John L. R. Rubenstein - US grants

DESCRIPTION (Adapted from applicant's abstract):Many severe psychiatric disorders of childhood such as autism, mental retardation, and childhood schizophrenia are caused by abnormalities in brain development. In some of these cases, the abnormal neural development is due to a genetic defect. The goal of the research proposed is to identify and study genes that regulate the development of the mammalian brain, in particular the telencephalon. It is likely that study of the basic genetic mechanisms that control neural development will provide a basis for understanding developmental psychiatric disorders. Subtractive hybridization between cDNA libraries will be used to enrich for genes that are preferentially expressed during development of the mouse telencephalon. Genes that are homologous to transcriptional regulators will be identified using PCR and hybridization. Using these methodologies, several cDNAs were cloned in the last year. Highest priority will be given to analysis of one of these genes called TES-l. Preliminary evidence shows that TES-l encodes a homeodomain- containing protein that is transiently expressed during development of the ventral forebrain, which suggests that TES-l encodes a transcriptional activator that may regulate differentiation of the ventral forebrain. Analysis of TES-l will include DNA sequencing, in situ hybridization, and characterization of the genomic DNA.Functional studies will include construction of transgenic mice which express the LacZ gene under the control of the TES-l promoter, and construction of strains of mice containing mutations in TES- l.

PROJECT SUMMARY Telencephalic GABAergic neurons in the basal ganglia and cerebral cortex have central roles in cognition and emotion. Dysfunction of these neurons contributes to some types of epilepsy, intellectual deficiency, autism and schizophrenia. During development, subpallial progenitors generate most of telencephalic GABAergic neurons, including basal ganglia projections neurons and cortical interneurons6. The Dlx1,2,5&6 homeodomain transcription factors (TF) have central roles for this process7-24. Understanding the genetic circuitry upstream and downstream of the DLX TFs is essential for elucidating the basic mechanisms of telencephalic GABAergic development. To elucidate the genetic circuitry driving the development and function of telencephalic GABAergic progenitors and neurons, we must define the TFs, REs (enhancers and promoters) and the coding regions that they control. We hypothesize that the DLX homeodomain (TF) are at the core of transcriptional circuits, which we call the ?Dlx Pathway?, that regulate the development of most telencephalic GABAergic neurons, including basal ganglia projections neurons and cortical interneurons. We propose experiments aimed at elucidating the network of TFs in the Dlx Pathway that directly regulate genes controlling development of cells generated in the embryonic basal ganglia (ganglionic eminences, GEs). We will use chromatin immunoprecipitation followed by whole genome sequencing (ChIP-Seq) to elucidate in vivo genomic binding sites for TFs upstream and downstream of Dlx1&2 (Aim 1). Analysis of changes in RNA expression in the GEs of Dlx1/2 mutants (Aim 2) will provide evidence for the genes whose expression depends on Dlx function. Histone ChIP-Seq and ATAC- Seq (Aim 3), in conjunction with TF ChIP-Seq, will provide evidence for the locations of regulatory elements (REs; enhancers and promoters) used by Dlx Pathway. Final we will assay RE activity using transgenic methods (Aims 4&5). Elucidating transcription circuits driving telencephalic GABAergic development provides a fundamental framework for understanding the genetic pathways, including the REs, which generate inhibitory neurons. !

DESCRIPTION: This proposal is driven by the hypothesis that regional specification of the forebrain is largely controlled by a hierarchy of genes encoding transcriptional regulators. A large portion of the proposal depends on gene knockout technology which will be used to inactivate the Dlx-1 and Dlx-2 genes, both individually and simultaneously. The phenotype of mice that are heterozygous and homozygous for the mutations will be analyzed histologically and biochemically.

[unreadable] DESCRIPTION (provided by applicant): The frontal cortex arguably houses the most important set of integrators of cognition, emotion and motor control in the mammalian brain. Therefore, understanding the genetic mechanisms that control development of the prefrontal and motor cortex have clear ramifications for understanding neuropsychiatric disorders. I hypothesize that one can gain fundamental insights into this subject through the analysis of how Fgf8 signaling regulates development of the telencephalon. Fgf8 signaling is now firmly established as a central mechanism that controls molecular regionalization and morphogenesis of the rostral telencephalon, including the frontal neocortex. In this grant application I describe experiments aimed at testing the hypothesis that analysis of mice bearing mutations in Fgf agonists (Fgf8, 15 and 17), FGF antagonists (Sprouty 1 and 2) and transcription factors that are regulated by Fgf8 (COUPTF1 and Emx2) will elucidate mechanisms that regulate the size, nature and function of the frontal cortex. [unreadable] [unreadable] [unreadable]

DESCRIPTION (Adapted from applicant's abstract): There is abundant evidence to suggest that neuropsychiatric disorders such as schizophrenia and autism are caused in many cases by genetic abnormalities that affect development and function of forebrain neural systems involved in cognition and emotion. The largest structures of the forebrain are the cerebral cortex and the striatum; both have been implicated as having a role in neuropsychiatric disorders. The goal of my research is to understand how genes regulate development of the striatum. To this end, my laboratory has identified the Dlx genes, which encode a family of homeodomain transcription factors that are candidates for having a central role in striatal development. There are four known Dlx genes that are expressed in the embryonic forebrain. The aims of the experiments proposed in this grant application are focused on (1) elucidating the sequence of these genes and their encoded proteins, (2) characterizing the biochemical properties of the DLX proteins, (3) determining whether the DLX proteins are transcriptional regulators, (4) identifying proteins that interact with and modulate the function of the DLX proteins, (5) determining the intracellular location of the DLX proteins, (6) determining the temporal and spatial patterns expression of the Dlx RNAs and proteins in the prenatal and postnatal forebrain, and (7) beginning to determine where the Dlx genes are in the genetic hierarchy that regulates development of the forebrain using ectopic expression experiments.

DESCRIPTION (Adapted from applicant's abstract): There is abundant evidence to suggest that neuropsychiatric disorders such as schizophrenia and autism are caused in many cases by genetic abnormalities that affect development and function of forebrain neural systems involved in cognition and emotion. The largest structures of the forebrain are the cerebral cortex and the striatum; both have been implicated as having a role in neuropsychiatric disorders. The goal of my research is to understand how genes regulate development of the striatum and other forebrain structures. To this end, my laboratory has studied the Dlx genes, which encode a family of homeodomain transcription factors. There are four known Dlx genes that are expressed in the embryonic forebrain. This application is for renewal of a grant in which I proposed to study the function of Dlx-1 and -2 using gene targeted mutagenesis in mice (1 R01 MH51561-01A1). We have accomplished the aims set out in that grant and have discovered that mice lacking both Dlx-1 and -2 have a severe abnormality of basal ganglia differentiation. The aims of the experiments proposed in this grant application are focused on (1) fully characterizing the phenotype of the Dix-1, Dlx-2 and Dlx-1 and -2 mutant mice; (2) identifying and characterizing genes that are dysregulated in the Dlx-mutant mice; (3) studying the genetic interactions of the Dlx, Gsh and Mash genes; and (4) making mutations in Dlx-5, Dlx-6 and Dlx-5 and -6.

DESCRIPTION (Adapted from applicant's abstract): This grant is aimed at studying the roles of three mouse Nkx homeodomain transcription factors, Nkx2.1, 2.2 and 6.1, in development of the basal plate of the CNS. The genes are expressed in distinct and overlapping expression patterns in the basal plate at different anterior-posterior positions. Work of Rubenstein and others have shown that all three genes are required for aspects of ventral CNS development. Dr. Rubentsein proposes to further study these three genes by carrying out a similar set of experiments for each gene. He will characterize null mutants by marker gene analysis to determine whether specification of certain cell types is altered and analyze cell proliferation, cell death and cell migration in the affected regions. He will complement these studies with gain- of-function studies by ectopically expressing the genes in early chick brain to determine whether they are sufficient to induce ventral cell types. He will also study double mutants to address overlapping gene functions and analyze in more detail expression patterns of these genes later in development and in the adult. A collaborator, Dr. Puelles, will visit the lab twice, for two months each, to help analyze the expression patterns and morphological mutant phenotypes.

DESCRIPTION (provided by applicant): Among the most common and debilitating human birth defects are those that affect craniofacial tissues. The last decade has witnessed the identification of numerous candidate regulatory genes that are expressed in regionally restricted patterns in the craniofacial primordia. Among these are homeobox transcription factors that include the D1x gene family. In mammals there are three Type A D1x genes (2,3,5) and three Type B D1x genes (1,6,7). These genes are expressed in nested patterns in the primordia of the branchial arches as well as the olfactory and otic apparati. We have made loss-of-function mutations of D1x1, D1x2, D1x1&2 and D15 in the mouse and found that these genes are essential for normal skeletal morphogenesis of the jaw apparatus and teeth, as well as the nasal and otic capsules. Comparison of the D1x-expression patterns with the morphological defects seen in the D1x mutants suggests that there is a D1x combinatorial code that specifies regional morphogenesis of the branchial arches and olfactory and otic apparati. To evaluate our combinatorial model of D1x function, we are studying craniofacial molecular and tissue patterning in D1x compound mutants. In addition, we will study the cellular and molecular mechanisms through which the D1x genes regulate craniofacial development.

Among the most common and debilitating human birth defects are those that affect craniofacial tissues The last decade has witnessed the identification of numerous candidate regulatory genes that are expressed in regionally restricted patterns in the craniofacial primordia Among these are homeobox transcription factors that include the Dlx gene family In mammals there are three Type A Dlx genes (2,3,5) and three Type B Dlx genes (1,6,7) These genes are expressed in nested patterns in the primordia of the branchial arches as well as the olfactory and otic appatati We have made loss-of-function mutations of Dlxl, Dlx2, Dlxl&2 and Dlx5 in the mouse and found that these genes are essential for normal skeletal morphogenesis of the jaw apparatus and teeth, as well as the nasal and otic capsules Comparison of the Dlx-expression patterns with the morphological defects seen in the Dlx mutants suggests that there is a Dlx combinatorial code that specifies regional morphogenesis of the branchial arches and olfactory and otic apparati To evaluate our combinatorial model of Dlx function, we are studying craniofacial molecular and tissue patterning in Dlx compound mutants In addition, we will study the cellular and molecular mechanisms through which the Dlx genes regulate claniofacial development

DESCRIPTION (provided by applicant): The cortex, striatum and pallidum are three key components of cortico-basal ganglia circuits - these circuits regulate limbic, associative and sensorimotor learning. The embryonic basal telencephalon generates subcortical nuclei and cortical interneurons that are required for the function of these circuits. As such, developmental defects of basal telencephalic development can have a profound influence on cognition, emotion and movement. Defects that alter sensorimotor learning can result in motor phenotypes, as exemplified by chorea, tremor and rigidity seen in disorders such as Huntington's disease and Parkinson's disease. Defects that alter limbic and associative learning can result in affective and cognitive defects that may underlie disorders such as Tourette's, Schizophrenia and addiction. The embryonic basal telencephalon primarily consists of the medial ganglionic eminence (MGE);it produces GABAergic and cholinergic projection neurons of the globus pallidus, nucleus basalis and adjacent regions, and GABAergic and cholinergic interneurons that disperse throughout the striatum and cortex. Thus, the basal telencephalon has a central role in generating components of cortical-basal ganglia circuits. An approach to elucidate the genetic underpinnings that regulate the basal telencephalon is to study the function of transcription factors that control the development and function of the neurons that are produced in this region. In this proposal, I describe experiments that study the functions of four transcription factors: Nkx2.1 (Aim 2), Lhx6 (Aim 3&4), Lhx7/8 (Aims 3&4) and Ldb1 (Aim 5). We hypothesize that combinatorial and unique functions of these four proteins participate in specifying the identity and properties of neurons generated by the embryonic basal telencephalon;the following schema provides the outline of our hypothesis. Aim 2 tests Nkx2.1 function in SVZ progenitors, pallidal projection neurons, and in the VZ of the most ventral regions of the basal telencephalon. Aim 3 studies how Lhx6 regulates MGE differentiation. Aim 4 tests whether Lhx6/Lhx7/8 coordinately regulate MGE development, and Aim 5 tests the function of Ldb1, and whether its phenotypes resemble Lhx6/7(8) mutants. In addition, we will perform fate mapping studies of cells produced in the embryonic basal telencephalon, using Cre-expressing alleles (Aim 1);these alleles will also be useful genetic tools for generating conditional mutants, such as in Aims 2 and 5. PUBLIC HEALTH RELEVANCE: The results from the proposed studies will provide basic information regarding the genetic and developmental mechanisms that control formation of brain regions that control cognition and movement. Disruption of these mechanisms can cause psychiatric and neurological disorders that include mental retardation, autism, schizophrenia, movement disorders and addiction.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The telencephalon is divided into many functionally distinct areas. These areas are characterized by different combinations of gene expression, cytoarchitecture, and pattern of connections. A major question in the field is how these areas are specified. Recent work has identified evolutionary conserved enhancer elements that regulate gene expression in sharply demarcated regions in the ventricular zone of the E11.5 mouse cortex. The spatial extent of these regions is regulated by multiple transcription factors. Our lab is interested in identifying these transcription factors using mass spectroscopy. Our approach involves applying nuclear lysate from specific forebrain regions to enhancer elements. The proteins that specifically bind the enhancer will be isolated and identified using mass spectroscopy.

? DESCRIPTION (provided by applicant): Molecular definitions of neural cell types largely depend on the expression of RNAs or proteins as assessed by in situ hybridization, RNA array and sequencing, and immunohistochemistry. However, recent studies are demonstrating that gene regulatory elements, such as enhancers, can have highly specific spatial and temporal activity patterns in the developing brain. Thus, enhancer activity can be used to define neural cell types, and importantly, also have other broad applications. First, they can be used as tools to drive gene expression in specific cell types, which can then be used to visualize and/or purify the cells (GFP), modify gene expression in the cells (Cre), modify electrical activity (channel rhodopsin), and visualize electrical activity in the cells (GCaMP). Secondly, knowledge about the nature and position of enhancers enables geneticists to identify disease alleles that map in extra-exonic genomic space. Herein, we will focus on identifying enhancers that are active in cortical interneurons, during development and in the mature state. We choose to study cortical GABAergic interneurons because of their central role in cortical function and diseases. We will focus on these GABAergic neurons derived from the medial ganglionic eminence (MGE), which generates the majority of cortical interneurons. Our aim is to identify novel enhancers that are active in cortical interneurons using three assays: 1) histone (H3K27Ac) ChIP-seq (marker of active enhancers); 2) transcription factor (TF) ChIP- seq using TFs that regulate cortical interneurons development and function (Arx, Dlx2 and Lhx6); 3) a newly developed in vivo assay of enhancer function based on viral transduction of the enhancer driving Cre and GFP in immature cortical interneurons. We will then use a series of methods to define the interneuron subtypes that have enhancer activity. Together, our unique approach should define interneuronal cell types (developmental and adult), and simultaneously generate a powerful toolkit that will enable new ways to assess neural function and disease.