Marianne E. Bronner - US grants

9722460 Bronner-Fraser This Americas Program award will support Dr. Marianne Bronner- Fraser, California Institute of Technology, in a research collaboration with Dr. Roberto Mayor, Universidad de Chile. The investigators plan to study the effect of expression of a newly isolated gene, NP-1, on the initial formation of neural crest cells. The new gene, isolated and sequenced from avian embryos by Dr. Bronner in collaboration with two other researchers, has no obvious similarities to other known gene families. However, although the chick system has many advantages for experimental embryology, it is difficult to ectopically express gene products in birds. Therefore, the researchers have initiated studies of the Xenopus system, which represents an ideal system in which to pursue ectopic expression of molecules in early development. To that effect. they plan to isolate the Xenopus homologue of NP-!. Their research will combine molecular biological approaches with experimental embryology, with the long-term goal to characterize and understand the function of the gene in neural crest formation. The collaboration will benefit greatly from the combined expertise and research intereqt of both researchers. The proposed experiments will take place in Dr. Mayor's laboratory, who is an expert on the Xenopus system and has a long standing interest in neural crest development. . ***

The long term goal of the Caltech Center for Excellence in Genomic Science (CEGS) is to develop and deploy the tools needed for the high-throughput creation of functional fusion proteins that tag endogenous gene products with a fluorescent protein, for the creation of marked mutant alleles of each protein, for the multiplex in situ hybridization of several different gene products in normal and perturbed embryos, for the analysis of the resulting patterns of expression with in toto imaging, and for the rendering/analysis of labeled cell positions and movements. Under the support of the parent grant, the development of each experimental tool has advanced to the point that they have been validated. This supplement requests support for the final steps in refining these technologies, through the support of four key senior post-doctoral fellows, including one involved in data management and establishment of a public interface. The supplement requests support for the supplies and small equipment needed to increase the rate at which data can be collected. The requested supplement will play a major role in advancing the goals of the Caltech CEGS grant into its production mode, enabling the advances made to date by key post-doctoral research to reach the scientific community in a much faster rate. Given the difficult job market today, these funds also will allow these talented young individuals the necessary time to secure appropriate positions at top-tier universities.

By adapting approaches that have been applied with great success to testing the sea urchin developmental gene regulatory network, we propose to perform a detailed analysis of the gene interactions involved in specifying vertebrate neural crest cells. Our aim is to understand the genomic control of this process at a systems level by revealing most/all of the inputs into the system and methodically functionally perturbing them to examine interactions amongst players. In other words, we propose to test a putative neural crest gene regulatory network (NC-GRN) at a systems levels in a single vertebrate. These efforts will be greatly facilitated by the advent of new, high speed technologies that will significantly increase the rate of data acquisition and interpretation as well as novel bioinformatics tools to interrogate genomic information. We will draw heavily on methodologies and concepts developed in the Davidson lab. The goal is to apply these to a vertebrate system at moderate to high throughput. This represents a huge leap forward in both the scale and depth of what can be tested. The recent availability of the chick genome affords a rich tool for discovery of genes and regulatory regions. In addition as an amniote, chick development is similar to humans and, unlike mammals, is accessible to imaging at early stages since the embryo develops outside the mother. We will test linkages in the chick neural crest gene regulatory network, identify regulatory elements and test direct interactions. Aim 1: Examine effects of loss-of-function of known neural plate border and neural crest specifiers. By introducing morpholino antisense oligonucleotides into the prospective neural plate border or closing neural tube. Effects on potential downstream targets will be examined by in situ hybridization and QPCR. Aim 2: Test the function of newly identified transcription factors in the NC-GRN We will test the role and position additional transcription factors in the network and we will continue to attempt to identify transcription factors that feed into the NC-GRN. Aim 3: Isolate regulatory regions of neural crest specifer and downstream targets. We will isolate putative regulatory regions of neural crest specifier genes, initially for Sox 10 and then other specifiers, via comparative sequence analysis. Candidate regions will be electroporated into early chick embryos to identify neural crest regulatory elements. Aim 4: Establish direct relationships within the network by identification of transcription factor binding sites within regulatory regions of downstream genes. We will interrogate the regulatory regions of neural crest enhancer elements for critical sequences responsible for binding of neural plate border specifiers genes and/or neural crest specifier genes. We will assay for direct binding interactions within the network using chromatin immunoprecipitation assay, electrophoretic mobility shift assays and mutational analysis.

DESCRIPTION (provided by applicant): Evolution of vertebrates has been intimately linked to the advent of the neural crest, a migratory and multipotent cell population that gives rise to many defining characters of vertebrates, including a well-defined head and peripheral ganglia. These multipotent progenitor cells form at the border of neural and non-neural ectoderm in vertebrate embryos. The regulatory interactions predicted to underlie neural crest formation involve inductive signals (e.g. Wnt, BMP, FGF) that establish the neural plate border, by up-regulation of border specifier genes like Msx1/2, Pax3/7, and Zic. These border genes in turn regulate neural crest specifier genes like Slug/Snail, FoxDS and the SoxE family. Finally, neural crest specifiers turn on specific downstream targets that render the neural crest migratory and multipotent. The goal of the proposed study is to address whether the neural crest gene regulatory network of traditional vertebrate models is conserved to the base of vertebrates. Data from non-vertebrate chordates suggest this network is a vertebrate novelty and that neural crest evolution involved cooption of several transcriptional regulators to the neural plate border of the vertebrate ancestor. We will compare the neural crest gene regulatory network of traditional vertebrate models with that of sea lamprey, jawless fish that represent the most primitive extant vertebrates. Our preliminary results suggest that many neural crest derivatives, early migratory routes and some components of the neural crest gene network are conserved in lamprey. We will test for conservation at the level of deployment of these molecules at the neural plate border as well as ability to carry out similar functions. To explore events that led to the evolution of this important cell type and thus to the origin of vertebrate features, this proposal will address the following specific aims: 1) Examine whether key genes that function as neural plate border and neural crest specifiers are conserved in sequence and distribution between jawless and jawed vertebrates. 2) Establish connections within the network by morpholino-mediated knock-down of selected transcription factors;establish epistasis by examining the consequences on expression of other genes in the network and their ability to rescue the loss-of-function phenotype. 3) Isolate regulatory regions of amphioxus and lamprey "specifier genes."

DESCRIPTION (provided by applicant): The vertebrate inner is the most complex of the sense organs responsible for hearing and balance. Yet during development it forms from a simple epithelium, the otic placode. During development of the ear, multipotent progenitor cells become progressively restricted in their potential. This process is controlled by regulatory genes whose temporal and spatial expression patterns are tightly regulated in an orchestrated gene regulatory network (GRN). We and others have established a hierarchy of events controlling the specification and determination of inner ear progenitors and identified some of the regulatory genes involved. Based on this information, we have established a preliminary network controlling these processes and designed a molecular screen to identify novel otic specifiers. We have defined co-regulated groups of genes that reflect different stages of ear specification. We will now harness genome sequence information, new technology to monitor changes in the expression of more than 100 genes simultaneously and newly developed bioinformatics tools to build up and verify the preliminary GRN. Specifically we will: identify new genes responsive to otic inducing signals establish the epistatic relationships between ear specific transcription factors and signaling components isolated and characterize enhancers controlling transcription factor expression in the ear and examine direct inputs predict common upstream regulators for genes in each synexpression group using newly developed algorithms and test the predicted regulators in vivo. This will uncover the basic GRN controlling the specification of inner ear progenitors together with its terminal target genes. In the future, this GRN will serve as a basis for studying protein-protein and protein-DNA interactions to build up a complete network, for quantitative analysis and mathematical modeling of this process, as well as a platform to discover new candidate genes for human disease affecting hearing and balance.

DESCRIPTION (provided by applicant): The peripheral sensory nervous system in the vertebrate head has a dual origin from cranial ectodermal placodes and neural crest cells. Cranial placodes arise from regions of thickened ectoderm in the embryonic head that invaginate and/or delaminate to give rise to portions of the cranial ganglia as well as sensory structures like the ear, lens, and nose. While peripheral ganglia of the trunk are exclusively neural crest-derived, those arising at cranial levels have a dual origin from both neural crest and placodes. Despite their importance to peripheral sensory innervation of the head, comparatively little is known about the early development of the placodes and how they become specified to adopt different lineages. This proposal aims to further characterize the cellular and molecular events underlying placode development, focusing on the olfactory placode domain, and how these cells obtain their prospective fates rather than becoming neural crest. Appropriate acquisition of placode fate is critical for proper craniofacial development. 1) Single cell lineage analysis of olfactory/lens primordium. We have shown that precursors to both lens and olfactory structures arise from a common territory next to the anterior neural plate and that they segregate over time by directional movements. Single cell lineage analysis will be performed to resolve whether there is a common lens/olfactory precursor or whether cells already know their fates within this common domain. Surprisingly, preliminary data indicate that these lineages may be segregated even within the preplacodal domain. 2) Environmental control of cell fate in the anterior neural folds: olfactory versus neural crest fate. The anterior neural fold is the only region of the neural tube that does not form neural crest, but rather forms olfactory placode. We will test whether olfactory placode can form neural crest when grafted caudally and if more caudal neural folds can form olfactory placode if grafted rostrally. 3) Epigenetic control of cell fate within anterior neural folds: the role of PHD12, a histone deacetylase complex member, in olfactory versus neural crest fate. PHD12 is selectively expressed in anterior neural folds and its knock-down causes anterior expansion of neural crest specifier genes in this domain. We will test whether PHD12 expression in the neural folds alters after heterotopic grafting and whether its gain- or loss-of-function modulates the olfactory/neural crest fate switch. 4) Transcriptional control of cell fate in anterior neural folds: the role of Pea3 transcription factor in olfactory versus neural crest fate. The transcription factor Pea3, which is a known downstream effector of FGF signaling, is expressed in olfactory and otic placodes at early stages. Preliminary data suggest that knock-down of Pea3 in the olfactory territory up-regulates the neural crest marker, Sox10. We will test whether this and other factors may act as switch points between placodal versus neural crest fate.

DESCRIPTION (provided by applicant): Neural crest (NC) is a transient embryonic multipotent group of cells that arises within the dorsal neural tube, to forms cartilage and bone of the craniofacial skeleton, among other derivatives in vertebrate embryos. Therefore, craniofacial abnormalities are usually attributed to problems in neural crest cell development and depending of which phase of neural crest cell development is disrupted, very different craniofacial anomalies can manifest. Epithelial-to-mesenchymal transition (EMT) is one of the first events before initiation of the bona fide neural crest program, and the disruption of this step can result in very severe craniofacial anomalies. EMT is accompanied by changes in expression of members of the cadherin family molecules, including the down-regulation of Cad6B prior NC delamination. This process is regulated by Snail family members, which directly bind the Cad6B promoter and repress its transcription. However, the mechanisms underlying the role of repressive and activating signals in EMT are likely to be complex and involve multiple and interconnected factors. Here, we explore the possible role of epigenetic modification in this process and specifically the role of PHD12 (named for homology to plant homeodomain 12), a gene discovered as upregulated during neural crest induction, in cooperation with Snail2. Different studies have demonstrated that both Snail and PHD12 can recruit Sin3A/HDAC complex. This large multiprotein complex is able to deacetylate the histones located on the proximity of promoters to repress the target gene. Although Snail2 can bind to the E-boxes of Cad6B, we hypothesize it requires a partner able to read the epigenetic marks and recruit the repressive complex Sin3A. The goal of this application is to demonstrate that the direct interaction between Snail2 and PHD12 makes possible to recruit the repressive complex Sin3A/HDAC to complete shutdown Cad6B expression via histone deacetylation. This application involves three specific aims that include: Aim 1, characterize the role of PHD12 in NC EMT. Our preliminary data show that PHD12 is expressed in a time and location appropriate to be associated with NC prior their delamination. We hypothesize and will test whether the presence of PHD12 affects the NC EMT related genes. In the Specific Aim 2, we will determine the mechanistic analysis of Snail2, PHD12 and Sin3A interaction, and their role in Cad6B repression. Our preliminary ChIP experiments provide evidence that PHD12 interacts with Cad6B locus. We hypothesize and will test the interaction between Snail2-PHD12-Sin3A as well as the requirement of this interaction to bind to the Cad6B locus to repress it via promoter deacetylation. Finally, in the Specific Aim 3 will determine the role of PHD12 in the migration of NC cells and the formation of craniofacial derivatives. We will focus on the role of PHD12 in long term effects on neural crest migration to the branchial arches and subsequent differentiation. To extend this to later times when neural crest cells are forming critical craniofacial derivatives, chick embryos will be allowed to develop in ovo to later stages (e.g. branchial arch stages; cartilage and bone formation; palatal shelf formation) to test if early knockdown leads to later problems in migration and/or formation of facial derivatives. Taking together we will systematically address the epigenetic role of PHD12 on NC EMT and it implication on the formation of craniofacial derivatives.

DESCRIPTION (provided by applicant): The neural crest is a multipotent embryonic cell population that contributes to diverse derivatives, including peripheral ganglia, cartilage and bone of the face, and melanocytes. We have proposed and tested a multistep gene regulatory network (GRN), comprised of a logical series of distinct regulatory steps that act in concert to imbue the cranial neural crest with its defining traits. However, there are significant differences in developmental potential and migratory pathways of different neural crest populations arising at different axial levels. Here, we propose to explore GRN differences along the neural axis, focusing on premigratory neural crest cells from two distinct regions: cranial versus trunk. Our preliminary transcriptome analysis reveals many transcription factors and signaling molecules specific to the cranial but not trunk neural crest or vice versa. Our goal is to determine the position of these genes in the cranial versus trunk GRNs. This systems level strategy will provide understanding of why neural crest GRNs produces a particular regulatory state for use in preprogramming these cells to a different state. The aims are: Aim 1: Multiplex perturbation analysis of GRN connections at cranial and trunk levels. With the genome-wide representation of the active transcriptome of premigratory cranial and trunk neural crest in hand, we will perform loss-of-function experiments to perturb gene function and quantitate subsequent global transcriptional changes in putative target genes in single embryos using Nanostring analysis. Aim 2: Phylogenomic and functional analysis/dissection of neural crest enhancers. We will identify cis-regulatory elements that mediate expression of key GRN factors in cranial versus trunk neural crest populations. We will perform multidimensional modeling that incorporates results of transcriptome data and active enhancers with functional perturbation results into representational models of neural crest GRNs. Aim 3: Reengineering of the trunk neural crest program to test skeletogenic potential. Using GRN information, we will challenge the fate of trunk NC by reengineering their regulatory circuits and observing if misexpression/deletion of key GRN subcircuits affects their identity and ability to contribute to cartilage.

The neural crest is a multipotent embryonic cell population that contributes to diverse derivatives, including peripheral ganglia, cartilage and bone of the face, and melanocytes. We have proposed and tested a multistep gene regulatory network (GRN), comprised of a logical series of distinct regulatory steps that act in concert to imbue the cranial neural crest its defining traits. However, there are significant differences in developmental potential and migratory pathways of different neural crest populations arising at different axial levels. Here, we propose to explore GRN differences along the neural axis, focusing on premigratory neural crest cells from two distinct regions: cranial versus trunk. Our preliminary transcriptome analysis reveals many transcription factors and signaling molecules specific to the cranial but not trunk neural crest or vice versa. Our goal is to determine the position of these genes in the cranial versus trunk GRNs. This systems level strategy will provide understanding of why NC GRNs produces a particular regulatory state for use in preprogramming these cells to a different state. The aims are: Aim 1: Multiplex perturbation analysis of GRN connections at cranial and trunk levels. With the genomewide representation ofthe active transcriptome of premigratory cranial and trunk neural crest in hand, we will perform loss-of-function experiments to perturb gene function and quantitate subsequent global transcriptional changes in putative target genes in single embryos using Nanostring analysis. Aim 2: Phylogenomic and funcfional analysis/dissection of neural crest enhancers. We will identify cisregulatory elements that mediate expression of key GRN factors in cranial versus trunk neural crest populations. We will perform multidimensional modeling that incorporates results of transcriptome data and active enhancers with functional perturbation results into representational models of neural crest GRNs. Aim 3: Reengineering ofthe trunk neural crest program to test skeletogenic potenfial. Using GRN informafion, we will challenge the fate of trunk NC by reengineering their regulatory circuits and observing if misexpression/deletion of key GRN subcircuits affects their identify and ability to contribute to cartilage.

DESCRIPTION (provided by applicant): Evolution of vertebrates is intimately linked to the advent of the neural crest, a migratory and multipotent cell population that gives rise to many defining vertebrate characteristics, including a well-defined head and peripheral ganglia. Jawless fish (including the lamprey Petromyzon marinus) have bona fide neural crest cells, but do not have the full complement of neural crest derivatives, lacking jaws, sympathetic and possibly enteric ganglia. Here, we will test the hypothesis that the novel deployment of transcription factors in premigratory neural crest cells may have conferred new developmental potential onto this cell population, potentiating evolution of selected neural crest derivatives. Consistent with this hypothesis, two transcription factors, Twist and Ets1, are expressed in premigratory neural crest cells of gnathostomes but not lampreys. To test regulatory connections that may have facilitated production of novel vertebrate cell types, we will utilize RNA-seq together with novel transgenesis approaches to perform the following aims: Aim 1: Transcriptome analysis of lamprey premigratory cranial, vagal and trunk neural crest: To investigate the differences observed between cyclostome and gnathostome neural crest, we will perform RNA-seq analysis of cranial, post-otic (i.e. vagal) and trunk dorsal neural tubes containing premigratory neural crest cells from embryonic lamprey. We will confirm expression patterns of differentially expressed genes by in situ hybridization in lamprey and chick to find differentially expressed between agnathans and gnathostomes. Aim 2: Phylogenomic and functional analysis/dissection of conserved regulatory elements mediating expression of neural crest genes of lamprey and other vertebrates: We will identify regulatory regions of lamprey neural crest genes, initially focusing on Ets1 and Phox2b since they are differentially expressed between lamprey and gnathostomes. These will be tested by transgenesis in both lamprey and chick embryos. Aim 3: Utilize conserved regulatory elements to misexpress gnathostome neural crest genes in lamprey: We will utilize cross-species and/or endogenous enhancers to ectopically express missing transcription factors in lamprey to see if they promote formation of cell types that lamprey lack.

DESCRIPTION (provided by applicant): This is an application for renewal of a Program Project now in its 14'^ year. Here we propose to build in novel directions on the large success we have had in solving and authenticating gene regulatory networks (GRN) for development. GRNs provide causal explanations for developmental processes in the terms of the genomic regulatory code, in which all species-specific developmental processes are ultimately programmed. A developmental GRN serves as a conceptual, system-level logic map, which we have shown to possess direct predictive power. Thus GRNs bridge between functional genomic DNA sequence of regulatory significance and the biology of embryogenesis and body plan formation. They do this by specifying the regulatory interactions which causally drive the progression of regulatory states in diverse cellular territories. During recent years, this Program has been responsible for the experimental solution of the most advanced developmental GRN yet available for any developing animal organism. This is the GRN underlying the specification of the endomesodermal territories of the sea urchin embryo. Recently proof of the principle that as a GRN approaches completion it indeed provides explanation of all the observed biological, functions has been obtained in this work. We now intend to capitalize on the growing suite of successful technological and conceptual approaches to GRN analysis that we have developed, to confront challenges that heretofore were inaccessible, or could not even have been defined. Current or soon to be completed sea urchin embryo GRNs include all but one of the major domains of the embryo, from the earliest zygotic genomic activity (at the beginning of cleavage) to just before gastrulation. In addition, in the current period of the Program Project, an advanced GRN has been successfully constructed for the cranial neural crest of the chicken using the intellectual and technological approaches pioneered by this Program Project. The DAVIDSON COMPONENT (Project I) of the sea urchin embryo GRN will now expand GRN analysis to Include the whole of the embryo in a single GRN model such that every input to every part of RENEWAL The only way medical practice will advance beyond elegant forms of band aids and single molecule drug targets will be by interventions at the level of organization that life system actually operate, particularly the control systems. This Project concerns the most advanced example of genomic control systems biology we have at present. Its successful conclusion will show what the structure of these systems is; how to think about intervening in them; and directly inform considerations of the role of developmentally active regulatory gene mutations in the many forms of human developmental genetic disease we have become aware of. The medical research community is well aware of these points and the PI's of this application are frequently asked by forward looking members of it for collaborations, consultations, symposium presentations etc.

The objecfive of this Core Unit is to assure tight administrative coordinafion of the whole Program Project and to organize systematically continuing inter-laboratory communication.

The objective of Core A is indicated by its title. Its functions are defined as the provision of technologies, services and research materials commonly used by the individual Research Project components. These are services or materials which are difficult or expensive to generate, which can be provided by a central source, and indeed would be ridiculously wasteful for each lab to provide for itself. The emphasis here is on high technology aspects, and materials that require special facilities or know-how to produce.

Neural crest are a population of multipotent stem cells that originate in the dorsal part of the forming neural tube. Around the time of neural tube closure, they lose their epithelial characteristics and delaminate, by process known as epithelial-to-mesenchymal transition (EMT), acquiring migratory ability. Once neural crest cells reach their destination, they differentiate into numerous derivatives including neurons and glia of sensory ganglia. In a manner that is essentially the reverse of EMT, the formation of sensory ganglia requires the coalescence of neural crest cells via a process of mesenchymal-to-epithelial transition (MET). Both embryonic EMT and MET bear similarities to the molecular pathways and cellular changes undertaken by cancer cells during metastasis and establishment of secondary tumors. In recent years, there has been demonstrated that epigenetic-miRNA circuitries regulate EMT in various type of cancerous cells. However, the in vivo study of MET in cancer cells is very complex and unpredictable. In contrast, neural crest development is highly regulated and predictable, since the migratory pathways and process of condensation into sensory ganglia are well characterized. Based on this, we hypothesize that reversible epigenetic-microRNA regulatory networks may occur during transitional states of neural crest cells delamination (EMT) and sensory ganglia coalescence (MET). In this context, the goal of Aim 1 is to identify a core miRNA signature necessary for neural crest delamination and sensory ganglion condensation. Here we propose to perform using Ago2-HITS-CLIP assays, the first transcriptome-wide map of miRNA targeting events associated with epithelial plasticity during neural crest delamination and sensory ganglia condensation. Then, we will perform gain and loss-of-function experiments in chick embryos to validate the role of selected miRNAs in vivo. Next, we propose to determine the epigenetic regulation of core miRNAs expression (Aim 2). To this end, we will determine the DNA methylation status of selected miRNAs loci during transitional states of neural crest delamination and coalescence. Finally, Aim 3 will evaluate the epigenetic writers and erasers that control the core miRNA expression. Based on the proposed reversibility of the EMT and MET processes, we will examine the role of DNA methyltransferases (DNMT) and ten-eleven translocation (TET) as key regulators of DNA methylation/demethylation status of the core miRNAs necessary for neural crest delamination and sensory ganglion coalescence. Taken together, this study proposes to identify a core miRNA signature, their epigenetic regulation and regulators necessary for the EMT and MET during sensory ganglion formation. These studies hopes to lay the foundation for therapeutic intervention for certain types of neurocristopathies, neuropathies, and neural crest- derived cancers like neuroblastoma.

One of the most unique neural crest populations is the ?cardiac neural crest? that contributes to the outflow tract and outflow septum. Ablation of the cardiac crest in bird embryos causes a heart defect reminiscent of the human birth defect, persistent truncus arteriosus. In preliminary experiments, we have performed a transcriptome analysis of early migrating cardiac neural crest cells, isolated by enhancer-based cell sorting. The results reveal transcription factors (e.g. MafB, Krox20, Lhx1, Id1, Sall3) as well as signaling molecules and other factors that are selectively enriched in the early migrating cardiac neural crest compared to other cell populations. Here, we propose to explore the role of factors identified in our screen in the gene regulatory network that imbues the cardiac neural crest with its unique identify. Loss- and gain-of- function experiments will be used to functionally test the role of these factors and their position in a cardiac crest-specific gene regulatory module. In addition, we will perform cell lineage analysis using retrovirally encoded fluorophores to follow cell fate and gene expression of clonally related cardiac neural crest cells. The following specific aims will be performed: Aim 1: Testing regulatory connections of genes expressed in early migrating cardiac neural crest cells. With our preliminary genome-wide analysis of the active transcriptome of cardiac neural crest cells in hand, we will perform loss-of-function experiments to perturb gene function and establish the order of gene activity in the cardiac neural crest. Starting with MafB, we will perturb function of the transcription factors and analyze effects on expression of known neural crest genes as well as new genes uncovered in our screen. In this way, we can assemble a functional gene battery in the early migratory cardiac neural crest. Aim 2: Transcriptional profiling of individual cardiac neural crest cells using single cell RNA-seq and multiplex single molecule fluorescent in situ hybridization (smFISH). To gain a comprehensive view of the gene expression profile of individual cardiac crest cells, we will perform single cell RNA-seq on several hundred cells per time point sorted from the cardiac crest. To perform a similar analysis with the advantage of providing spatial information, we have devised an adaptation of smFISH called Spatial Genomic Analysis (SGA) that will be performed on tissue sections of carefully staged embryos, enabling simultaneous analysis of the expression of 35 probes selected from cardiac crest genes identified in our transcriptome dataset. Aim 3: Retrovirally mediated clonal analysis coupled with Spatial Genomic Analysis (SGA) to examine the cell lineage and fate of individual chick cardiac neural crest. To determine the developmental potential of individual cardiac neural crest cells to contribute to the cardiovascular system, we will perform multi-color clonal analysis of the cardiac neural crest region of chick embryos using a mixture of recombinant replication incompetent avian retroviruses (RIA) encoding different fluorescent proteins to label individual clones with distinct colors. By coupling clonal analysis with SGA, we will determine at single cell resolution which cells co-express transcription factors and signaling molecules identified in our screen. These specific aims are designed to define the molecular and cellular mechanisms underlying cardiac neural crest development. The ultimate goal is to provide important insights into the pathogenesis of septal defects that will lead to development of novel strategies for the prevention of neural crest-related heart defects.

A major question in developmental biology is how precursor cells give rise to diverse sets of differentiated cell types. This proposal tackles the question of multipotency and migratory behavior of neural crest cells, focusing on the cranial neural crest due to its broad ability to contribute to numerous and diverse cell types, as distinct as neurons and cartilage. Although classical grafting experiments have elucidated the derivatives of the neural crest, comparatively little is known about the developmental potential of individual cranial neural crest cells in vivo. Here, we propose to use replication incompetent avian retroviruses encoding different fluorescent fluorophores to label dorsal neural tubes in order to perform clonal analyses. The goal is to examine the developmental potential, movement and morphogenesis of individual or small populations of cranial neural crest cells. Experiments will be performed on avian embryos because of several advantages. Chick embryos are easily accessible to retroviral infection and experimental perturbation at early stages of development, allowing temporally and spatially controlled manipulation. Birds like humans are amniotes but, unlike mice, develop outside the mother. Therefore, they are much more accessible at early stage, while developing in a manner that is morphologically nearly identical to human embryos at comparable stages. Aim 1: Retrovirally mediated clonal analysis of the chick cranial neural crest: The cranial neural tube of chick embryos will be infected with replication incompetent avian retroviruses that encode four different fluorophores. Clonality will be established by visual observation of single cells a few hours after infection. We will then follow the long term fate of clonally related cells as a function of time by examining their localization and differentiation using antibody markers characteristic of various cell fates. Aim 2: Coupling lineage analysis with single molecule Fluorescent In Situ Hybridization to examine multiplex gene expression of clonally related cells. We will couple lineage analysis with a novel adaptation of smFISH that we have recently developed that allows multiplex analysis of gene expression at single cell resolution. Spatial Genomic Analysis (SGA) enables simultaneous analysis of the expression of 35 or more genes on tissue sections at migratory and post-migratory stages. We will combine clonal analysis with SGA to determine the genes co- expressed by clonally related cells using markers of various lineages together with neural crest and pluripotency genes to characterize the transcriptional profile of clonally related genes. Aim 3: Analysis of migratory interactions between clonally related cells: We will examine the migratory behavior of clonally related cells both in whole mount, using in ovo imaging, as well as in slice tissue sections to visualize interactions between sister cells and unrelated neighbors. Once normal migratory patterns and cell interactions are established, we will examine the effects of perturbing cell-cell interactions in individual clones migrating through an otherwise normal environment.

Bronner, M.E. In the early embryo, the neural plate border region contributes to diverse cell fates, ranging from neural crest cells and ectodermal placode cells to neurons of the central nervous system. Despite extensive studies of the neural crest and ectodermal placodes at post-neurula stages, surprisingly little is known about how these populations become distinct from one another within the early neural plate border. Based on our preliminary data, we hypothesize that many neural plate border cells are multipotent as evidenced by their concomitant expression of markers characteristic of several fates. We will test this hypothesis by: 1) conducting a detailed analysis of the emerging neural plate border region by multiplex protein and gene expression profiling coupled with cell lineage analysis and 2) examining how perturbation of transcription factor levels affects expression profiles and lineage allocations of individual neural plate border cells. The significance of this proposal is that it will be the first to test how and when ectodermal placode precursors are segregated from neural crest and neural precursors at the neural plate border. The following aims will be performed: Aim 1: High resolution analysis of protein expression of neural plate, neural crest, placode and other ectodermal markers in the neural plate border as a function of time. We will examine co-expression of transcription factors associated with neural crest, placode, neural plate and ectodermal lineages quantitatively and at single cell resolution in chick gastrula to neurula stages to determine their degree of overlap and if/when a discrete separation occurs between them in the neural plate border. To take this to a multiplex level, we will then perform single molecule fluorescent in situ hybridization analysis (smFISH) at similar stages with 35 or more probes selected from known genes and new candidates from our single cell RNA-seq dataset. Aim 2: Molecular dissection of regulatory interactions that mediate gene expression and cell fate choice at the neural plate border. We will examine the consequence of perturbing individual transcription factors (e.g. Pax7, Sox2, Six1) on expression of others neural plate border genes at the population and single cell level. To examine inputs that regulate neural plate border formation, we will dissect novel enhancers for Pax7, Six1 and other genes to determine direct regulatory inputs. Finally, we will examine how balancing levels of transcription factors may influence other factors in the neural plate border region. Aim 3: Single cell lineage analysis of cells at the neural plate border. To definitively test whether individual cells at the neural plate border have restricted or broad developmental potential, we will carry out single cell lineage analysis by performing iontophoretic injection of lysinated rhodamine dextran into individual neural plate border cells. We also will use enhancers for Pax7, Sox2, or Six1 as well as photoconversion of individual cells to follow the long term fate of neural plate border cells and examine how blocking individual transcription factors affects cell lineage allocation. 1

The neural crest is a uniquely vertebrate cell type that is thought to have played an important role in vertebrate evolution by forming peripheral ganglia and jaws, thus facilitating predation and expansion of the brain. We recently identified a ?cranial-specific? neural crest transcriptional subcircuit in jawed vertebrates (gnathostomes) that imbues this neural crest population with the unique ability, absent from trunk neural crest, to form craniofacial cartilage that is critical for jaw formation. Here, we propose to examine whether homologous genes are expressed in the cranial premigratory neural crest of lamprey, a jawless basal vertebrate (agnathan). Our preliminary results suggest that many of these genes are ?missing? from the lamprey's premigratory cranial neural crest, thus challenging the hypothesis that invention of neural crest in vertebrates gave rise to a ?New Head?. Accordingly, we hypothesize that there was progressive expansion of neural crest derivatives during the course of vertebrate evolution. This likely occurred by addition of new enhancer elements into the premigratory neural crest that conferred novel developmental potential onto this cell population. As case in point, lamprey lack a vagal neural crest that in gnathostomes forms the enteric nervous system. To test this hypothesis, we will perform lineage analysis, analyze transcription factors and regulatory regions of selected genes across agnathan and gnathostomes. We will identify putative enhancers, dissect their regulatory inputs, and test the ability of these regions to drive reporter expression in lamprey as well as cross-species, in the zebrafish. The results promise to elucidate how new cell types arose during vertebrate evolution under the umbrella of the neural crest.

Emergence of the vertebrate lineage was accompanied by the advent of the neural crest and its formation of novel derivatives, which allowed for the elaboration of the chordate body plan. While all vertebrates have neural crest cells, the neural crest is not a single population but rather comprised of distinct subpopulations, cranial, vagal, trunk and sacral, that arise at different levels of the body axis. Our preliminary results suggest that emergence of vertebrates may have occurred prior to regionalization of the body into four distinct neural crest subpopulations, since lamprey lack a discrete intermediate ?vagal? neural crest population. Thus, we hypothesize that addition of novel transcription factors via new enhancer elements in the premigratory neural crest may have conferred novel developmental potential onto this cell population, potentiating progressive expansion of the head and evolution of selected neural crest derivatives in jawed vertebrates (e.g. sympathetic, enteric neurons, jaws). To test this hypothesis and examine regulatory connections that may have facilitated production of axial level specific neural crest subpopulations, we will utilize transcriptomics, epigenomics and transgenesis approaches to perform the following aims: Specific Aim 1: Identification of lamprey homologues of ?cranial crest specific subcircuit? genes using a candidate approach. In chick, we have identified a gene regulatory subcircuit, unique to the cranial neural crest and lacking in trunk, that imbues them with the developmental potential to form craniofacial cartilage. We will test whether a homologous subcircuit exists in lamprey by isolating homologues of these genes and examining their expression pattern at premigratory, migratory and post-migratory stages. As our preliminary data suggest that several genes of this subcircuit may be ?missing? from the lamprey cranial premigratory neural crest but active later in the branchial arches, we will test the effects of ectopically introducing this subciruit at an earlier stage. Specific Aim 2: Transcriptional profiling of lamprey neural crest subpopulations along the body axis. We will perform whole population and single cell RNA-seq analysis of cranial, post-otic, trunk and sacral neural crest cells isolated by FACS from embryonic lamprey expressing crestin-enhancer mediated GFP for comparison with existing chicken and zebrafish neural crest transcriptome data sets for these axial levels. The goal is to identify transcription factors and/or genetic circuits that are absent or unique to lampreys and provide a snapshot of the transcriptional state of basal neural crest cells. Specific Aim 3: Identification of lamprey enhancers and testing their conservation via cross-species transgenesis. We will perform ATAC-seq at post-otic, trunk and sacral levels to identify open chromatin regions of lamprey neural crest cells, similar to our existing cranial ATAC dataset. For enhancer identification, we will start by testing peaks in the vicinity of 9 neural crest genes (FoxD3, Snail2, tfAP2, Ets1, Twist1, Dmbx, Lhx5, Brn3, Axud1) and expand this list as time allows. Putative enhancer regions will be tested for their ability to drive expression in the lamprey neural crest and dissected to identify their regulatory inputs. We have already identified SoxE and HoxA2 enhancers functional in the lamprey neural crest. Conserved functions will be examined via cross-species transgenesis to compare enhancer activity in lamprey and zebrafish embryos.