The limb is one of the most evolutionarily diverse structures in vertebrate morphology. Our lab focuses on the lesser Egyptian jerboa, Jaculus jaculus, as an opportunity to identify the mechanisms that reshape the limb as a consequence of natural selection. The jerboa is a bipedal rodent with elongated hindlimbs and three toes on feet that lack intrinsic foot muscles. During my thesis work, I sought to understand the progression and mechanism of muscle loss in the context of its relationship with the motor nerve. We initially tested two alternate hypotheses: either jerboas never develop foot muscles or muscle fibers form and are lost over time. RNA in situ hybridization to MyoD and immunofluorescence detection of myosin heavy chain determined that jerboas are born with muscles in their feet, which are virtually nonexistent by post-natal day 7. I examined the structure of the nerve and its associated neuromuscular junctions to evaluate if proper innervation occurs at early critical stages and if that innervation is maintained during the earliest stages of myofiber degeneration. I utilized immunohistochemistry to examine the integrity of the nerve structure and to identify structural synapses. I also injected neonatal jerboas with CTB-555 labeling dye to retrograde trace motor neurons of the foot back to their origin in the spinal cord. I discovered that the muscles in the feet of the jerboa receive proper innervation and that this innervation is maintained for several days after birth, even after the myofibers begin to disorganize and degenerate. These results indicate it is unlikely a developmental innervation failure is responsible for muscle loss in the jerboa but rather itself degenerates concurrent with loss of its innervation target.

The vertebrate skeletal system exemplifies the intricate relationship between genetic regulation and evolutionary adaptation. This thesis investigates the genetic mechanisms underlying the evolution of skeletal proportion and development, focusing on the Lesser Egyptian Jerboa (Jaculus jaculus), a rodent with extremely elongated hindlimbs. By integrating comparative genomics, developmental biology, and advanced gene editing technologies, we aimed to better understand the complex cis-regulatory landscapes driving these morphological changes. Here, I present work that identified thousands of putative cis-regulatory regions exhibiting opposing but complimentary evolutionary patterns in the vertebrate limb, underscoring the scale and complexity of evolution within cis-regulatory sequences driving the development of different skeletal forms. I also explore the feasibility of CRISPR/Cas9-mediated gene conversion to facilitate the study of complex genetic traits. Despite challenges in achieving high conversion efficiency, our findings emphasize the importance of precise temporal control of Cas9 activity in the rodent germline. Additionally, we leveraged a conditional knockout strategy using hemizygous transgenes and tissue-specific Cre systems, focusing on the Bone Morphogenic Protein (BMP) signaling pathway. This approach reveals the complexity of genetic redundancy and compensatory mechanisms, providing new insights into skeletal maintenance and homeostasis. This work collectively advances the understanding of macroevolutionary processes and genetic complexity, offering powerful tools for studying the genetic basis of trait evolution. The integration of these methodologies not only enhances our knowledge of vertebrate skeletal diversity but also holds potential for practical applications in conservation biology and biomedical research. This thesis emphasizes the importance of viewing evolutionary biology through the lens of comparative genomics and functional genetics, which will undoubtedly facilitate future discoveries in the field.

Vertebrate species use the musculoskeletal system as the framework for stability and locomotion; differing environmental conditions and organismal needs select for the optimal, relevant morphology. For about 55 million years since the divergence of the jerboa and house mouse from their last common ancestor, jerboas have evolved disproportionately elongated hind feet to help them traverse their environment and lost all muscle within these feet. These morphological changes occurred due to selection on phenotypic variance, and these same phenotypes are also sensitive to environmental variance. As such, my lab is interested in understanding if environmentally sensitive systems share a genetic basis with the evolutionary genetics of musculoskeletal morphology. I first addressed the development of skeletal proportion by determining that mice are unfortunately not an appropriate model to test if nutritionally sensitive genes are among the genes that drove the evolution of skeletal proportion. In chapter two, I built upon the work of a previous PhD student to gain a better understanding of the molecular mechanisms underlying jerboa foot muscle loss. Using a more complete genome and new tools for improved orthology annotation, I identified a set of key genes and pathways likely related to jerboa foot-muscle loss. In the final chapter, I developed an image processing tool that can automatically quantify cell aggregates from images such as chondrogenic micromasses. This new tool and the knowledge gathered from my thesis research will assist future studies to determine the genetic framework of limb development.

Over the course of millions of years, modifications in mammalian genomes have produced an incredible variety of phenotypes, from the antlers of deer, to the breathing capacity of whales, to the dexterous fingers of a human. This remarkable diversity is perhaps nowhere more evident than in the limb of the mammal, which, despite its underlying structural similarities, exhibits specialized morphologies that look strikingly different in different species. Thus the hands typing this abstract are structurally similar but morphologically and functionally divergent from the fins of a manatee, the wings of a bat, or the legs of a horse. Comparing the genetic and regulatory underpinnings of this divergence (and indeed, of the similarities between species) allows us to simultaneously reconstruct genomic evolution and delve into the developmental mechanisms that produce limbs. The lesser Egyptian jerboa, Jaculus jaculus, is genomically similar to the common laboratory mouse, Mus musculus, but has highly divergent derived characteristics in the hindlimb. The hindlimb of the jerboa features a striking elongation of metatarsals and tibia, the loss of two digits, and the fusing of metatarsals into one central bone. By engineering ‘jerboanized’ mouse models, where regions of the mouse genome are replaced with their jerboa homologues to produce animals with jerboa-esque phenotypes, we can elucidate the mechanisms of divergence and development in the jerboa in a well-established and highly manipulable model system. Here, I present work on two genetic engineering techniques that promise to transform interspecies comparisons in rodent systems. First, I introduce a large interspecies conversion that simultaneously deletes a ~26kb region of the mouse genome and replaces it with ~31kb of homologous sequence from the jerboa. Second, I present the first demonstration of active genetics, a technique that makes use of the CRISPR Cas9 system to convert genotypes, in a rodent model. Active genetics in the rodent may be used to increase the rate of inheritance of an allele, which has profound implications the future of genetic engineering in both laboratory and applied settings. Together these tools may be used to develop complex mouse models of jerboanized phenotypes to probe the specific genomic changes responsible for the evolution of the development of the jerboa morphology.

Many species that run or leap across sparsely vegetated habitats, including hooved animals like horses and deer, evolved the severe reduction or complete loss of foot muscles as skeletal elements elongated and digits were lost. Although entire groups of muscles were lost repeatedly throughout vertebrate evolution, the developmental mechanisms remain unknown. Here, we investigate the cellular and molecular mechanisms underlying the natural loss of intrinsic foot muscles in a small bipedal rodent, the lesser Egyptian jerboa (Jaculus jaculus). Although adults have no muscles in their feet, myoblast migration and fusion proceed normally to form multinucleated myofibers in newborn animals that rapidly disappear soon after birth. We were therefore surprised to find no evidence of apoptotic or necrotic cell death and no stimulation of a local immune response during stages of peak myofiber loss, countering well-supported assumptions of developmental tissue remodeling. We instead see hallmarks of muscle atrophy, including an ordered disassembly of the sarcomere apparatus associated with upregulation of the E3 ubiquitin ligases, MuRF1 and Atrogin-1. Transcriptome profiling of newborn jerboa foot muscle showed differentially expressed genes significant overlap with models of muscle loss such as cancer-induced cachexia and botulinum-toxin-induced muscle loss. We propose that the natural loss of muscle, which remodeled foot anatomy during evolution and development, involves cellular mechanisms that are typically associated with disease or injury.