Kristin Bruk Artinger, Ph.D. - US grants

During neural development, cellular differentiation of precursor cells generate a precise pattern within the embryo that is required for proper function. Cells are specified by receiving a series of inductive signals from the environment or within the cell itself, that instructs the cell toward a specific program of differentiation. A model system for studying the molecular events of neural development in the embryonic zebrafish. Due to the ability to perform large scale genetic screens, anamniotic mode of development, [d generation time and optical lucidity offers ideal conditions to study events in early embryogenesis invertebrates. The process of dorsal neural tube and neural crest formation and migration is a ideal system studying Patterning events during neural development. The specific goal of this proposal is to identify characterize genetic mutants that have specific defects in the dorsal neural tube. To this end, a large e genetic screen will be initiated to specifically screen using a battery of molecular probes, for dorsal neural tube mutants. Once these mutants are identified, it is the long term goal of this proposal to initiate cloning strategies and embryological techniques to determine the role of these genetic loci during development.

DESCRIPTION: (provided by applicant) Neural crest cells serve as progenitors for many important cell types, including neurons and glia of the peripheral nervous system and cartilage of the craniofacial skeleton. Elegant embryological studies have revealed key insights into the tissue and cellular interactions that control neural, crest cell specification ,migration and differentiation. Little is known, however, about the genetic mechanisms that control these processes. The goal of this proposal is to define molecular mechanisms controlling specification and differentiation of cranial neural crest cells in the developing vertebrate nervous system and craniofacial skeleton. Zebrafish will be used primarily for these studies since it is an advantageous system for studies involving both genetics and embryolqgy. I have recently determined that novel cell autonomous zebrafish mutation, narrowminded (nrd), is an essential regulator for the development of cranial neural crest cells. This proposal aims to molecularly characterize nrd, which will serve as a starting point in the isolation of additional regulators of the neural crest. Positional cloning of nrd, and the identification of candidate genes for he mutation has been initiated, both with the end goal of cloning the gene. Once the gene has been cloned, the further characterization the nrd gene product will be initiated in cranial neural crest cell patterning. This will include the analysis of the craniofacial phenotype of nrd by expression analysis, along with perturbation of endogenous nrd expression. To understand how nrd affects cell fate, lineage analysis will be done to correlate cell fate with morphological structure and localize patterns of gene expression. The results will lay the foundation for future experiments involving cranial neural crest cells. Finally, aim 3 will evaluate the role of other cell autonomous factors, such as Dlx3, that function downstream of nrd in neural crest cell formation and differentiation. Preliminary data using dominant negative and active mutants of Dlx3 suggest that it is crucial for correct patterning of the neural/non-neural ectodermal border region, from where neural crest, placodal cells and sensory neurons originate. The further characterization of nrd and downstream genes involved in cranial neural crest cell development will add to our understanding of this highly migratory population with the hope of preventing congenital disorders within craniofacial and other neural crest derived tissues, such as in Pierre Robin and DiGeorge syndromes.

[unreadable] DESCRIPTION (provided by applicant): In order to generate a functional nervous system, neural progenitors need to adopt one specific fate verses another. It is the long-term goal of my laboratory to determine the molecular mechanisms that regulate cell fate determination of neural crest and Rohon-Beard sensory neurons. Both neural crest cells and Rohon- Beard sensory neurons arise from the lateral portion of the neural plate and while neural crest cells migrate extensively to form all of the peripheral nervous system (PNS; among other derivatives), Rohon-Beard sensory neurons remain within the central nervous system (CNS). The specific objective of this application is to determine the location of the progenitor population for neural crest and RB sensory neurons, to determine whether prdml actively regulates cell proliferation and/or differentiation of neural crest and RB sensory neurons and to identify the components of the molecular pathway of prdml regulation on border cell fates. Our hypothesis is that prdml is crucial for formation of neural crest and RB sensory neurons at the lateral neural plate. A series of experiments to test this hypothesis is proposed. We propose to test the hypothesis that neural plate border progenitors are spatially segregated and restricted in their innate cell competence and their ability to respond to the environment. In addition, we will test the hypothesis that prdml acts to specify cell fate by promoting the differentiation of precursor cells at the neural plate border, without affecting cell proliferation or cell death. Lastly, we will address the hypothesis that prdml acts as a transcriptional represser to repress the target genes required for the maintenance of the precursor fate and thus promoting differentiation. Towards these goals, we will take advantage of the zebrafish system, Danio rerio, because it has well-established experimental and genetic methods make a detailed analysis of this process feasible. As an outcome of these studies, it is expected to determine that the prdml transcription factor plays critical role in the specification of neural tube and crest cells. This will be important in determining the genetic pathways that are important for neural tube and crest development, which in turn will be an important step in preventative measures in human congenital defects, such as spinal bifida and cleft lip and palate. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): In order to generate the craniofacial skeleton, neural crest progenitors need to adopt one specific fate verses another. It is the long-term goal of my laboratory to determine the molecular mechanisms that regulate cell fate determination of neural crest cell and their derivatives. Neural crest cells arise from the lateral portion of the neural plate and migrate extensively to form the craniofacial skeleton as well as other derivatives. The specific objective of this application is to determine the genetic requirement for prdm1 in the branchial arches of zebrafish and mouse, to determine the genetic interactions of prdm1 in the branchial arch, and to determine whether prdm1 actively regulates cell proliferation and/or differentiation in neural crest derived mesenchyme in the branchial arches of zebrafish and mouse. Our hypothesis is that prdm1 is crucial for formation of neural crest cell differentiation. A series of experiments to test this hypothesis is proposed. We propose to test the hypothesis that prdm1 required within the branchial arch for differentiation of the neural crest derived mesenchyme into ceratobranchial skeleton. In addition, we will determine the genetic hierarchy around prdm1 to test the hypothesis that prdm1 acts downstream of key craniofacial regulators and at the same level as hand2. Lastly, we will test the hypothesis that prdm1 acts to promote cell differentiation by promoting the proliferation of precursor cells, without affecting cell fate and is required within the neural crest derived mesenchyme in both zebrafish and mouse. Towards these goals, we will take advantage of the zebrafish and the mouse systems, because of their well-established experimental and genetic methods make a detailed analysis of this process feasible. As an outcome of these studies, it is expected to determine that the prdm1 transcription factor plays critical role in the differentiation of neural crest cells. This will be important in determining the genetic pathways that are important for neural crest development, which in turn will be an important step in preventative measures in human congenital defects, such as cleft lip and palate.

? DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of how defects in chromatin remodeling proteins, methyltransferases and acetyltransferases are causative for human craniofacial phenotypes. This represents an important problem, because craniofacial defects occur frequently in the human population, 1 in every 1000 live births annually in the U.S. (CDC, 2011) and many are associated with epigenetic regulators of the genome. Our long-term goal is to better understand the function of chromatin remodelers during cranial neural crest (cNCC) development. The objective of this application is to determine the mechanism by which two families of epigenetic regulators, KAT2a lysine acetyltransferase and PRDM lysine methyltransferases that regulate each other and act to modify the same H3K9 residue on histone 3, function in zebrafish and mouse cNCC development. We will use two excellent developmental model systems and combine genetic tools with live cell imaging of zebrafish and mouse cNCC behaviors and transcriptional studies to tackle the question of why mutations in Kat2a and Prdms lead to craniofacial abnormalities. The overall hypothesis is that these chromatin modifying enzymes act as opposing transcriptional regulators and function cell autonomously to regulate cNCC proliferation and migration. The rationale for this research is that understanding the mechanism of how KAT2a and PRDMs regulate cNCC development will have the potential to translate into a better understanding of the pathogenesis of craniofacial defects due to mutations in epigenetic regulators, including cleft lip and palate and various syndromes such as Kabuki and SBBYSS that affect the human population. From strong preliminary data, we have designed 3 specific aims: 1) Determine the developmental function of KAT2A and PRDMs in cranial neural crest development, 2) Examine the genetic interaction and regulation of gene targets by KAT2A and PRDMs, and 3) Determine the enzymatic regulation and chromatin state of KAT2A and PRDMs target genes. Under the first aim, we have determined that Prdm1, Prdm3, Prdm16 and Kat2a have craniofacial defects in both mouse and zebrafish. We have the tools and expertise to determine the specific craniofacial defects and to define abnormalities in proliferation and migration of cNCCs. For the second aim, we have generated and obtained most of the zebrafish and mouse strains, and performed transcriptional profiling in both zebrafish and mouse, demonstrating feasibility. For aim three, we have shown analysis of acetylation and methylation states in both tissue and biochemically. Our approach is conceptually innovative in testing a novel hypothesis and technically innovative in the use of live cell imaging and the interplay between two species that model human craniofacial development. The proposed research is significant because it is expected to advance an understanding of how cNCCs form the craniofacial skeleton, which has the potential to inform the treatment of neural crest associated birth defects and craniofacial syndromes.

Summary Rhabdomyosarcoma (RMS) accounts for ~3% of all pediatric cancers. Unfortunately, the overall 5-year survival rate for children diagnosed with metastatic RMS is less than 30%. Sarcoma patients experience higher rates of morbidity and mortality than other cancer patients, and this is particularly evident in children. As a result of their therapies, 42% of childhood cancer survivors experience severe, disabling, or life threatening conditions (including secondary tumors). Thus, there is clearly a need to develop new, more targeted treatment strategies for pediatric tumors; treatments that inhibit tumor progression yet confer limited side effects. The pro-metastatic transcription factor SIX1 is overexpressed in RMS, where it is plays a critical role in metastasis. During embryonic development, Six1 promotes precursor cell activation and migration to enable proper formation of muscle, kidney and the inner ear. However, after development is complete, Six1 expression is silenced or reduced in most tissues. Thus, Six1 is upregulated in the setting of RMS, where it is known to be important for progression of the disease. These data suggest that gaining an understanding of the mechanisms controlling Six1 downregulation during muscle development may provide a novel means by which to target Six1 in the setting of RMS. Such targeting may be anticipated to inhibit the tumor, yet have limited toxicity due to the paucity of Six1 expression in adult tissue. Throughout embryogenesis and myogenesis, microRNAs (miRs) have been shown to coordinate complex temporal and tissue-specific patterns of protein expression, including regulation of many homeobox genes. In tumor models, miRs have been shown to inhibit RMS tumor growth, and miR-mediated downregulation of Six1 can prevent kidney tumor progression. In zebrafish, there are two orthologs of Six1, six1a and six1b. Our recently published data demonstrate that miR30a negatively regulates both six1a and six1b in zebrafish muscle development, and that miR30a-mediated downregulation of Six1 is required for proper muscle development to occur. Thus, in this proposal we will test the following Hypothesis: Upregulation of Six1 is required for RMS tumor onset and progression. We will further test whether zebrafish will be an ideal model to rapidly and inexpensively test whether inhibitors of Six1 (beginning with miR30a, but in the future adding in additional miRs or novel Six1 small molecule inhibitors) will inhibit RMS progression. Specific aims: 1) To investigate the role of the Six1 transcriptional complex in RMS initiation and progression in zebrafish models, and 2) To identify potential therapeutic options for RMS through downregulation of Six1. The second of these aims will test, as proof of principal, whether miR30a has the potential to inhibit RMS progression. This R21 mechanism, by allowing us to develop the zebrafish models, will set the stage for testing novel small molecule inhibitors that target the Six1 transcriptional complex in the context of a whole organism. As we are currently developing such inhibitors, these models will allow for more efficient screening of a large number of compounds in vivo than could be done in other model systems.

Summary A fundamental question in developmental biology and genetics is how defects in cell fate specification and differentiation results in specific birth defects. This represents an important problem, because defects in limb and neural crest development underlie many human congenital birth defects including split hand foot malformation (SHFM). SHFM is a devastating congenital birth defect that presents with hands and feet that have a median cleft as well as craniofacial abnormalities that occurs in about 1:18,000 live births. Because there is limited information regarding genetic loci for SHFM, there is a critical need for clinical scientists to partner with basic developmental biologists to identify novel genetic loci and provide a mechanistic basis for this malformation such that treatments and genetic counseling can be developed. Here, we will identify novel loci for SHFM including the newly identified linkage with PRDM1. We hypothesize that PRDM1 functions as a transcriptional and epigenetic regulator required for NCC and limb development and when mutated, results in SHFM. The rationale for the proposed studies is that an in depth understanding of specific genetic loci responsible for SHFM will provide insights into both basic biology and the etiology of congenital birth defects. We will test this hypothesis in the following specific aims: 1) Determine the comprehensive patient phenotype and causative genetic loci for SHFM in humans. 2) Test the hypothesis that PRDM1 functions to regulate craniofacial and limb development and when mutated is causative for SHFM. Together, these studies will identify and test the function of new genetic loci for SHFM and once identified, determine the cellular and molecular mechanisms by which specific variant mutations function and the basis of the variability in phenotype. These data will provide a foundation for the design of therapeutic strategies for neural crest associated birth defects.

How cells become specified and differentiate at the correct time and place is a fundamental question in developmental biology. Cranial neural crest cells (cNCCs) are an excellent model system to understand this process due to the multipotent nature of the progenitor cells, generally unrestricted developmental potential with known lineage and derivatives, and defined gene regulatory networks. In addition to the gene networks, epigenetic regulators can affect the expression of numerous target genes and may help to explain the differences in penetrance and phenotype between individuals with the same genotype. This is important since defects in neural crest development underlie many human congenital birth defects, such as cleft lip with or without palate and many craniofacial syndromes. Thus, understanding the genetic and epigenetic regulators in cNCC development is key to understanding how cell fate is determined. We hypothesize that PRDM paralogs regulate global gene expression by regulating downstream targets oppositely, including Wnt pathway components, to control the timing of cartilage/bone differentiation within the cNCC lineage. The rationale for the proposed studies is that an in depth understanding of normal cNCC development will provide insights into normal biology and the etiology of neural crest-associated birth defects, many of which are thought to arise from cNCC abnormalities. We will test this hypothesis in the following specific aims: 1) Test the hypothesis that PRDM proteins act upstream of Wnt signaling to control the timing of cNCC differentiation into chondrocytes. We will test the hypothesis PROM paralog activity is required in cNCCs cell autonomously upstream of Wnt signaling to promote differentiation of chondrocytes. 2) Test the hypothesis that Prdm3 and Prdm16 genetically interact to regulate cNCC gene expression and chromatin accessibility. In Aim 2, hypothesis that Prdm3 and Prdm16 genetically interact to control gene expression via regulating transcription and chromatin modification specifically at cNCC and Wnt gene targets. 3) Test the hypothesis that Prdm3 regulates global gene expression by controlling the timing of genomic accessibility of Prdm16. In Aim 3, we will test the hypothesis that loss of Prdm3 leads to global alterations in chromatin state at cNCC progenitor genes via changes in binding of Prdm16 throughout the genome, which controls the liming of cNCC differentiation into chondrocytes. Together, these studies will reveal basic information of how cNCCs differentiate into specific cell types during development. The results of this proposal have the potential to reveal important new insights into cNCC development and how these processes go wrong in disease, with the hope of providing a foundation for the design of therapeutic strategies for neural crest associated birth defects.