Kimberly L. Cooper - US grants

[unreadable] DESCRIPTION (provided by applicant): The interest in small interfering, non-coding microRNAs has exploded due to emerging discoveries of their diversity and prevalence across animal species. For all of the enthusiasm that they have garnered in the field, very little is known of their individual target genes and biological functions. The goal of this proposal is to determine the function of a set of microRNAs expressed in the developing vertebrate limb bud by combining molecular biology, computational bioinformatics, and classical embryology. Specific Aim 1 will test the hypothesis that let-7e functions in the limb to inhibit inappropriate chondrogenesis. Specific Aim 2 will use similar experimental methods to investigate the functions of five additional microRNAs expressed strongly in the vertebrate limb. Specific Aim 3 will focus on elucidating the requirement for the microRNA assocated protein Argonaute 2 and its cognate microRNAs by generating a conditional null allele of Ago2 in the mouse and performing a microarray analysis. The results of these investigations will reveal functions of microRNAs in the developing embryo and will open avenues to research that may allow development of antisense RNA technology for therapeutic applications. [unreadable] [unreadable]

Millions of Americans are impacted by muscle loss ? as an effect of disease, injury, or aging ? and yet there is currently no cure for any form of muscle degeneration. Consistent with the mission of the National Institute of Arthritis and Musculoskeletal and Skin Diseases, it is therefore essential to understand muscle development and degeneration in the broadest context. The Cooper lab has identified naturally occurring muscle loss during development of a bipedal desert rodent, the lesser Egyptian jerboa. Aspects of the cellular process, which occurs early and rapidly after birth of the animal, defy predictions based on decades of muscle research in traditional model organisms and highlight gaps in the current state of understanding. Most surprising, despite the rapid and complete loss of muscle structural protein expression, there is no detectable evidence of cell death or an immune response in the jerboa foot. Early stages of muscle maturation appear to proceed normally, but nascent muscle structure subsequently disassembles by an as yet unknown mechanism. Muscle progenitor cells persist until late in the phase of muscle cell loss, but they are insufficient to restore muscle. A deep understanding of this remarkable phenotype stands to transform our understanding of the cellular and molecular mechanisms of sarcomere disassembly and to potentially identify unexpected developmental plasticity of neonatal muscle cells. Specifically, the First Aim will address the perplexing observation that no characteristic features of multiple mechanisms of cell death are detected concurrent with widespread and rapid muscle cell loss. We will apply an electroporation-mediated cell tracking approach to follow the fate of the muscle lineage after muscle cells can no longer be identified by expression of muscle proteins. The Second Aim will implement an RNA sequencing approach to identify the cellular and molecular processes unfolding at the initiation of muscle loss. Each Aim investigates an aspect of jerboa foot muscle cell loss that potentially intersects with human muscle degenerative disorders yet here occurs in the context of normal development of the organism. The experiments outlined in this proposal are essential first steps toward a broader goal of understanding the molecular mechanisms that underlie the striking anatomic specificity of hindfoot muscle loss in the jerboa. Since the fundamentals of cell and tissue function are conserved across species, or indeed traditional model organisms would have no value, answers to the questions outlined in this proposal will inspire explorations of new dimensions of cell biology in a variety of tissues and contexts.

The current standard of Mendelian breeding strategies in the laboratory mouse means that certain genotypes comprised of multiple loci and/or two or more linked genes are impractical. This is a significant obstacle to solving a variety of problems, including production of better animal models for drug discovery and to understand complex genetic diseases and evolutionary traits. The CRISPR-Cas9 based gene drive system recently developed in insects catalyzes the conversion of heterozygous genotypes to homozygosity and biases the inheritance of a preferred allele. Despite the potential to more broadly transform basic and applied genetics, especially in rodent models, this technology has not yet been developed and implemented in a vertebrate species. The proposed objectives will leverage the innovative design of a transgenic mouse with an ?active genetic element? to establish evidence for the feasibility and efficiency of CRISPR gene drive in rodents. The CopyCat element disrupts a pigmentation gene in the mouse genome. The insert encodes a guide RNA (gRNA) designed to recognize the homologous allele in a heterozygous animal. When crossed to a mouse that expresses the Cas9 transgene, gRNA and Cas9 protein will combine to cleave DNA at the target recognition site. The resulting double strand break can then be repaired by non-homologous end joining to produce a mutation or by recombination with the homologous chromosome to convert a heterozygous CopyCat genotype to homozygosity. Gene drive efficiency will be measured as the frequency of inheritance of a copied CopyCat allele on a genetically marked target chromosome. As the first steps toward implementing CRISPR-Cas9 mediated gene drives in rodents, the proposed work will assess the frequency of inheritance of a converted gene drive allele under conditions that vary the timing of Cas9 expression. Aim 1 of this proposal seeks to assess the efficiency of gene drive when Cas9 protein is expressed in the newly fertilized egg or in the embryo. Aim2 will assess the efficiency of CRISPR gene drive when Cas9 protein is expressed during male and female gametogenesis when homology directed repair is naturally favored. Even if unsuccessful, these efforts will catalyze the next phase of research using the CopyCat tool to further optimize conditions that favor gene drive allele transmission in mice. If successful, this work will initiate a new era of laboratory applications for gene drives in rodents and in other vertebrates.

The length of each skeletal element changes independently during development and evolution to transform an embryonic skeleton with similar sized cartilages into a diverse array of adult forms and functions. Loss of function mutations of many genes produce proportionately dwarfed skeletons that suggest a common ?toolkit? is required for elongation of all of the long bones. Far less well understood, however, are the mechanisms that establish the specific rate and duration of elongation at each growth plate, which together determine adult limb skeletal proportion. What are the genes that define skeletal proportion? Is differential growth controlled by modular enhancers that locally tune expression of genes common to all growth plates and/or by genes that function only in subsets of growth plates? Our laboratory is positioned to answer these profoundly important questions about how vertebrate limbs acquire form and function using two uniquely suitable species: the laboratory mouse and the lesser Egyptian jerboa. Among the nearest mouse relatives, the jerboa has the most extremely different hindlimbs with extraordinarily long feet, but its forelimbs are similar to the mouse. These similarities and differences coupled with high genome sequence homology enable the identification of genetic mechanisms that locally control skeletal growth rate. RNA-Seq analysis of mouse and jerboa forelimb and hindlimb elements revealed that 10% of orthologous genes are differentially expressed correlating with relative growth rates within and between species. These include 40 genes with strong evidence for enhancer modularity in both species. Aim 1 will implement comparative ATAC-Seq and mouse transgenesis to identify and functionally test modular enhancers in the mouse and jerboa genomes. We predict that some of these 40 genes are controlled by radius/ulna enhancers that are conserved between species and by distinct metatarsal enhancers that functionally diverged in jerboa and allowed the uncoupled evolution of jerboa hindlimb proportion. Our expression data also provides a valuable opportunity to fill critical gaps in our understanding of the genes that regulate limb skeletal growth and proportion in all vertebrates. We previously showed that IGF1 signaling is required in mice for hypertrophic chondrocyte size differences in growth plates that elongate at different rates. Although IGF1 has a well-established role in whole organism and organ growth, it is unclear how the pathway is locally regulated to modulate differential growth. In Aim 2, we will biochemically test the hypothesis that elevated protease expression in rapidly elongating skeletal elements cleaves IGF binding proteins thus freeing bioactive IGF1 protein for signaling to accelerate growth. Although six other high priority candidate genes are also expected to be critical regulators of skeletal growth, they have not yet been attributed growth plate functions. Aim 3 will implement a powerful overexpression approach in chicken embryos to test the hypothesis that each of these genes is sufficient to accelerate or inhibit limb growth rate.

A deep understanding of the genetic mechanisms of musculoskeletal development and disease requires study of genes that are frequently pleiotropic and/or highly redundant. Currently, the common strategy to study such complex genetic traits in mice is to combine multiple homozygous conditional (floxed) alleles with a tissue-specific and/or temporally inducible Cre recombinase transgene. However, such breeding strategies to understand three or more loci require an extraordinary investment of money, time, and mice to obtain just a few animals of the desired genotype, often limiting enquiry to a particular tissue and developmental stage. Here, we propose an innovative approach in mice using just three hemizygous transgenes to conditionally induce multi-gene, bi-allelic, loss-of-function mutations to study complex genetic control of development and maintenance of the limb skeleton. We previously demonstrated highly efficient CRISPR/Cas9 mutagenesis from genetically encoded elements that included the Rosa26-LoxStopLox-Cas9 transgene. Crossing these mice with a tissue-specific and/or temporally-inducible Cre transgenic line provides an opportunity to control the timing and/or location of CRISPR/Cas9 activity. The third transgene required for this proposed system is a `polycistronic tRNA-gRNA' (PTG) array of CRISPR guide RNAs interspersed with tRNA sequences. These transcripts recruit endogenous RNases to cleave apart gRNAs, and processed gRNAs can complex with Cas9 protein to induce sequence-targeted double strand breaks in DNA. PTG arrays have been effective in rice, Drosophila, zebrafish, and cultured human cells, but they remain untested in mice. Using this strategy, we plan to engineer and validate two models of skeletal development. In Aim 1, we will engineer a Smad1, Smad5, and Smad8 conditional knockout strategy to target loss of BMP/GDF signaling at a critical bottleneck. This will be a valuable model to thoroughly assess pathway function during limb patterning, digit condensation and interdigital cell death, chondrocyte specification and maintenance, and osteoblast specification and maintenance. In Aim 2, we will engineer an aquaglyceroporin (AQP3, AQP7, and AQP9) conditional knockout strategy. This model will allow us to test the hypothesis that these membrane channels that increase plasma membrane permeability to water and small uncharged osmolytes are necessary for growth plate hypertrophic chondrocyte swelling and maintenance of articular cartilage. Since the purpose of this R21 proposal is the engineering and validation of new technology and animal models, our primary emphasis will be to determine the efficiency and reproducibility of such strategies and to establish a set of guidelines for future implementation. Successful outcomes of this work will provide a wealth of opportunities to understand the importance of these two complex genetic systems, and the strategy has broad potential to accelerate research and reduce the costs associated with mouse models of development and disease.