal Cord Development. PLoS ONE 6(6): e20940. doi:10.1371/ journal.pone.0020940 (PMCID: PMC3116860).

Ellisor D, Zervas M (2010) Tamoxifen dose response and conditional cell marking: Is there control? Mol. Cell Neurosci. 45:132-138. Selected scientific image featured on cover (PMID: 20600933).

Brown A, Brown B, Ellisor D, Hagan, N, Normand, E, Zervas, M (2009) A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo. J Vis Exp, 43: pii: 1687, doi: 10.3791/1687 (PMCID: PMC2846818).

Ellisor D, Koveal D, Hagan, N, Brown, A, Zervas M (2009) Comparative analysis of conditional reporter alleles in the developing embryo and embryonic nervous system. Gene Expr Patterns, 9:475-489 (PMCID: PMC2855890).

Zervas M (2007) Genetics, Neurobiology, and Translational Medicine: The Future of Schizophrenia Research. White Paper, Johnson & Johnson Pharmaceutical Research and Development. 133pp., Role: researcher, author.

Joyner AJ, Zervas M (2006) Genetic inducible fate mapping in mouse: establishing genetic lineages and defining genetic neuroanatomy in the nervous system. Dev. Dynamics 235:2376-2385 (PMID: 16871622).

Zervas M, Blaess S, Joyner AJ (2005) Classical embryological studies and modern genetic analysis of midbrain and cerebellum development. Curr. Topics Dev. Biol., (Neural Development), 69:101-138. Invited review; Selected scientific image featured on cover (PMID: 16118800).

Zervas M, Opitz T, Edelmann W, Wainer B, Kucherlapati R, Stanton P (2005) Impaired Hippocampal Long-Term Potentiation (LTP) in Microtubule-Associated Protein 1B-deficient Mice. J. Neurosci. Res. 82:83-92 (PMID: 16243598).

Zervas M, Millet S, Ahn S, Joyner AJ (2004) Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1. Neuron 43:345-357 (PMID: 15294143).

Zervas M, Somers KL, Thrall MA, Walkley SU (2001) Critical role for glycosphingolipids in Niemann-Pick disease type C. Curr. Biol. 11(16):1283-1287 (PMID: 11525744).

Zervas M, Dobrenis K, Walkley SU (2001) Neurons in Niemann-Pick disease type C accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations. J. Neuropathol. Exp. Neurol. 60(1):49-64 (PMID: 11202175).

Walkley SU, Zervas M, Wiseman S (2000) Gangliosides as modulators of dendritogenesis in normal and storage disease-affected pyramidal neurons. Cereb. Cortex 10(10):1028-1037 (PMID: 11007553).

Zervas M and Walkley SU (1999) Ferret pyramidal cell dendritogenesis: changes in morphology and ganglioside expression during cortical development. J. Comp. Neurol. 413(3):429-448 (PMID: 10502250).

Walkley SU, Siegel DA, Dobrenis K, Zervas M (1998) GM2 ganglioside as a regulator of pyramidal neuron dendritogenesis. Annals of the NY Academy of Science 845:188-199 (PMID: 9668352).

Edelmann W, Zervas M, Costello P, Roback L, Fischer I, Hammarback JA, Cowan N, Davies P, Wainer B, Kucherlapati R (1996) Neuronal abnormalities in microtubule-associated protein 1B mutant mice. Proc. Natl. Acad. Sci. USA 93:1270-1275 (PMID: 8577753).

2011 Invited to co-organize the Northeast Regional Meeting of the Society for Developmental Biology

2009 Tuberous Sclerosis Travel Award

2006-Present Manning Assistant Professorship

2003-2006 NIH Ruth L. Kirschstein National Research Service Award (F32HD43533)

Member, Society for Developmental Biology
Member, Society for Neuroscience

Teaching
Course Leader
BIOL1310 and BIOL2310: Analysis of Development

Course Leader
BIOL2320A: Current Topics in Developmental Biology: Cell Fate and Lineage Decisions in Neural Development and Neurological Diseases

Mentoring
Mentoring is an important part of my teaching. Four undergraduates in my lab have completed multiyear independent study projects, culminating in senior honors theses and authored publications. My undergraduates have been awarded a total of five Brown University Undergraduate Teaching Research Awards, a RI-INBRE Summer Undergraduate Research Fellowship, and a Brown University Galkin Award. I also mentored students participating in the UMBC MARC U*STAR Summer Intern program, the BP-ENDURE at Hunter/NYU Neuroscience Research program, and a high school student during her senior year. I trained five graduate students with three having earned their Ph.D. and two that are in the final year of their thesis work. My graduate students have earned the Reismann Fellowship, Kaplan Summer Graduate Research Award, and Brain Science Graduate Research Awards. My graduate students have given talks at meetings, won poster prizes for their efforts, and are postdocs at Harvard and Johns Hopkins. Student related publications are highlighted in my biosketch.

My teaching philosophy focuses on connection and transformation. My primary pedagogical goal is to establish with students, a deeper understanding of the genetic and molecular architecture that drives morphological diversity. I teach an upper level undergraduate class "Analysis of Development" and since becoming the sole instructor I redesigned the course with an emphasis on modern developmental genetic tools and the integration of development and disease mechanisms. I teach a diverse array model experimental systems and concepts that include, but are not limited to gene regulation, lineage restriction, cell fate specification, development and disease, stem cell programming and therapeutic approaches, environment and development, and evolution and development. This is an unusual undergraduate course because it draws all lecture material from primary literature, which the students learn to read, critically evaluate, and discuss. I coordinate, oversee, and teach a cluster of labs designed to augment the lecture material. The labs include Mammalian Developmental Genetics: Mouse, Neural Crest/Compartments: Axolotl, and Chick Embryonic Development. This course also has a seminar component where students give a ten minute slide presentation on a developmental biology topic that was not directly covered in the lectures; seminars were concomitant with a ten page research paper. Notably, exam questions were drawn from student seminars to foster student participation and ownership by linking their presentation and course material. I also taught "Topics in Developmental Biology: Genetic Control of Cell Fate Decisions" and "Current Topics in Developmental Biology: Cell Fate and Lineage Decisions in Neural Development and neurological diseases." These two graduate level seminar courses focused on cell signaling and transcriptional regulation in programming cell identity, and explored complex neurological diseases.