1990 — 1991 |
Selleri, Licia |
F05Activity Code Description: To provide collaborative research opportunities for qualified non-immigrant alien scientists who hold a doctoral degree or its equivalent in one of the biomedical or behavioral sciences. |
Molecular Characterization of the T(11;22) of Ewing Sarc @ Salk Institute For Biological Studies |
0.909 |
1995 — 1998 |
Selleri, Licia |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Resources @ University of Texas SW Med Ctr/Dallas
transfection /expression vector; genetic library; biomedical facility; human genetic material tag;
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0.904 |
2004 — 2008 |
Selleri, Licia |
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. |
Genetic Control of Skeletal Development by Pbx1 @ Weill Medical College of Cornell Univ
DESCRIPTION (provided by applicant): Perfect coordination of the extent and timing of cellular proliferation with terminal differentiation is a genetically controlled process, fundamental for the development of all tissues and organ systems. Deregulations of this tightly regulated process can cause human birth defects and neoplastic transformation. Pbx1 is a homeodomain protein that collaboratively binds DNA with Hox proteins to modulate their DNA binding specificities and is a homolog of Drosophila extradenticle (EXD), whose function in patterning the fly body plan has been demonstrated genetically. Our ongoing efforts and long-term goals utilize genetically modified mouse models to assess the contributions of the Pbx family of Hox cofactors to mammalian patterning and morphogenesis. In embryos, the lack of Pbx1 (Pbx1-l-) results in late gestational lethality, widespread patterning defects of the axial and appendicular skeleton, homeotic transformation of second branchial arch neural crest cell-derived skeletal structures, markedly diminished chondrocyte proliferation, accompanied by precocious chondrocyte hypertrophy, and premature ossification of bone. Unlike Pbx1-l-, both Pbx2 -l-and Pbx3-l- mice do not display gross abnormalities either in patterning or in skeletal development/maturation. Nonetheless, Pbx1-l-; Pbx2-l- mutants die earlier in utero and show drastic exacerbation of the skeletal defects. The goal of this proposal is to finely dissect the genetic control of patterning and skeletal development by the homeobox gene Pbx1, through the following specific aims: 1) genetically uncouple the early roles of Pbxl in patterning from its later roles in cartilage proliferation, differentiation and endochondral ossification, through the generation of knockout mice where Pbxl is inactivated in a tissue-specific manner in neural crest and chondrocytes, by utilizing available Cre mice; 2) characterize the role of Pbx1 in chondrocyte proliferation by using genetically modified (Pbx1-l-) mesenchymal cells in culture, such as Mouse Embryonic Fibroblasts (MEFs), which show a striking growth defect, and Micromass Mesenchyme Cultures; 3) identify unique and overlapping functions of Pbx1 with the related family member Pbx2 in skeletal development and also exclusively in the genetic control of chondrogenesis. Completion of these studies will advance our understanding of the genetic regulation of patterning and skeletal development by Pbx1. Under a broader perspective, this work will shed light on the dramatic effects of the perturbations of skeletal development and hopefully impact on our understanding of the pathogenesis of human birth defects that affect the development of the craniofacial, axial and appendicular skeleton.
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1 |
2008 — 2009 |
Selleri, Licia |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Cloning of the Hol Mutation @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): Our laboratory uses genetic and developmental approaches, exploiting the mouse as a model system, to study skeletal patterning and morphogenesis during development. To this end, we are performing a phenotype- based forward genetic screen by ethylnitrosourea (ENU) mutagenesis in the mouse to uncover novel genes important in mammalian craniofacial development. Within the past two years, the screen produced four mutant mouse lines with skeletal defects. Two of these lines exhibit remarkable craniofacial malformations. Specifically, mutants from line 04/014 develop a short mandible, a hypoplastic or absent hyoid, and rudimentary neck cartilages. Also, their palate is cleft and the basisphenoid is fused to the basioccipital bone. Additionally, 04/014 mutants exhibit low-set and hypoplastic ear pinnae and their middle ear ossicles are severely affected. Finally, the cartilage primordium of the petrous part of the temporal bone is absent, leaving a hollow space (therefore this line will be defined hereafter as "Hollow ear", or Hol), and the Otic Capsule (part of the lateral chondrocranium, containing the developing inner ear apparatus) is hypoplastic and dysmorphic. Overall, the Hol craniofacial mutation phenocopies the Tbx1 homozygous mutation in the mouse, which models DiGeorge Syndrome (DGS). We propose that the Hol mutation disrupts a gene essential for vertebrate craniofacial and ear development. Indeed, by exploiting high-resolution genetic mapping, we discounted that the Hol mutation maps to either the Tbx1 or Crkl loci on chromosome 16. Furthermore, we mapped the Hol mutant gene to an interval of approximately 8 Mb on mouse chromosome 11, by exploiting novel mapping technology based on whole genome scanning using single nucleotide polymorphisms (SNP) panels. Finally, since the last amended submission of this proposal, we have further narrowed the Hol-bearing interval to approximately 3.9 Mb, by high-resolution mapping and polymorphic markers. We hypothesize that the Hol gene acts in the Tbx1 path- way in craniofacial development, but Hol is not Tbx1 or Crkl. We plan to uncover the molecular basis of this mutant phenotype through the following specific aims: 1) Identify Hol candidate genes, by performing further high-resolution mapping and microarray analysis for "gene finding";and 2) Identify the Hol gene, by conducting analysis of candidate genes and identification of the Hol molecular lesion by sequencing of candidate genes. Completion of these studies will advance our understanding of the genetic regulation of craniofacial patterning and morphogenesis, as well as lead to the discovery of a new gene that likely acts in the Tbx1 pathway. Under a broader perspective, this work will have an impact on the pathogenesis of human congenital disorders that affect craniofacial and ear development and function, such as DiGeorge Syndrome (DGS). PUBLIC HEALTH RELEVANCE: Knowledge of the patterning and morphogenesis of craniofacial and ear structures and of the genes implicated in their developmental processes is still elementary. Completion of the studies proposed here will represent a step forward in our understanding of the genetic regulation of patterning and morphogenesis of the vertebrate cranium. Furthermore, this work will uncover a novel gene that likely acts in a genetic pathway together with Tbx1 and Crkl and is required for critical craniofacial developmental processes. Under a broader perspective, these studies will have an impact on our knowledge of the pathogenesis of human congenital disorders that affect normal craniofacial and ear development and function, in particular with regard to the malformations of DiGeorge Syndrome.
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1 |
2010 — 2014 |
Selleri, Licia |
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. |
Genetic and Transcriptional Control of Spleen Development @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): Organogenesis begins with the specification, positioning and assembly of the cell types specific to an organ into the organ primordium (anlage). Active cell proliferation also takes place to build a critical mass for organ morphogenesis and expansion to occur. In this proposal, we will use the vertebrate spleen as a model to investigate these fundamental steps. The complex architecture and functions of the spleen result from intimate interactions among different cell types: mesenchymal cells (basic parenchyma), invading endothelial cells and colonizing hematopoietic cells. In humans, the spleen has critical roles in early hematopoiesis, immunity and blood filtering and its absence (as in congenital asplenia, an under-diagnosed disorder often recognized only at autopsy) results in a high risk for life-threatening bacterial infections in newborns and children. Our long-term objective is to identify genetic pathways that control the successive stages of spleen development: i.e. morphogenesis, expansion, and influx of hematopoietic and endothelial cells, since these interrelated organogenetic processes are of utmost importance to spleen function and yet mostly unknown. Using genetic approaches and asplenic mouse strains, we defined key steps in the genetic pathways that govern early spleen development. We reported that the homeobox gene Pbx1 is required for spleen cell fates and is a hierarchical co-regulator of Nkx2.5 and Hox11 (which are also essential for spleen formation). We also found that Pbx1 expression commences earlier than that of both Nkx2.5 and Hox11 in the Lateral Plate Mesoderm (LPM). Additionally, we uncovered that Pbx1 is expressed in the endothelium of the developing spleen anlage. In view of these findings, our hypothesis is that a distinct sub-population of Pbx1-positive progenitor cells within the LPM is required for spleen parenchyma specification, morphogenesis, and expansion and that Pbx expression in the endothelium also contributes to its function in spleen morphogenesis and expansion. In addition, we hypothesize that both an intact mesenchymal anlage and endothelium are essential for normal spleen hematopoietic colonization and function. Using available lines of gene-targeted and transgenic mice, we will test our hypothesis through embryologic, genetic, and molecular approaches. First, we will establish genetic and molecular pathways that control spleen morphogenesis and expansion. To this end, we will characterize the spleen morphogenesis and cellular proliferation defects in a mouse line with conditional inactivation of Pbx1 in the spleen mesenchymal parenchyma, but not in the endothelium. We will further utilize immortalized cell cultures generated from these embryonic spleens to determine the roles of Pbx in cell cycle regulation. Second, we will assess whether an intact endothelium is essential for spleen morphogenesis and expansion by characterizing a mouse line in which only the endothelium is altered by genetic inactivation of Pbx1. Also, by Pbx1 inducible inactivation, we will establish Pbx temporal requirements in the spleen endothelium. Third, we will genetically dissect the role of the mesenchyme and endothelium, respectively, in spleen hematopoietic colonization, development, and function. Our studies will shed light on novel genetic and molecular networks that underlie the development of the spleen, a neglected organ in regard to its ontogeny. In light of the intimate interactions among the mesenchymal spleen anlage, invading endothelial cells and hematopoietic cells, the new knowledge generated from this work will have a deep impact on the understanding of spleen function. Lastly, our studies aspire to provide a better comprehension of the pathogenesis of congenital asplenia, as we put forth the prerequisite basic genetic background towards prenatal molecular diagnosis of this condition.
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0.976 |
2010 |
Selleri, Licia |
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. |
Genetic Control of Limb Development by Pbx @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): Hox homeodomain transcription factors play essential roles in vertebrate limb patterning and morphogenesis, but the mechanisms of Hox regulation remain elusive at the molecular level. The Pbx1 TALE homeodomain protein is a homolog of Drosophila extradenticle (Exd), which has critical roles in patterning of the fly body. While the fly has only one TALE-encoding gene, Exd, the mouse has four Pbx genes (Pbx1-4). Based on molecular and biochemical analyses, for the last fifteen years the prevailing view has been that Pbx/Exd primarily helps Hox proteins execute their developmental programs. Hence, Pbx proteins have been named as "Hox cofactors". However, it has been unclear whether Pbx proteins exert their roles more broadly than Hox ancillary factors in vivo. Our objectives are to: 1) use the limb as a tractable system to delineate genetic and molecular networks controlled by TALE proteins in developmental programs;2) establish whether regulation of Hox "collinear" expression in the limb, an elegant but mysterious developmental phenomenon, is mediated by Pbx TALE homeodomain proteins. Using the mouse as a system, we have established that different Pbx genes share partially overlapping roles in various developmental processes, including limb patterning. Accordingly, Pbx1/Pbx2 double homozygous (Pbx1-/-;Pbx2-/-) embryos lack limbs altogether, while Pbx1-/-;Pbx2 mutants exhibit dramatic limb truncations. Most significantly, we have uncovered that during limb morphogenesis Pbx1/Pbx2 control the onset and spatial distribution of 5'Hox expression in the autopod. Thus, Pbx proteins hierarchically govern Hox gene expression in this system. In view of these unanticipated findings, our current working hypothesis proposes that Pbx homeoproteins do not function exclusively as Hox ancillary factors in the limb, but that they control Hox genes and possibly regulate 5'HoxD expression at the transcriptional level in the limb bud. We will test our hypothesis in the mouse using genetic and molecular approaches. First, by tissue-specific and inducible genetic ablation, using our new Pbx1 conditional allele (on a Pbx2-deficient background), and available Cre lines (one of which is inducible in the mesenchyme), we will dissect Pbx1/Pbx2 spatial and temporal requirements in limb field and bud mesenchyme. We will then determine when Hox expression is first affected by Pbx1/2 loss in the mesenchyme. By molecular approaches, we will subsequently determine whether Pbx1/2 regulate 5'HoxD transcription by direct control of the HoxD GCR, a genomic region that controls HoxD collinear expression in the autopod. Furthermore, we will test whether Pbx binding to the HoxD GCR has functional bearings on transcription by both transient transfections in cell culture and transient transgenesis experiments in vivo in the mouse. Finally, we will analyze Pbx1/Pbx3 phenotypic interactions in mouse forelimb (FL) development and assess Pbx1/Pbx3 control of early Hox expression in the FL field. We will specifically focus on FL patterning since Pbx3, which is not expressed in hindlimbs, acts as a FL-specific marker. Completion of these studies will shed light on Pbx- controlled programs in limb development and establish novel regulatory networks that govern transcription of Hox genes, key architects of the body plan. Also, this work will bear directly on our understanding of human genetic limb malformations. More broadly, given the involvement of HOX genes in human chromosomal translocations that perturb HOXD13 or HOXA9 expression resulting in acute leukemia, our studies will inform general comprehension of Hox gene function also in other contexts, as human neoplastic transformation. PUBLIC HEALTH RELEVANCE: Homeodomain proteins control patterning and morphogenesis of the appendicular skeleton within genetic and molecular networks that are poorly understood. We have established critical genetic roles for the TALE-class of homeoproteins Pbx1 and Pbx2 in limb development. The studies proposed in this application will provide novel insight into the control of HoxD gene expression by Pbx in the mesenchyme of the distal limb, as well as strengthen our new model wherein Pbx do not act solely as Hox ancillary factors in limb development, but govern Hox gene expression. Furthermore, the proposed experiments will shed light on the unique and partially overlapping contributions of Pbx1/Pbx2 in limb bud mesenchyme in skeletal development. Finally, the planned studies will establish as yet unknown roles for Pbx3, a forelimb-specific marker, in patterning anterior and proximal forelimb bud mesenchyme together with Pbx1, as well as uncover novel molecular networks that are perturbed when Pbx1/Pbx3 are concomitantly lost. Direct involvement of Hox genes in human congenital malformations has been reported as a result of genetic mutations. Accordingly, a broader impact of this work will be the generation of new knowledge on the pathogenesis of human congenital disorders, including those that affect limb skeletal development and function. Indeed, mutations in 5'HOX genes result in severe limb abnormalities, including synpolydactyly, monodactyly, club foot malformation, hand-foot-genital syndrome, and brachydactyly, among others. Additionally, common forms of human acute myelogenous leukemias with fatal outcome involve chromosomal translocations that join HOXD13 or HOXA9 to NUP98, thereby resulting in perturbed and ectopic expression of the HOX gene. Lastly, perturbed expression of HOX genes in humans has been reported in several types of cancers, including lung cancers, gliomas, neuroblastomas, and breast and ovarian cancers. While this proposal focuses on Pbx functions in the limb and potential regulatory roles of Pbx on Hox transcription in this system, perturbation of Pbx-ruled developmental processes in the mouse leads to a variety of other abnormalities (in addition to craniofacial, axial, rib cage, limb, and girdle defects) that closely model a broad range of human congenital diseases, including congenital heart defects, diabetes mellitus, congenital asplenia. Therefore, it is of high social and medical relevance to conduct basic research that will establish as yet unknown Pbx-controlled networks in organogenesis as well as novel mechanisms for Hox transcriptional regulation and collinear expression. Deeper comprehension of these basic processes in the limb will be applicable to other contexts within the developing embryo and ultimately aid our understanding of the pathogenesis of human congenital diseases as well as neoplastic transformation.
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0.976 |
2012 |
Selleri, Licia |
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. |
Mechanisms of Pbx-Directed Genetic &Transcriptional Control of Limb Development @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): In vertebrates, Hox genes play major roles in the formation of most vital organs. It has been proposed that the exquisite DNA-binding specificities that allow different Hox proteins to regulate specific target genes, thus instructing the identity of distinct body structures, depend on interactions with other homeoproteins, which act as Hox cofactors. For the last fifteen years, based on molecular and biochemical analyses, the prevailing view has been that TALE homeodomain proteins, which comprise the products encoded by the Pbx gene family, act as ancillary cofactors for Hox. Pbx1 is a homolog of Drosophila extradenticle (exd), which has critical roles in patterning of the fly body. While exd is the sole Pbx-encoding gene in the fly, the mouse has four such genes (Pbx1-4). Despite their paramount roles in organogenesis and patterning of the body and limb axes, the molecular mechanisms of Hox regulation remain elusive. Our objectives are to use the mouse limb as the most tractable and established system to delineate whether regulation of Hox collinear expression, a basic and mysterious biological phenomenon, is governed by Pbx. We have established that different Pbx genes, similarly to Hox genes, share overlapping roles in limb patterning and outgrowth. Accordingly, Pbx1/Pbx2 double homozygous (Pbx1-/-;Pbx2-/-) embryos lack limbs altogether, while Pbx1-/-;Pbx2 mutants exhibit limb truncations similar to those of HoxA/D mutants. Additionally, we have found that Pbx1/Pbx2 control the onset and spatial distribution of 5' HoxA/D expression in limb mesenchyme. These findings establish that Pbx proteins hierarchically govern 5' HoxA/D gene expression in the limb. In view of these new findings, our hypothesis proposes a novel mechanism for Hox gene regulation, whereby 5' Hox expression is directly controlled at the transcriptional level by Pbx in the bud mesenchyme for limb morphogenesis and digit formation. We will test our hypothesis using embryologic, genetic and molecular approaches in the mouse. First, by molecular methods, we will determine whether Pbx1/2 regulate 5' HoxD transcription by direct control of the HoxD GCR, a genomic region that governs HoxD collinear expression in the autopod. We will then test whether Pbx binding to the HoxD GCR has functional bearings on transcription by both transient transfections in cell culture and transient transgenesis experiments in the mouse. Moreover, by tissue-specific and inducible genetic ablation, using our new Pbx1 conditional allele (on a Pbx2-deficient background), and available Cre lines (one of which inducible in the mesenchyme), we will dissect Pbx1/Pbx2 spatial and temporal requirements in the limb field and bud mesenchyme. By this approach, we will determine when Hox expression is first affected by Pbx loss in the limb bud. Completion of these studies will define novel regulatory networks that govern transcription of Hox genes and will directly contribute to the understanding of human congenital limb malformations. Broadly, given the involvement of human HOX genes in leukemias and solid tumors, our studies will inform general comprehension of HOX regulation also in human neoplasia. PUBLIC HEALTH RELEVANCE: Hox proteins play essential roles in the formation of many critical organs in mammals, including the limb; however, the molecular mechanisms underlying Hox gene regulation remain elusive. The proposed studies will use mouse models to provide novel insights into the control of Hox regulation by Pbx proteins in the limb. Accordingly, a broad impact of this work will be the generation of new knowledge on the pathogenesis of human congenital malformations, including those that affect limb skeletal development and function, occurring in approximately 1 of 500 human live births.
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0.976 |
2013 — 2016 |
Selleri, Licia |
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. |
Mechanisms of Pbx-Directed Genetic & Transcriptional Control of Limb Development @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): In vertebrates, Hox genes play major roles in the formation of most vital organs. It has been proposed that the exquisite DNA-binding specificities that allow different Hox proteins to regulate specific target genes, thus instructing the identity of distinct body structures, depend on interactions with other homeoproteins, which act as Hox cofactors. For the last fifteen years, based on molecular and biochemical analyses, the prevailing view has been that TALE homeodomain proteins, which comprise the products encoded by the Pbx gene family, act as ancillary cofactors for Hox. Pbx1 is a homolog of Drosophila extradenticle (exd), which has critical roles in patterning of the fly body. While exd is the sole Pbx-encoding gene in the fly, the mouse has four such genes (Pbx1-4). Despite their paramount roles in organogenesis and patterning of the body and limb axes, the molecular mechanisms of Hox regulation remain elusive. Our objectives are to use the mouse limb as the most tractable and established system to delineate whether regulation of Hox collinear expression, a basic and mysterious biological phenomenon, is governed by Pbx. We have established that different Pbx genes, similarly to Hox genes, share overlapping roles in limb patterning and outgrowth. Accordingly, Pbx1/Pbx2 double homozygous (Pbx1-/-;Pbx2-/-) embryos lack limbs altogether, while Pbx1-/-;Pbx2 mutants exhibit limb truncations similar to those of HoxA/D mutants. Additionally, we have found that Pbx1/Pbx2 control the onset and spatial distribution of 5' HoxA/D expression in limb mesenchyme. These findings establish that Pbx proteins hierarchically govern 5' HoxA/D gene expression in the limb. In view of these new findings, our hypothesis proposes a novel mechanism for Hox gene regulation, whereby 5' Hox expression is directly controlled at the transcriptional level by Pbx in the bud mesenchyme for limb morphogenesis and digit formation. We will test our hypothesis using embryologic, genetic and molecular approaches in the mouse. First, by molecular methods, we will determine whether Pbx1/2 regulate 5' HoxD transcription by direct control of the HoxD GCR, a genomic region that governs HoxD collinear expression in the autopod. We will then test whether Pbx binding to the HoxD GCR has functional bearings on transcription by both transient transfections in cell culture and transient transgenesis experiments in the mouse. Moreover, by tissue-specific and inducible genetic ablation, using our new Pbx1 conditional allele (on a Pbx2-deficient background), and available Cre lines (one of which inducible in the mesenchyme), we will dissect Pbx1/Pbx2 spatial and temporal requirements in the limb field and bud mesenchyme. By this approach, we will determine when Hox expression is first affected by Pbx loss in the limb bud. Completion of these studies will define novel regulatory networks that govern transcription of Hox genes and will directly contribute to the understanding of human congenital limb malformations. Broadly, given the involvement of human HOX genes in leukemias and solid tumors, our studies will inform general comprehension of HOX regulation also in human neoplasia.
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0.976 |
2015 — 2021 |
Selleri, Licia |
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. |
Pbx-Directed Control of Cellular Behaviors That Drive Midface Morphogenesis @ University of California, San Francisco
? DESCRIPTION (provided by applicant): Clefts of the lip and/or palate (CL/P) are the most common human craniofacial birth defect (1/700 births). The mouse offers a suitable model to study human craniofacial morphogenesis and its abnormalities, which remain poorly understood. It was reported that a unique cellular behavior known as Epithelial Mesenchymal Transition (EMT) mediates craniofacial prominence fusion in the chick. However, the underlying mechanisms were not determined and it remains unknown whether EMT controls upper lip morphogenesis in mammals. Our evidence indicates that: 1) in the mouse embryo, in addition to apoptosis, epithelial plasticity at the prominence seam mediates tissue remodeling and fusion; 2) this process is dependent upon Pbx transcription factors (TFs); 3) epithelial cells at the Pbx mutant seam lose expression of Snail1 and nuclear Smad3/4, critical EMT mediators; and 4) Pbx TFs bind to Snail1 and Smad3 potential regulatory elements, as demonstrated by Chromatin immunoprecipitation (ChIP) on midface tissue. Moreover, whole-genome sequence analysis of Pbx1-bound regions in the murine face identifies additional potential Pbx target genes associated with epithelial plasticity, EMT, cell adhesion, and migration. In view of these results, we posit that, in addition to controlling apoptosis, Pbx TFs act as novel regulators of epithelial plasticity or EMT at the seam. We also posit that Pbx TFs concomitantly control closely related cellular behaviors, namely cell adhesion and migration, in mouse midface morphogenesis. First, we will determine whether local epithelial plasticity or complete EMT mediates tissue remodeling and prominence fusion at the seam. To this end, we will define the requirement for Pbx in the control of epithelial plasticity in the embryonic head using our Pbx-deficient mouse lines. Then we will investigate whether Pbx TFs are necessary and also sufficient to drive epithelial plasticity or complete EMT using NMuMG epithelial cells, a classic model of EMT. Second, we will establish whether Snail1 and Smad3 are regulated by Pbx TFs in midface morphogenesis. To achieve this, we will perform transient transfections in embryonic cells and transient transgenesis in the mouse. Third, we will delineate a comprehensive regulatory network of Pbx-directed effectors of epithelial plasticity, EMT, cell adhesion and migration in murine face morphogenesis. To this end, we will determine which Pbx1-bound enhancers identified by ChIP-Seq on midface tissue regulate Pbx target genes by transcriptome assays on wild type and Pbx-mutant cephalic epithelium. We will validate selected novel Pbx-regulated genes in vivo, with priority based on current findings for candidates Zeb1 & Zeb2 and Zpo1 & Zpo2, all of which are effectors of EMT, cell adhesion, or migration. This re- search will identify novel craniofacial cis-regulatory elements and Pbx-directed networks driving interconnected cellular behaviors that are critical for face morphogenesis, as well as revealing new Pbx target genes for pre- natal diagnostics of CL/P. Investigations on basic mechanisms underlying epithelial behaviors in head development will have applications to other processes that rely on the same cell behaviors, e.g., tumor invasion.
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0.976 |
2019 — 2021 |
Bush, Jeffrey Ohmann Selleri, Licia |
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. |
Phenotype-Driven Approach to Understanding the Function of Craniofacial Regulators Using Impc-Generated Mouse Strains @ University of California, San Francisco
ABSTRACT Many of the datasets resulting from genome-wide approaches lack functional validation in living organisms. While the laboratory mouse is often used as an experimental model, a large number of mouse genes have unknown functions. The International Mouse Phenotyping Consortium (IMPC) is building the first catalogue of mammalian genome function by generating knockout (KO) mouse strains for every protein-coding gene in the genome. Taking advantage of this opportunity, the two co-PIs have designed this proposal in response to NIH PAR-17-005 for phenotyping IMPC embryonic and perinatal lethal KO mouse lines. Our focus is on mutations that affect the craniofacial complex, based on our expertise in modeling craniofacial malformations in the mouse. While craniofacial defects represent one third of all human birth defects, our knowledge of the underlying cellular and molecular mechanisms remains poor. To select mutant mouse lines for in-depth phenotyping of craniofacial abnormalities, we generated algorithms to intersect the current list of IMPC lethal /subviable KO strains exhibiting craniofacial defects with: 1) all genes present in transcriptomes of mouse embryonic craniofacial domains that were either generated in our labs or available in the FaceBase database; and 2) all significant ChIP-Seq peaks for binding of the enhancer-associated protein p300 in mouse embryonic whole faces present in the FaceBase database. By searching the Mouse Genome Informatics (MGI) database for phenotypic data and by prioritizing genes that have unknown or poorly defined roles in craniofacial develop- ment, we restricted the number of chosen genes to N=30. These comprise regulators of various cellular functions that cause embryonic lethality and craniofacial defects when disrupted in the mouse. Preliminary phenotyping of the KO mouse line for Zfhx4, a gene that exhibited highest maxillary enrichment in our RNA- Seq dataset, revealed that all Zfhx4 KO embryos present cleft palate and maxillary hypoplasia, providing proof of concept for the effectiveness of the proposed strategy. Accordingly, we will characterize 30 mutant lines (15 per lab over 5 years) via the following specific aims: AIM 1: Examination and classification of 30 IMPC mutant mouse lines for craniofacial phenotypes. Employing a two-stage phenotyping pipeline, we will categorize the craniofacial defects for each line and narrow the time of onset. AIM 2. Deep characterization of craniofacial phenotypes. Based on three phenotyping platforms, we will uncover the function(s) of the 30 chosen genes in craniofacial development through in-depth analyses. Platform A will characterize early craniofacial anomalies including defects in branchial arch patterning, as well as primary palate morphogenesis and fusion (E8.5-11.5); Platform B will characterize later abnormalities of secondary palate fusion (E11.5-15.5); and Platform C will dissect perturbations of craniofacial shape/morphology and skull ossification. Shedding light on the cellular and molecular processes controlled by genes that are essential for embryonic development and cause human birth defects when disrupted will be vital to the health of the fetus before and long after birth.
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0.904 |
2020 — 2021 |
Marcucio, Ralph S [⬀] Selleri, Licia |
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. |
Transcriptional Regulatory Landscapes Underlying Fez Formation @ University of California, San Francisco
Summary: Morphogenesis of vertebrate tissues and organs occurs through well-orchestrated tissue interactions. During development of the upper jaw, signaling interactions among the forebrain, the facial ectoderm, and the intervening neural crest cells regulate development of the upper jaw. Our research has revealed that signals from the brain and the neural crest cells act together to induce expression of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ), a signaling center located in the surface ectoderm of the Frontonasal Process (FNP) that directs patterned growth of the upper jaw anlagen. Our preliminary data also suggest that Pre-B-cell leukemia homeobox (PBX) transcription factors are involved in inducing and maintaining the pattern of SHH expression in the FEZ by an interacting genetic network. The research proposed in this application is designed to uncover the gene regulatory mechanisms that are activated by signals from the forebrain and the neural crest, as well as, to investigate the role of PBX transcription factors in regulating SHH expression in the FEZ. Specifically, we hypothesize that 1) SHH signaling from the forebrain induces ?competence? in surface ectoderm cells to express SHH; 2) subsequently, upon arrival, neural crest cells induce transcription of SHH in competent cells of the FEZ, and 3) PBX transcription factors participate in regulating expression of SHH in the FEZ. We will test this hypothesis in three specific aims using avian and murine embryos. In each Aim, we will use chick embryos because they allow us to manipulate the signaling interactions between the brain and the FEZ (Aim 1), the neural crest cells and the ectoderm (Aim 2), or expression of PBX1 and PBX3 (Aim 3). We will use a variety of genomic approaches (ATAC-seq, ChIP-seq, GRO-seq) to evaluate changes in chromatin and activated transcriptional regulatory elements (TREs) at the SHH locus. In Aim 3, we will also assess morphological, cellular, and molecular outcomes of altering PBX gene expression, and we will use mouse embryos to take advantage of data already generated in the Selleri laboratory. The results of the work proposed in this application will shed light on the molecular aspects of facial development, and will aid understanding of disease processes that occur in this region. Allelic variants in SHH, or SHH pathway members, are associated with human dysmorphologies of the craniofacial complex in diseases such as Holoprosencephaly and cleft lip with our without cleft palate. Recently, PBX genes have been identified as risk variants in patients with craniofacial dysmorphology. Hence, our innovative approach combining studies with a solid foundation in developmental biology with chromatin analyses will yield information that is directly applicable to understanding human dysmorphology.
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0.904 |