2001 — 2011 |
Clouthier, David E. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Endothelin-a Signaling in Craniofacial Development @ University of Colorado Denver
DESCRIPTION (provided by applicant): The goal of the proposed study is to examine the role of endothelin-A (ETA) receptor signaling during cephalic neural crest cell development. Our preliminary results indicate that ETA receptor signaling is likely required for multiple aspects of neural crest development. We hypothesize that ETA receptor signaling is required by a subset of cephalic neural crest cells for both their migration into the arches and subsequent patterning of specific arch subregions during craniofacial development. Mice homozygous for a targeted mutation in the ETA gene are born with numerous craniofacial birth defects resulting from aberrant neural crest development. Our preliminary data show that loss of ETA signaling 1) results in absence of ETA-/- cells in the distal pharyngeal arches of ETA-/- +1+ chimeras, 2) prevents normal post-migratory neural crest cell proliferation and 3) disrupts development of neural crest-derived mesenchymal cells within the pharyngeal arches, but only in a region-specific manner. We have three specific hypotheses: 1) ETA receptor signaling is required for both cephalic crest cell migration into the pharyngeal arches and post-migration proliferation, 2) This dual action represents two distinct requirements for ETA signaling and 3) ETA expression in different arch subregions initiates or maintains distinct signaling cascades involved in the development of specific craniofacial structures. Our planned experiments include elucidating the fate of ETA-/- neural crest cells by following their migration and proliferation using a genetic marking system. We will investigate the timing of ETA action during neural crest development by transient blockade of ETA signaling in pregnant mice, using a potent and specific ETA receptor antagonist. Finally, we will investigate the regional significance of ETA receptor signaling within the first mandibular arch by inactivating the ETA gene in arch subregions using Cre/loxP technology. Understanding the mechanisms by which ETA receptor signaling contributes to cephalic neural crest cell development will be an important advance in understanding the genetic and cellular control of craniofacial development and the etiology leading to specific human craniofacial birth defect syndromes.
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2002 — 2006 |
Clouthier, David E. |
K02Activity Code Description: Undocumented code - click on the grant title for more information. |
Endothelin a Signaling in Craniofacial Development @ University of Louisville
DESCRIPTION (provided by applicant): Most bone and cartilage structures of the jaw and throat are derived from cephalic neural crest cells. These cells, arising from the hindbrain, migrate to the pharyngeal arches, where they undergo a proliferation and differentiation program culminating in the formation of craniofacial structures. While numerous signaling cascades regulate this developmental program, the effectors of these cascades are poorly defined. This Independent Scientist Award application requests salary support for the candidate to elucidate the downstream effectors of endothelin-A receptor signaling that control development of cephalic neural crest cells and their derivatives. The candidate will use a variety of techniques to accomplish this aim, including analysis of genomic changes using custom cDNA microarrays and analysis of proteomic changes using 2D-gel electrophoresis and mass spectroscopy. Career goals of the candidate are centered on developing an active research laboratory dedicated to understanding the molecular and cellular basis of normal and abnormal craniofacial development. This lab will serve the additional goal of training pre- and post-doctoral students in craniofacial research. An ISA will enhance the candidate's development as an independent investigator by enabling specific expansion of expertise and research activities. Immediate plans include traveling to an external laboratory to develop expertise in the production and analysis of large-scale custom cDNA microarrays, as well as learning modern techniques in protein profiling and identification at the Core Proteomics Laboratory and the Biomolecular Mass Spectroscopy Core Laboratory, both at the University of Louisville. To take full advantage of the array and proteomic data, the candidate will also attend a bioinformatics course designed to enable researchers to write programs to manage genomic and proteomic research data. The Institution's development plans include: provision of significant start-up funds; guaranteed salary support beyond the period of time requested for K02 support; permission for the candidate to spend essentially full-time conducting research: minimal departmental duties; funds to travel to laboratories as outlined in the candidate's plans; provision of funds to hire a post-doctoral fellow to contribute to the growing research program in the candidate's laboratory: participation in monthly inter- and intra-departmental discussion groups designed to facilitate the development of the candidate's research program. The research environment offers excellent opportunities for scientific interactions that foster scientific growth and development. Many senior investigators direct active research programs at the University of Louisville, offering many potential interactions. Importantly, the candidate's chairman is a well-established investigator studying the molecular and cellular basis of facial birth defects. Both his personal interaction as well as his continuing efforts to recruit individuals with active research programs in the areas of craniofacial development and genetics underscores the commitment to build a strong research group, further helping the candidate build his own research program.
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2009 — 2016 |
Clouthier, David E. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Hand2 Function and Regulation During Craniofacial Development @ University of Colorado Denver
DESCRIPTION (provided by applicant): The goal of this project is to define the function of the basic helix-loop-helix transcription factor Hand2 during mammalian lower jaw development. Our hypothesis is that Hand2 contributes to the development of most lower jaw structures by establishing a distal morphogenetic domain in the mandibular arch. Within this boundary, Hand2 confines genes involved in mandibular pharyngeal arch development while also repressing the expression of genes normally observed outside this domain. Further, we hypothesize that Hand2 expression and function is at least partially regulated by Twist1 through both transcriptional repression and genetic interactions. In both mouse and zebrafish, Hand2 is expressed in cranial neural crest cell (NCC)-derived cells within the mandibular pharyngeal arch, from which bone and cartilage of the lower jaw arise. Targeted inactivation of the Hand2 gene in mice results in early embryonic lethality (by E10.5). However, hand2-mutant zebrafish are viable for five days;in these mutants, extensive malformations in craniofacial cartilages are accompanied by positive and negative changes in gene expression. We have shown in term mouse embryos that Hand2-daughter cells compose most structures derived from the mandibular arch. In our Preliminary Data, we show that loss of Hand2 in NCCs resultst in craniofacial defects indicative of NCC mispatterning. In this proposal, we will address the role of Hand2 in facial morphogenesis in three Specific Aims. In Aim 1, we will define the cellular and molecular changes in facial development following conditional inactivation of the Hand2 gene in cranial neural crest cells. In Aim 2, we will use hand2 zebrafish mutants to examine whether Hand2 acts as both a transcriptional activator and repressor and use Hand2 conditional mouse mutants to identify targets of Hand2 action. In Aim 3, we will define the role of Twist1 in the Hand2 mandibular arch domain and determine whether Twist1 and Hand2 interact genetically within this domain. By coupling our cellular and molecular analysis of Hand2 conditional knockout mice with our functional analysis of Hand2 action in mouse and zebrafish, we expect to uncover novel regulatory mechanisms governing fate and identity of NCCs that ultimately lead to facial development and whose disruption can lead to human facial birth defects, including micrognathia. PUBLIC HEALTH RELEVANCE: Craniofacial birth defect syndromes occur in 1 out of every 250 live births and represent a large financial and social burden within our society. While numerous mouse developmental genetics studies have elucidated the basis of some of these syndromes, the cause of many more remains unknown. The phenotype of the hand2 mutant zebrafish indicates that Hand2 may be involved in establishing or maintaining a developmental domain necessary for mandibular arch development, though early lethality prevents analysis of the role of Hand2 in later arch patterning events and bone formation. Our study will directly address the function and regulation of Hand2 during lower jaw development using a combination of model systems. Together, our studies will help define how regional developmental domains necessary for facial development are established and how different domains may antagonize each other to produce a final facial plan. Such knowledge can be subsequently combined using a systems biology approach to build a more comprehensive "gene map" involved in facial formation, which could lead to significant advances in future tissue engineering approaches to treat human craniofacial anomalies.
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2009 — 2013 |
Artinger, Kristin Clouthier, David E. Postlethwait, John H. (co-PI) [⬀] |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Idenfication of Mirnas Involved Midfacial Development and Clefting @ University of Colorado Denver
DESCRIPTION (provided by applicant): The goal of this project is to identify microRNAs (miRNAs) involved in vertebrate mid-face development and to determine their function in this process. Our hypothesis is that a limited number of miRNAs play crucial roles in the patterning, proliferation and differentiation of the mid-face through precise spatial and temporal expression of specific protein-coding genes. Further, we hypothesize that both loss- and gain-of-function of these miRNAs leads to mid-facial and orofacial defects/clefting. In humans, defects in mid-facial development, including cleft lip/palate, account for the largest number of birth defects annually. Alone, over 400 syndromes result in cleft lip and palate, which has an occurrence rate of 1 in 800 live births. Our laboratories have a long-standing interest in determining the gene regulatory networks that control normal orofacial development, many of which are causative in animal models of mid-facial clefting. In our Preliminary Data, we show that microarray technology can generate comprehensive miRNA expression profiles and that bioinformatic analysis can define miRNAs whose expression patterns are conserved across vertebrates. In this proposal, we will investigate the identity and function of miRNAs expressed in the developing mid-face in three Specific Aims. In Aim 1, we will determine and validate the temporal and spatial expression patterns of miRNAs in the developing mouse maxillary/frontonasal prominences using miRNA microarray technology. In Aim 2, we will determine the in situ expression patterns of identified miRNAs in both mouse and zebrafish embryos to define those that show a conserved pattern of expression. In Aim 3, we will use zebrafish to determine the regulatory function and morphogenetic mode of action of the miRNAs by loss- and gain-of-function analysis. We propose using both mouse and zebrafish systems because their complementary well-established experimental and genetic methods make a detailed analysis of this process feasible. By defining miRNAs involved in mouse mid-facial development and then quickly and efficiently determining miRNA function in zebrafish, we will be able to elucidate the miRNAs involved in vertebrate mid-facial development. This is crucial to further our understanding of genetic events leading to human mid-facial birth defect syndromes. PUBLIC HEALTH RELEVANCE: Mid-facial birth defects represent a significant social and medical problem throughout the world. By defining the miRNAs involved in mid-facial development, we expect to uncover novel genetic regulatory mechanisms involved in normal and abnormal mid-face development. Our characterization of miRNAs in the mid-face will provide potential targets for the development of novel therapeutics, with the goal of repairing and preventing mid-facial human congenital defects.
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2014 — 2018 |
Clouthier, David E. Nie, Qing (co-PI) [⬀] Schilling, Thomas F (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Defining An Integrated Signaling Network That Patterns the Craniofacial Skeleton @ University of Colorado Denver
DESCRIPTION (provided by applicant): Much of the craniofacial skeleton arises from the pharyngeal arches, 3D structures that undergo complex changes in shape and gene expression over time. However, detailed analyses of early gene expression profiles in the arches have not been performed in vivo. Without such information, it is impossible to predict how gene expression changes will affect subsequent skeletal development. Our labs have extensively studied the endothelin1 (Edn1), bone morphogenetic protein (Bmp), Wnt and Jag/Notch signaling pathways in the arches, as these four pathways pattern the dorsal-ventral (D-V) axis of the facial skeleton. We have shown that Edn1 and Bmp signaling initially promote ventrally- expressed genes and later subdivide the arches into separate D-V sub-domains. Our preliminary data suggest that Wnt signaling controls competence to respond to Edn1/Bmp and that these three pathways are opposed by Jag/Notch signaling. The pathways regulated by these four signals are highly dynamic, containing multiple feedback loops and crosstalk that create a robust system resistant to perturbation. With a collection of mouse and zebrafish mutants in all four signals, we are in a unique position to assess conservation of gene expression across species in sufficient detail for computational modeling. Our goal is to use these models to predict in silicon facial defects observed following genetic perturbations of these signals. Our dual-species approach will identify new candidate genes and generate models that are clinically relevant to human craniofacial genetics. To address these goals we will pursue two specific aims. In Aim 1, we hypothesize that while Edn1, Bmp, Wnt and Jag/Notch signaling are all critical for establishing the initial identities of skeletal progenitor in the arches along the D-V axis, they each play distinct roles. We will address this hypothesis by using high-throughput RNA sequencing to define early changes in gene expression in mutants of all four pathways. These Early Response Profiles (ERPs) will be used to produce models that integrate gene expression changes across mutants to understand both the unique roles of each factor and crosstalk between signals. In Aim 2 we hypothesize that core sets of enhancers are responsible for the ERP for each signal, some of which mediate crosstalk between or feedback within a signaling pathway, as well as insulating pathways from one another. We will address this by isolating enhancers for genes identified in Aim 1 and testing their activities in both mice and zebrafish. These will be incorporated into our mathematical models to understand enhancer sensitivity and how this regulates sharpness of gene expression boundaries. Our long-term goal is to build a comprehensive model for a craniofacial gene regulatory network that can be amended as new data are available.
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2014 — 2018 |
Clouthier, David E. Niswander, Lee A. (co-PI) [⬀] Williams, Trevor J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Phenotyping Embryonic Lethal Knockout Mice With Neural Crest and Neural Defects @ University of Colorado Denver
DESCRIPTION (provided by applicant): Large-scale high-throughput mouse gene knockout studies have generated considerable data with respect to adult phenotypes of viable homozygous mouse strains. However, many strains do not survive into the post- natal period. The current KOMP2 initiative provides a tremendous opportunity to determine how the disruption of numerous genes impacts embryonic development and to provide insight into the gene networks responsible for normal embryogenesis and fetal development. These studies are especially relevant to the investigation of the underlying causes of human pregnancy loss and structural birth defects. Importantly, some of the most frequent developmental abnormalities observed in large-scale mouse knockout studies are also common human birth defects. Defects in neural tube closure and neural crest formation account for a high proportion of birth defects in humans and these defects can have severe consequences on viability. This reinforces how knowledge gained from the study of mouse models can impact our understanding of human pathology. This proposal brings together three experts - Williams, Clouthier, and Niswander - who have extensive and collaborative experience in the detailed characterization of embryonic mutations that disrupt neural tube closure, craniofacial formation, and heart development. Our experience in forward genetic screens and high- throughput characterization makes us cognizant of the necessity for rapid analysis of mutant strains in order to minimize the shelf-time of the live colonies. Our goal in Aim 1 is to expand and complement the phenotyping information for the KOMP2 strains by providing a detailed histological assessment of strains that would not otherwise undergo this gold standard of analysis at KOMP2. All data will be uploaded and available through a publicly accessible website, with annotations and ontologies established by IMPC, MGI and KOMP2. Aims 2 and 3 will delve much deeper into the cellular and molecular mechanisms responsible for the defects seen in neural tube (Aim 2) and neural crest (Aim 3) formation in a select number of KOMP2 strains. Aim 2 is relevant for understanding the causes of human neural tube defects such as exencephaly and spina bifida (1 in 1000 births). Molecular assays and genetic interaction studies will provide pathway information to elucidate the genetic networks that orchestrate NT closure. Innovative live-imaging will explore how changes in gene function alter the cell behaviors that underlie neural tube morphogenesis. Aim 3 is relevant to major abnormalities associated with neural crest pathology include craniofacial defects (1.5 in 1000 births), heart defects (1 in 125 births), and enteric nervous system defects (1 in 5000 births). Aim 3 will study formation, migration and differentiation of the neural crest ad its interactions with surrounding tissues. Furthermore, tissue-specific knockout studies will determine if the observed defects are autonomous to the neural crest or if they are caused by defective signaling interactions with other tissues, particular the ectoderm. Overall these proposed studies will provide biological insight into the function of new genes arising from the KOMP2 centers.
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2019 — 2021 |
Clouthier, David E. Firulli, Anthony B. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Gene Regulatory Networks That Establish Mandible and Maxilla Patterning @ University of Colorado Denver
Abstract Craniofacial abnormalities affecting the mandible, maxilla and jaw joint are commonly encountered birth defects, most of which require surgical correction to establish quality of life and in some cases survival. While one or more organizing centers within the developing pharyngeal arches, from which the face arises, may hold the key to understanding the etiologies of these defects, the existence of such centers has never been proven, leading to a poor understanding of how gene regulatory networks are regulated and integrated during facial development. This limiting knowledge stifles new approaches to efficaciously treat these deformities. Endothelin1 (through DLX proteins) and BMP (through SMAD proteins) signaling establishes positional and structural identity of neural crest cells within the ventral mandibular arch. This is primarily achieved by induction of the basic bHLH transcription factor HAND2. HAND2 and BMP synergy is required for induction of the bHLH factor HAND1 within the ventral-most ?cap? of the mandibular arch. Interestingly, altering HAND1 dimer partner choice (thus altering HAND1-mediated signaling) results in pronounced mid-facial clefting, even though Hand1 is not expressed in the mid-face structures. Our data shows that loss of either BMP or HAND2 activity disrupts the establishment of the ventral cap. These findings establish our hypothesis that the intersection of BMP and HAND2 activity establishes a ventral cap-signaling center, which acts in both cell and non-cell autonomous manners to drive upper and lower jaw development. Additionally, we hypothesize that DLX activity antagonizes BMP/HAND2 synergy. This proposal takes advantage of the craniofacial development expertise of Dr. David Clouthier, the bHLH signaling expertise of Dr. Anthony Firulli and a number of novel mutant mouse alleles to test these hypotheses in two Aims. In Aim 1, we will use single cell (sc) RNA-seq to define the gene regulatory networks that are initiated by the coordinated action of both HAND2 and BMP that act either in an autonomous (ventral cap) or non-cell autonomously (more dorsal first arch areas) manner. Following analysis of scRNA-seq data, the top HAND2/BMP effector candidates will be functionally evaluated in loss-of-function and genetic studies. In Aim 2, the role of DLX proteins in confining ventral cap size will be examined using a novel gain-of- function Dlx5 mouse allelefollowed by functional testing of DLX action by creating a Hand1 mouse mutant lacking DLX cis-element inputs. Together, these novel approaches will provide the first direct evidence that the mandibular arch ventral cap is a signaling center required for facial development. Relevance: Craniofacial abnormalities are common and require intensive reconstructive surgical corrections. HAND2 and BMPs play key roles in patterning the neural crest cells that form the face. Gaining insight into the molecular mechanism of this understudied developmental process could have great potential for initial development of non-surgical treatments for congenital craniofacial defects in patients.
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2020 — 2021 |
Clouthier, David E. Schwartz, David Albert Yang, Ivana V (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genes and Transcripts That Interact With Muc5b in Pulmonary Fibrosis @ University of Colorado Denver
ABSTRACT The overall goal of our proposed research is to identify genes and transcripts associated with MUC5B expression that contribute to the development of idiopathic pulmonary fibrosis (IPF). Rare mutations (16- 21) and common variants (22-26) are associated with IPF and account for at least 40% of the risk of developing this disease. In the past 5 years, we have found that: 1) a gain-of-function MUC5B promoter variant rs35705950 is the strongest risk factor for the development of IPF (22, 26-34); 2) epigenetic mechanisms affect the expression of MUC5B (35); 3) IPF is a complex genetic disease with rare and common variants contributing to the development of this disease, including pronounced changes in DNA methylation (36) and transcriptional subtypes (37); and 4) MUC5B appears to be involved in the pathogenesis of IPF (27, 38-40). However, there is no clear explanation for the low penetrance of the MUC5B promoter variant; while the minor allele of MUC5B is present in the heterozygous or homozygous state in ?19% of the general population (27), IPF occurs in far less than 1% of the population (41, 42). These observations lead us to postulate that the etiology of IPF will best be understood by identifying the genes and transcripts that control MUC5B expression and contribute to the development of pulmonary fibrosis. To generate a feasible experimental model to pursue this concept, we phenotyped the response to intratracheal bleomycin in the eight founder mouse strains that were used to create the Diversity Outbred (DO) mouse population (43) and found that although there is a clear relationship between Muc5b RNA and MUC5B protein expression and bleomycin-induced lung fibrosis, this relationship is not consistent across the eight founder DO mouse strains. These preliminary findings suggest that the DO mouse population can be used to integrate genetic variation with gene expression to create multi-scale models of bleomycin-induced lung fibrosis and then use this knowledge to understand the genetic basis of MUC5B-induced IPF. Based on these observations, we hypothesize that genes and transcripts that control bleomycin- induced Muc5b/MUC5B expression contribute to the risk of developing pulmonary fibrosis. In this project, we plan to phenotype and genotype 900 DO mice for their response to bleomycin to identify the genes that control MUC5B protein expression and contribute to the development of lung fibrosis (Aim 1), while also identifying transcriptional changes (including Muc5b transcript) associated with bleomycin-induced lung fibrosis (Aim 2). We will then determine whether these candidate genes and transcriptional changes identified in mice exposed to bleomycin are generalizable to IPF (Aim 3). The successful completion of these Aims will have broad impact, resulting in specific genetic targets and biologic pathways for use in the design of future preventive, mechanistic, and intervention studies of IPF.
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