Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 58-65).

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 85-120).

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 44-48).

Next Generation Sequencing (NGS) technologies have dramatically increased the throughput and lowered the cost of DNA sequencing. In this thesis, I apply these technologies to unresolved questions in skeletal development and disease. Firstly, I use targeted re-sequencing of genomic DNA to identify the genetic cause of the cartilage tumor syndrome, metachondromatosis (MC). I show that the majority of MC patients carry heterozygous loss-of-function mutations in the PTPN11 gene, which encodes a phosphatase, SHP2, involved in many signaling pathways. Furthermore, I show that cartilage lesions in MC patients likely arise following somatic second-hit mutations in PTPN11. Secondly, I use RNA-seq to identify gene expression changes that occur following genetic inactivation of Ptpn11 in mouse chondrocyte cultures. I show that chondrocytes lacking Ptpn11 fail to properly undergo terminal differentiation and instead continue to express genes associated with earlier stages of chondrocyte maturation. I validate these findings in vivo by examining markers of specific chondrocyte maturation stages in the vertebral growth plates of mice following postnatal mosaic inactivation of Ptpn11. Together, my results provide insight into the molecular mechanisms underlying the initiation and growth of cartilage tumors. In the third component of my thesis, I develop a method to map and clone zebrafish mutations by performing whole genome sequencing on pooled DNA. I apply this method to zebrafish mutants identified in a mutagenesis screen for adult phenotypes, including skeletal phenotypes, and determine that a nonsense mutation in bmp1a underlies the craniofacial phenotype in the wdd mutant. In summary, I show that NGS technologies can be successfully utilized to firstly identify the genetic cause of a human skeletal disorder, secondly investigate the molecular mechanisms regulating the maturation of skeletal cells, and thirdly expedite the process of mapping and cloning zebrafish mutants with skeletal phenotypes. Altogether, my research provides insight into the pathways and processes regulating skeletal development and disease.

Hematopoietic Stem Cells (HSCs) are characterized by their ability to both self-renew and give rise to all lineages of the blood system. A recent chemical genetic screen identified 17β-estradiol (estrogen) as a novel modifier of the expression of the conserved HSC markers runx1 and cmyb in the Aorta-Gonad-Mesonephros of developing zebrafish. Exposure to exogenous estrogen during the development of the hematopoietic niche impeded specification of hemogenic endothelium and the subsequent emergence of HSCs via antagonism of somitic-derived VEGF signaling. Conversely, inhibition of endogenous estrogen activity increased the number of functional HSCs present in the embryo and resulted in higher expression of VEGF target genes, suggesting that endogenous estrogen acts to define the ventral limit of VEGF activity and hemogenic endothelial specification. In contrast, when embryos were exposed to estrogen after niche specification, markers of HSCs were increased, indicating that estrogen has a biphasic effect on HSC formation; this effect appears to be at least partially mediated by enhanced cell cycling of the HSC population. Estrogen exposure during primitive erythropoiesis likewise increased the number of erythroid progenitors in the embryo, but their maturation into functional erythrocytes was impaired. Inhibition of erythrocyte maturation is also conserved in a mammalian model of in utero excess estrogen, causing propensity for embryonic lethality. Treatment of adult zebrafish with exogenous estrogen after ablation of the hematopoietic system by irradiation revealed that elevated estrogen levels improved hematopoietic regeneration. Consistent with a role for hormonal regulation of HSC homeostasis, accelerated recovery of hematopoietic stem and progenitor numbers was observed in female fish compared to males, suggesting an endogenous difference in regenerative capacity between the sexes. Together, these data identify multiple distinct roles for estrogen in HSC biology and indicate it is a physiologically relevant regulator of HSC development and homeostasis.

Hemoglobin switching is a developmental process involving the dynamic transcriptional regulation of multiple globin genes. This molecular process involves multiple layer of complexity, and elucidating new mechanisms in this process will result in a more complete understanding of general gene regulation and will likely have direct clinical implications for hemoglobinopathies, such as sickle cell anemia. In this dissertation, I develop and characterize a new model for hemoglobin switching, the zebrafish. I defined and fully annotated the two zebrafish globin loci, termed major and minor loci. Both loci contain α– and β–genes oriented in a head–to–head fashion. Characterization of the globin expression pattern precisely defined the timing of normal switching and demonstrated that zebrafish, like humans, have two globin switches. The locus control region for the major locus was identified and in conjunction with a proximal promoter was able to generate robust, erythroid–specific expression in a transgenic line.

Sonic hedgehog (Shh) is a signaling molecule that is involved in patterning the embryo and regulates adult stem cell homeostasis. Patched1 (Ptch1) is the receptor for Shh and upon binding to Shh is endocytosed, allowing downstream signaling to occur. Ptch1 is critical to the cellular response to Shh because it is both a negative regulator of the Shh signaling pathway and a transcriptional target of the pathway. Therefore, the regulation of Ptch1 levels will directly affect the ability of cells to respond to Shh. Understanding this process requires the characterization of novel Ptch1-interacting proteins that regulate Ptch1 levels in the cell. This thesis investigated a role for the adapter protein Tom1 as a putative Ptch1-interacting protein involved in regulating Ptch1 levels through endocytic cycling. It was found that Tom1 overexpression did not regulate the patterning of vertebrate nervous system, but did play a role the sub-cellular localization of Ptch1.

In early rainbow trout development, a switch from high-affinity embryonic hemoglobin to lower-affinity adult hemoglobin occurs along with a turnover of round, embryonic erythrocytes to oval, adult erythrocytes. The objective of my thesis was to determine how the ontogeny of blood oxygen transport was affected by chronic hypoxia (30% of saturation) in rainbow trout. Hemoglobin-oxygen affinity, cooperativity, and the Bohr and Root effects were unaffected by hypoxia treatments, whereas hemoglobin content, erythrocyte counts, and hematocrit were significantly reduced. In hypoxia, larvae had higher concentrations of embryonic hemoglobin mRNA and embryonic erythrocytes than stage-matched normoxia-reared larvae. Overall, these results indicate that chronic hypoxia suppresses erythrocyte development prior to complete yolk absorption. Ultimately, this suggests that in early ontogeny rainbow trout conform to low oxygen conditions, rather than mounting the hypoxia response observed in oxygen-regulating adult trout.

<p>Roots are developmentally plastic and highly dependent on the immediate environment. By studying root responses to abiotic stress, we have identified novel regulators of developmental modulation. When roots are deprived of phosphate (Pi), developmental programs are modulated to slow primary root growth and expand surface area through emergence of root hairs. By focusing on exposure time-periods of less than two days, we have described very early changes to root development in response to this condition that may reveal new mechanisms of root hair specification and emergence. Also, using transcriptomic analyses with high spatial resolution, we identified a kinase that is specifically induced in root vascular tissue within three hours of exposure and acts to modulate aspects of root development in response to deprivation of Pi. These data suggest that individual tissues play unique roles in whole organ development, and that interpretation of Pi -deprivation responses may change as we develop methods with resolution necessary to understand these roles. Beyond Pi, we compared transcriptomic data for four additional stresses and identified a novel stress-responsive transcription factor that modulates expression of a cell expansion protein. This putative network connection demonstrates the value of using high-dimensional data for inference of regulatory relationships. Overall, we have combined "-omics" approaches with reverse genetics to identify novel developmental regulators and described a phenotypic frame-work with resolution at which cellular mechanisms can be studied.</p> / Dissertation

Thesis (M.S.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Christa Merzdorf. Includes bibliographical references (leaves 51-55).