Catherine Dulac - US grants

DESCRIPTION (adapted from the applicant's abstract) The studies proposed here will investigate the molecular mechanism of sensory transduction in the vomeronasal organ (VNO). Convergent lines of evidence have shown that transduction cascades elicited by olfactory signals in the main olfactory epithelium (MOE) and by pheromone signals in the VNO are different. However, functional and molecular characteristics of the VNO signal transduction machinery are still largely unknown. In particular, both molecular and physiological approaches have failed, so far, to determine the nature and the mode of activation of the conductance that is activated by pheromone signals, and leads to generation of action potentials. We developed a cloning strategy based on differential screening of cDNA libraries constructed from individual VNO neurons, which led to the isolation of two independent families of genes likely to encode mammalian pheromone receptors. We intend to use a similar strategy in order to identify signal transduction components involved in pheromone mediated signaling. This cloning effort will not make any assumption about the nature of the genes encoding signal transduction components, but will rather take advantage of the different signaling mechanisms that are expressed in MOE and VNO neurons, and that distinguish subpopulations of VNO neurons. Direct analyses of the responses of VNO neurons to physiologically relevant stimuli have been very difficult as few pheromones have been characterized. We intend to use the powerful tools of mouse genetics to develop a transgenic mouse model in which to study the physiological response of VNO neurons to known ligands. This will allow the functional dissection of signal transduction pathways that translate pheromone signals into neural activity, leading to the generation of stereotyped behaviors and neuroendocrine changes.

Studies conducted under this grant are aimed at characterizing brain circuits underlying pheromone- evoked behaviors and endocrine responses in the mouse. Recent data has demonstrated the involvement of both olfactory and vomeronasal (VNO) systems in pheromone processing and the key role of VNO inputs in ensuring the sex specificity of behavioral responses. These findings raise two fundamental questions: how are olfactory and VNO inputs integrated in order for the animal to achieve physiologically relevant responses, and what is the respective role of each chemosensory system and associated receptors in pheromone processing. We will investigate the hypothesis that specific neuropeptide networks integrate both olfactory and VNO pheromone signals throughout the brain in order to mediate reproduction and aggressive responses. Conditional pseudorabies virus (PRV) and genetic manipulations will be used to trace circuits associated with peptidergic neurons involved in the control of reproduction and aggression, respectively, and to identify specific olfactory and vomeronasal areas and sensory receptors involved. The functional contribution of olfactory and VNO inputs to the uncovered circuits will be further investigated by targeted genetic ablations. These studies will provide critical insights into the mechanisms by which the brain integrates distinct sensory information to regulate the neuroendocrine axis and the expression of appropriate behaviors.

DESCRIPTION (provided by applicant): The rapid pace of discovery in the life sciences requires that we constantly examine and improve the ways in which we prepare graduate students for productive careers. In response to these recent developments, the current Training Program in Molecular, Cellular, and Chemical Biology (MCCB) has been substantially reconfigured in this proposal and is, in essence, a new program. Intended to provide an even more comprehensive and interactive training experience for students interested in research and discovery in the life sciences, this new curriculum includes a broad array of established and emerging disciplines in the life sciences, ranging from genetics and evolutionary biology, to systems and chemical biology, to molecular biology and neurobiology. The new Joint Training Program in Molecules, Cells and Organisms (MCO) represents the combined talents of faculty from Molecular &Cellular Biology, Chemistry and Chemical Biology, and Organismic and Evolutionary Biology at Harvard. We believe that the application of the tools of molecular biology and genomics to virtually every discipline, from evolutionary biology, to cell biology, biochemistry and biophysics has revealed a complexity of organization and function that few could have dreamed of 20 years ago. This training will involve an initial exposure to a broad sweep of fundamental problems at every level through a set of core courses, followed by in depth immersion in focused areas. The latter will be accomplished through additional course work and Ph.D. thesis research. The newly designed group of core courses, in-depth track requirements, quantitative training, and specialized nanocourses combine to provide a strong foundation from which trainees will go on to conduct laboratory research in an exciting, interactive, and supportive environment. The MCO Training Program is precisely the kind of initiative that will advance the science of the post-genomic era, as the collective attention of the life science community turns toward the daunting task of elucidating the function of genes and their roles in the vast physiological networks necessary to develop and maintain complex organisms. RELEVANCE (See instructions): The proposed training program is designed to provide both the breadth and depth of didactic and research training necessary to prepare life scientists for a new era of interdisciplinary and collaborative research.

DESCRIPTION (provided by applicant): Mammalian olfactory sensory neurons must "choose" to express a single chemoreceptor gene from among hundreds in the genome. In addition to determining the odors (or pheromones) to which a neuron responds, the chosen receptor plays several critical roles during the life cycle of the cell. Activity of the receptor participates in controlling the position within the brain to which the axon is guided, refining axonal projections, and controlling how long the neuron will live. Variable neuronal longevity among olfactory neurons is thought to facilitate refinement of neuronal connections, elimination of damaged neurons, and, possibly, sensitization of an animal to odors and pheromones. Preliminary studies have led to the discovery of a mouse histone variant gene whose expression is detected only in olfactory sensory neurons in a pattern that is inversely related to neuronal activity. Histones are basic proteins that make direct contact with DNA and play critical roles in regulating gene expression. Elimination of the olfactory-specific histone gene from the genome caused mice to exhibit abnormal weight gain and significant defects in olfactory receptor gene expression as they aged. Two possible explanations for the observed defects in receptor gene expression are: 1) that the histone participates in choosing and/or maintaining receptor gene expression, or 2) that the histone participates in controlling neuronal longevity. These hypotheses will be tested in Aims 1 and 2. A hypothesis that the olfactory histone gene plays a role in axon guidance or refinement was born from observations that its elimination from the genome caused defects in the expression of genes involved in axon guidance, higher rates of cell death among immature neurons, and axonal projection defects in adults. A possible role for the histone in activity-dependent axon guidance or refinement will be investigated in Aim 3. Molecular, histological, genetic, and high-resolution imaging techniques will be used to complete the proposed aims. The proposed studies are anticipated to provide new mechanistic perspectives on how neuronal activity and epigenetics affect the development, refinement, and plasticity of the peripheral olfactory system, and may have important implications for other parts of the nervous system as well. Mechanistic issues regarding the choice of receptor gene expression, the guidance and refinement of axonal projections, and the control of neuronal longevity are central to the development and function of the olfactory system and remain particularly challenging. Understanding these processes will have major implications for olfactory dysfunction and related health problems in humans. PUBLIC HEALTH RELEVANCE: The role of an olfactory-specific histone variant in the activity-dependent control of neuronal longevity and axon guidance in mammals The human olfactory system contains hundreds of different types of sensory neurons, whose relative abundance and functional connection to the brain are controlled by processes that are poorly understood, but are critical to normal olfactory function. We have discovered a gene in mouse that is expressed only in olfactory tissues and that we suspect may play an important role in these processes. The study of this gene may have important implications for olfactory dysfunction in humans, including age-related olfactory decline and olfactory-related eating disorders.

PROJECT SUMMARY (See instructions): Genomic imprinting results in the preferential expression of the paternally, or maternally inherited allele of certain genes. Imprinted genes are highly expressed in the adult and developing brain, and this mode of epigenetic regulation of gene expression is essential for normal brain development and function. Moreover, defects in imprinted genes have been implicated in various mental illnesses including autism, psychosis, and mental retardation. We have recently developed a genome-wide sequencing strategy that led to the discovery of over 1300 new imprinted loci in the adult and developing brain and we have demonstrated that the repertoires of imprinted genes in the developing brain and adult male and female cortex are different. This suggests that genomic imprinting is a major and dynamic mode of epigenetic regulation, which has direct implications for the understanding of brain development and function, and of mental illnesses. The present proposal aims to unravel parental bias of gene expression in a genetically defined cortical cell population, the pan/albumin (PV) positive inhibitory interneurons that are thought to play major roles in the maturation of cortical circuits, and to be affected in various mental disorders. We will first optimize experimental strategies to purify PV cell-specific transcripts, and assess the nature of genes displaying parent of origin expression bias in mature PV interneurons. We will then compare the repertoire of imprinted genes in adult and developing PV cells, as well as in males and females. Finally we will investigate how the imprinted status of these gene is affected in mouse models of mental illness such as autism, schizophrenia and anxiety disorders. Our study will rely on the expertise and reagents from the Hensch group who has pioneered the study of PV-positive cortical interneurons function in the normal and pathological brain. In turn, our results will provide targets for further genetic, functional and circuit-wiring analysis by the Hensch and Lichtman's groups. The molecular and functional characterization of parental expression bias in PV cells will serve as a model for the experimental analysis of epigenetic controls of gene regulation in generically identified cortical cell types in the normal and pathological brain.

DESCRIPTION (provided by applicant): The goal of this project is to investigate how social chemosignals are processed by vomeronasal circuits in many species, the signals essential for guiding social interactions rely on the emission and detection of pheromones. Rodents strongly rely on the vomeronasal system to detect these cues and guide genetically pre-programmed social and defensive behaviors. We, and others, have recently made significant progress in understanding the molecular and celular basis of chemosensory detection by the vomeronasal organ (VNO) and in visualizing complex patterns of activity by its primary target, the accessory olfactory bulb (AOB). However, the information processing performed by downstream brain areas in order to elicit innate behavioral responses. encode behaviorally relevant signals has not yet been uncovered. This project draws on novel electrophysiological, genetic and optogenetic approaches to determine how units from the (MeA) transform social and defensive sensory cues into behavioraly relevant signals. The MeA occupies a critical position in the vomeronasal-sensorimotor transformation between the AOB and distinct nuclei of the hypothalamus that are involved in eliciting distinct behavioral responses. medial amydgala Proposed experiments will address 1- how the MeA processes the complex sensory representation from the AOB, in order to convey information reflecting the behavioral significance of the detected cues to centers in the hypothalamus. 2- the sensory representation of distinct genetically defined populations of neurons in the MeA, and more specifically the responses of two complementary populations of neurons expresing either the enzyme aromatase (Ar) or thyrotropin-releasing hormone (TRH that are located in distinct areas of the MeA reported to drive mating and defensive behaviors, respectively. 3- the contribution of elementary VNO signals to neuronal activation across the brain using transgenic mouse lines and optogenetic tools that enable the activation of discrete receptor populations corresponding to the detection of predators, as well as male and female conspecifics. In humans, defects in social recognition are the core of poorly understood and debilitating mental disorders such as autism and schizophrenia. Because of their central role in the coding and processing of environmental cues leading to appropriate behavioral responses, the neural principles of social and defensive recognition uncovered in these studies are largely applicable throughout the animal kingdom. Thus, our findings will inform the diagnosis and treatment of mental disorders, in which social and sensory communications are impaired.

DESCRIPTION (provided by applicant): The scale of biological studies is rapidly evolving and biological phenomena are now observed at both super-high resolution and with genome- and organism- wide perspectives. To rise to these challenges, we must train the new generation of students to develop a broad and interdisciplinary knowledge of modern biology and experimental approaches in a highly collaborative environment. The training program in Molecules, Cells, and Organisms (MCO) is a cross- departmental doctoral training program located in the Faculty of Arts and Sciences at the Cambridge Campus of Harvard University. This highly innovative program establishes the foundation for students to become the next- generation biologists. MCO faculty mentors represent all fields of modern biology, from biophysics, structural biology and microbiology, to developmental and regenerative biology, neurobiology and genomics. Collaborating departments include Molecular and Cellular Biology, Chemistry and Chemical Biology, Organismic and Evolutionary Biology, Stem Cell and Regenerative Biology, and Physics. Through its structure of core course requirements, quantitative biology courses, journal club, nanocourses, lab rotations, seminars, hands-on model systems workshops, scientific colloquia and retreats, the MCO program exposes its trainees to the full scope of research options available to modern biologists today, and helps them develop outstanding reasoning skills and creativity, as well as oral and written communication. As the advanced field of biological analysis is now attracting scientists from all disciplines, the MCO program is designed to meet the challenge of training that combines the methods of chemistry, physics, mathematics and informatics with new concepts of cellular and molecular biology.

? DESCRIPTION (provided by applicant): Many severe mental disorders with considerable disease burden such Autism Spectrum Disorders, Schizophrenia, and Major Depressive Disorder are characterized by profound social impairments. At present, there is little understanding of the origin of these social deficits, and efficient diagnosis and therapeutic options are lacking. Advanced molecular and genetic techniques make the discovery of specific neural circuits involved in social behavior possible, facilitating the development of diagnostics and novel therapeutic approaches specific to disorders with social deficits. We propose to take advantage of newly developed molecular and genetic tools to uncover and characterize the specific neural populations and circuits involved in parental care, a social behavior essential for the survival and well-being of the offspring. Male and female mice show either affiliative or agonistic behavior toward infants depending on prior social experience. In recently published work, we uncovered a specific subpopulation of hypothalamic neurons that are essential for the control of male and female parenting behavior. This finding provides us with a unique entry point to genetically dissect behavior circuits underlying parental care and their modulation by intrinsic and environmental factors. Using a combination of genetic and functional tools, we aim to characterize the circuit involved in parental behavior (Aim I) and uncover neuronal subpopulations driving agonistic behavior toward pups (Aim II). We will determine the functional role of these genetically defined neural populations and associated projections in parenting or agonistic behavior toward pups using cutting-edge molecular techniques by tracing inputs and outputs to genetically defined neuronal populations and manipulating their activity. In Aim III, we will perform an unbiased gene expression analysis to discover factors influencing the differential activity of the neurons and circuits associated with affiliative or agonistic behavior in males and females, and in different physiological circumstances.

Project Summary/Abstract The mammalian brain is a highly diverse structure in which large numbers of cell types, grouped into broad functional areas, serve defined functions according to their developmental origin, shape and connectivity, transcriptional program and intrinsic biophysical properties. A mechanistic understanding of how the brain works, and how dysfunctions lead to neurological disorders, will require a systematic characterization of neural cell types. In turn, such knowledge will transform our ability to analyze and experimentally manipulate specific neuronal populations in normal as well as diseased brains. Our project aims to examine the transcriptome of individual cells still embedded in brain tissue in order to take into account topographical features of individual cells that are lost during cell dissociation, such as information about the positions of cells in brain structures, and their participation in functional circuits. We propose here to develop an unbiased experimental approach and computational toolkit to classify cell types in situ, according to their gene expression profiles, and in the functional context of behaviorally relevant circuits. In Aim 1 of this grant, we propose to extend MERFISH (Multiplexed Error Robust Fluorescent In Situ Hybridization) to brain tissue, and to develop a computational pipeline to establish a spatially informed cell inventory. In Aim 2, we will use the methods of in situ single cell transcriptional profiling optimized in Aim 1 to create an inventory of cell types, first in an individual nucleus of the hypothalamus: the paraventricular nucleus (PVN), and next in the entire hypothalamus. We will then validate our approach in a different animal species by exploring the cellular composition of the PVN in the Common Marmoset. In Aim 3, we will use approaches developed in Aims 1 and 2 to uncover cell types across the hypothalamus that are associated with parenting behavior and infant-evoked aggression. We will further assess changes in transcriptional profiles of this hypothalamic behavior circuit in animals in different physiological states. In Aim 4, we will build on the approaches optimized in previous aims to characterize transcriptional profiles of neurons of a 3-relay circuit controlling parental behavior across the brain.

Project Summary/Abstract The function of multi-cellular systems emerges from the complex interactions between cells that have distinct behaviors and functions. Because cellular behaviors are in large part determined by their transcriptomes, the ability to quantify all transcripts in every single cell in a biological system would transform our understanding of a wide range of systems as well our ability to diagnose and treat diseases. Although single-cell transcriptomics methods based on high-throughput sequencing provide a powerful approach towards this goal, these methods requires dissociation of cells from their native tissue and extraction of RNA from the cells. As a result, it is difficult for these sequencing-based approaches to retain an important class of information that is crucial to a wide variety of biological processes: the spatial context of RNAs, i.e. where these RNAs are located within a cell and where the cells are located within the tissue. On the other hand, the spatial positions of RNAs within the cell can have a potent effect on their post-transcriptional fate and have been implicated in a diverse set of cellular behaviors from cell motility to cell polarization. Furthermore, the spatial organization of different types of cells within a tissue is of paramount importance to the tissue function: such spatial context modulates cell behavior, directs cell differentiation, and shapes the emergent behavior of the tissue as a whole. Therefore, a spatially-resolved approach to single-cell transcriptomics is in great demand and promises to transform many areas of biology. Here we propose to develop an imaging-based method that is capable of determining the precise copy numbers and spatial locations of most, if not all, RNA species (i.e. the whole transcriptome) within individual cells preserved in their native context. This approach functions by massively multiplexing single-molecule fluorescence in situ hybridization. In this approach, we will encode each RNA species in the cell with a barcode that is defined by a set of specially designed DNA probes that can specifically bind to and uniquely encode the target RNA. This barcode will then be read by a series of hybridization and imaging rounds, allowing us to determine the identity the RNA. Using an error-robust encoding scheme, we estimate that we should be able to image the entire mammalian transcriptome, i.e. several tens of thousands of RNA species, with high accuracy in just a few tens of imaging rounds or even fewer with multicolor imaging! This technique also promises a very high throughput of measuring hundreds of thousands of cells per single-day experiment. In this project, we will not only develop this technology and the above-mentioned capabilities, but also demonstrate the transformative impact of this technology in biology by mapping the spatial organization of the transcriptome inside individual cultured neurons and determining the number of transcriptionally distinct cell types and their spatial organization in a functionally important region in the mouse brain.

Program Abstract Biological science has rapidly evolved since the turn of the century; scientists are now able to routinely generate and analyze data with super-resolutions and at genome-, transcriptome-, proteome- and organism-wide scales. To rise to these challenges in the 21st century, the new generation of scientists must then be trained to have a broad and interdisciplinary knowledge of modern biology and experimental approaches in a highly collaborative environment. The training program in Molecules, Cells, and Organisms (MCO) is designed to train the next- generation biologists by allowing them access to faculty mentors representing all areas of modern biology? from biochemistry, biophysics, and structural biology to molecular, cell, neuro-, and developmental biology to genetics, genomics, and evolutionary biology to systems and computational biology. The training program is located at the Cambridge campus of Harvard University, and fifty-five faculty are drawn from five departments in the school of Faculty of Arts and Sciences, including, Molecular and Cellular Biology, Chemistry and Chemical Biology, Organismic and Evolutionary Biology, Stem Cell and Regenerative Biology and Physics, and faculty from the School of Engineering and Applied Science. Through its structure of core courses, quantitative biology courses, journal club, nanocourses, hands-on model systems workshops, lab rotations, seminars, scientific colloquia and retreats, the MCO program exposes its trainees from the start to a rigorous and multidisciplinary training, and helps them to develop outstanding reasoning, quantitative and creative skills required for modern biology, as well as oral and written communication. As biological sciences continue to attract scientist from all disciplines, the MCO program is designed to meet the challenge of training a new generation of biologists that can combine the methods of chemistry, physics, engineering, mathematics and informatics with new concepts in cellular, molecular and behavioral biology. The mission of the program is to equip trainees with the necessary technical and operational skills that are necessary to be successful in interdisciplinary research, as exemplified by first-author publications and timely graduation.

PROJECT SUMMARY ABSTRACT  During an infection, animals exhibit adaptive changes in behavior and physiology aimed at increasing survival.  Although  many  causes  of  acute  infection  exist,  a  similar  set  of  stereotyped  symptoms  occur,  which  includes  increased body temperature or fever, decreased appetite and increased lethargy. Both warm-­ and cold-­blooded  animals  generate  a  fever  in  response  infection  suggesting  that  fever  circuits  are  hard-­wired  and  highly  conserved, yet exactly how the nervous system alters body temperature and associated behavior in response to  infection remains unknown. We have identified a population of neurons in the preoptic area of the hypothalamus  that  are  highly  activated  following  administration  of  inflammatory  lipopolysaccharides  (LPS).  Due  to  the  close  proximity between  the  organum  vasculosum  of  the  laminae  terminalis  (OVLT),  where  inflammatory  cytokines  enter the brain to affect nearby cells, and neurons of the preotpic area regulating normal body temperature, and  our preliminary data, we propose that these newly identified LPS-­sensitive neurons control fever initiation during  an immune response. We will use chemogenetic activation and cell ablation approaches to demonstrate that this  population plays a role in increasing body temperature and in affecting other fever-­associated behaviors upon  LPS injection. Further, we have recently developed new approaches for molecular characterization of genetically  defined  cell  populations  in  situ  using  single-­cell  RNA  sequencing  (scRNA-­seq)  and  multiplex,  error-­robust,  fluorescent in situ hybridization (MERFISH) to generate a spatially-­resolved and functionally-­aware atlas of the  preoptic area. We will apply a similar strategy to characterize fever-­inducing neurons as well as surrounding non-­ neuronal  cell  types  that are  likely  to  play  a  role  in fever generation  through paracrine  mechanisms. Finally,  we  propose to use viral-­mediated tracing and functional tools to determine the direct and indirect circuit mechanisms  by  which  LPS-­sensitive  neurons  and  their  targets  exert  control  over  body  temperature  and  fever-­related  behaviors.  Our  data  will  lead  to  a  molecular  and  functional  characterization  of  LPS-­sensitive  neurons  in  the  preoptic area and to a better understanding of how inflammatory sickness symptoms, such as fever and related  behavioral  changes,  are  regulated  in  the  brain.  These  efforts  have  direct  implications  for  understanding  the  mechanisms underlying  human  sickness, and  may  inform  new therapeutic  strategies for the treatment  of fever  and associated symptoms.   

Project Summary/Abstract In order to control specific behavioral responses, transcriptionally distinct cell types assembled into dynamic brain circuits integrate environmental information with internal states and generate purposeful motor actions. While tools have been developed to independently measure the activity dynamics, connectivity and transcriptional profiles of individual neurons, it remains challenging to integrate this diverse information into a coherent model of behavior. To address this challenge, we aim to uncover the sensorimotor transformations leading to a complex naturalistic behavior, male and female parenting, by developing innovative molecular, imaging and systems-level approaches and by integrating multimodal information obtained from single neurons in behaving animals. In aim 1, we will develop new tools to uncover the activity and the transcriptional identity of neuronal cell types involved in infant-mediated behavior. In aim 2, we will explore in molecular, functional, and behavioral terms how olfactory and other sensory modalities underlie parenting behavior in males and females and in virgin versus mated states. In aim 3, we will investigate how specific hypothalamic cell types process information reflecting behavioral outcomes using a combination of spatial transcriptomic, functional imaging, circuit tracing and behavioral methods. In aim 4, we will combine cell-type based neuronal architecture with activity patterns across key brain regions to formulate predictive models underlying the neural control of parenting.

The transcriptional regulatory sequences communicate with each other dynamically in the 3D nuclear space to direct cell type specific gene expression. Currently, a major barrier to understanding the transcriptional regulatory programs is the lack of tools, models and maps to explore the chromatin architecture in diverse cell types and physiological contexts. We will address this pressing need by deploying transformative technologies to study the chromatin architecture in mammalian cells at an unprecedented resolution and scale. Specifically, we will generate navigable, cell-type-specific reference maps of chromatin architecture in the mouse, macaque and human brains by integrating high resolution and high throughput imaging and orthogonal single-cell-based genomic methods. We will also dissect the role of chromatin architecture in gene regulation through a set of controlled perturbation experiments in the mouse ES cells (ESC) and ESC-derived neural progenitor cells (NPC). We will develop structural models of chromatin organization with advanced polymer physics and statistical learning methods, and validate their predictive power in embryonic stem cells and in ex vivo brain slices. Finally, we will make the reference maps, analytical tools, visualization methods and structural models available to the broader community. The proposed research project will dramatically transform our ability to analyze the 4D Nucleome of complex tissues, and produce the much-needed maps, tools and models for understanding the gene regulatory programs encoded in the linear genome sequences.