Juhee Jeong, Ph.D. - US grants

DESCRIPTION (provided by applicant): Craniofacial abnormalities involving the first branchial arch derivatives, including the jaw, palate and teeth, are a major class of birth defects in humans. Understanding the molecular genetic mechanisms behind craniofacial development is vital to devising innovative methods for the diagnosis, prevention and treatment of human defects. The long-term goal of my research is to characterize the transcriptional network that governs development of the first branchial arch, by identifying transcription factors and cis-regulatory elements important in this process, and determining their hierarchical relationship. Previous gene expression analysis suggested that the Lhx6 and Lhxd genes, which encode homeodomain transcription factors, may be major regulators of first branchial arch development, but the functional evidence to support this idea was scarce. My preliminary analysis of mouse mutants lacking both Lhxd and Lhx8 activity indicates that they are essential for the normal development of the palate and teeth. Therefore, my short-term goal is to further define the function and regulation of Lhx6 and LhxS in the first branchial arch using the mouse mutant model, as well as biochemical and molecular biology methods. The specific aims of this proposal are 1) To characterize the phenotypes of Lhx6-/-8-/- mouse mutants at the anatomical, cellular and molecular level. Based on my preliminary data, I will focus on the secondary palate and tooth development. 2): To identify downstream targets of Lhxd and LhxS using genome-wide, unbiased approaches which combine transcriptional profiling and chromatin-immunoprecipitation. I will compare gene expression in the wild type and Lhx6-/-;8-/- first branchial arch using microarray analysis. In addition, I will perform chromatinimmunoprecipitation followed by DMA chip analysis (ChlP-chip) using an Lhx6 antibody to identify direct target genes of Lhx6 and the cis-regulatory elements that Lhx6 acts upon. 3) To identify enhancer elements that control Lhxd and Lhxd expression in the first branchial arch. I will identify candidate enhancers based on the evolutionary conservation of sequences. I will verify their activity in chick neural crest electroporation system and transgenic mice. I will then use bioinformatic tools to identify candidate regulatory proteins for these enhancers for further analysis. *. The results of my research will provide critical information on how disruption in Lhxd and LhxS gene function and expression can contribute to human craniofacial defects. In addition, the results will significantly enhance our overall knowledge on gene expression regulation during craniofacial and dental development, and thus facilitate identification and understanding of genetic defects behind human craniofacial birth defects.

DESCRIPTION (provided by applicant): The calvaria (upper part of the skull) comprises plates of bone and fibrous joints (sutures and fontanelles), and the balance between the two components is essential for normal development of the calvaria. Calvarial malformation is among the most common birth defects in humans; craniosynostosis (premature loss of suture(s)) leads to a dysmorphic skull and can also affect brain and orofacial development. Current treatment of calvarial defects often involves invasive and repetitive surgeries with relatively high risks for complications, and thus improving the methods of intervention is imperative. Comprehensive understanding of the molecular genetic regulation of calvaria development is crucial to devising innovative methods for diagnosis, treatment, and prevention of related birth defects. The long-term goal of our research is to characterize the gene regulatory network of calvaria development, by identifying factors important for this process and determining their relationship. Based on our preliminary results, LDB1 (LIM-domain binding protein 1), a transcription cofactor, plays an essential role in calvaria development; inactivation of Ldb1 in head mesenchyme and some of the surrounding tissues in mice led to ectopic bone formation at the vertex in place of a fontanelle and parts of the sutures, mimicking craniosynostosis. In addition, an earlier report showed a similar defect in mouse mutants of human Nail-Patella syndrome gene, Lmx1b (LIM homeobox transcription factor 1b), which suggests that LDB1 and LMX1B may be partners in this context. However, molecular and cellular mechanisms by which Ldb1 and Lmx1b regulate calvaria development remain unknown. Therefore, the goal of this research proposal is i) to characterize the function of Ldb1 in calvari development, and ii) determine whether Lmx1b is indeed the partner of Ldb1 for this specific role. We will use mouse mutant models as well as cell culture and organ culture systems to determine the morphological, cellular and molecular changes resulting from the mutation of Ldb1 and Lmx1b. The outcome of this research will provide novel insights into the genetic regulation of calvaria development, and establish Ldb1 and Lmx1b as important players in human calvarial birth defects such as craniosynostosis.

ABSTRACT The calvaria (upper part of the skull) comprises plates of bone and fibrous joints (sutures and fontanels), and the balance between the two components is crucial. Craniosynostosis (premature loss of suture(s)) occurs at a frequency of 1/2000 births, and it leads to a dysmorphic skull that can further affect brain and orofacial development. Current treatment of craniosynostosis often involves invasive surgeries at young ages, with risks for significant morbidity and even mortality. Therefore, improving the methods of intervention for this defect is of great importance to public health. The long-term goal of our research is to obtain comprehensive understanding of the molecular genetic regulation of calvarial development, which can lead to innovative strategies to treat and prevent related birth defects. During embryonic development, the head mesenchyme primordium of the calvaria completely encases the brain from early stages. Subsequently, the calvarial bone starts to develop from the mesenchyme on the lateral sides of the brain just above the eye (`supra-orbital' mesenchyme, SOM), and expands gradually toward the vertex. In contrast, the mesenchyme positioned at the vertex from the beginning (`early migrating' mesenchyme, EMM) does not initiate ossficiation, and contributes only to the soft tissue such as the sutures and the dermis. This spatial restriction in bone formation is crucial to making the properly patterned calvaria because it allows the vertex to be occupied by sutures and fontanels instead of bone. To date, little is known about the factors that underlie this regional difference in the developmental program within the head mesenchyme. Based on our preliminary data, we hypothesize that EMM is intrinsically programmed to resist osteogenic induction, and that LMX1B (LIM homeobox transcription factor 1b) is a key anti-osteogenic factor in this context. Therefore, the goal of this proposal is to elucidate the molecular mechanism controlling the osteogenic competence of EMM with a focus on LMX1B. We will define the spatial and temporal specificity of LMX1B function during calvarial development, and investigate the effect of LMX1B on the function of osteogenic signals. Furthermore, we will use genome-wide approaches to define the genetic programs specific to EMM and SOM, and identify Lmx1b-downstream genetic network that regulates this patterning of the head mesenchyme. The outcome of our research will provide crucial insights into the regulation of early stages of calvarial development and identify novel mechanisms and players that can contribute to the pathogenesis of craniosynostosis.