Development is the elegant and complex process by which a single celled embryo grows into a fully formed and functional adult. One of the major challenges in the process of development is that although each cell in an organism contains the same DNA, different cell types must express different sets of genes in order to perform their highly diverse functions. Furthermore, to form a functioning organism, an embryo must do more than simply form the correct tissue types. It is not sufficient to simply form bone for example; a functioning skeleton requires the correct number of bones, the correct size and shape of the bones, and the correct interaction of each of the bones with each other and with other tissues. Positional information has been proposed as the means by which a cell is able to achieve its appropriate fate by identifying its location within a field of cells in order to correctly pattern tissues as well as the embryo as a whole. Positional information is conferred by a combination of extrinsic signals such as through morphogen gradients and through intrinsic cues such as transcription factor expression. Throughout this work, I have taken advantage of two classic developmental systems, the chicken limb and the mouse axial skeleton, to investigate the diverse gene regulatory mechanisms cells use to interpret positional information in order to properly pattern complex tissues.
In chapter two of this dissertation, I explore the role of long noncoding RNAs (lncRNAs) in regulating chicken limb patterning. LncRNAs are an abundant class of transcripts similar in size to messenger RNAs, which are transcribed but not translated, and which play regulatory roles, as RNAs, in controlling gene expression. One important outstanding question is how and to what extent lncRNAs function in development and differentiation in vivo. In order to address this question, we first identified lncRNAs expressed in the developing chicken using RNA-Seq and a series of bioinformatic tools. We identified a total of 3,197 lncRNA genes, including 2,589 intergenic lncRNAs (lincRNAs), 537 antisense lncRNAs, and 71 lncRNAs acting as direct precursors for small RNAs. Using a whole mount in situ hybridization screen we identified lncRNAs with potential roles in limb development. One candidate, HOXA10-AS, was functionally tested by knockdown via in ovo electroporation, demonstrating a requirement for proper patterning of the limb proximal-distal axis.
In chapter three of this dissertation, I examine the role of charge-dependant gene repression via chromatin compaction by the Polycomb group protein CBX2 in patterning the mouse axial skeleton in vivo. Polycomb and Trithorax group proteins are epigenetic regulators which stably maintain gene silencing or activation respectively over many generations of cells. Mechanistic studies of Polycomb Repressive Complex 1 (PRC1) have mainly focused on its enzymatic function to ubiquitylate histones, but recent studies suggest that the ubiquitylation activity of PRC1 is dispensable for silencing. The ability of CBX2, a component of PRC1, to compact chromatin directly in a charge-dependant manner has been proposed as an alternative mechanism to explain the role of PRC1 in maintaining silencing. To investigate this possibility we generated three independent mouse lines in which six, 13, or 23 positive amino acids in the putative CBX2 compaction domain were mutated to alanine, demonstrating in a dosage-dependent manner that charge-dependent chromatin compaction by CBX2 is required for the maintenance of gene silencing and for properly patterning the mouse axial skeleton—likely through regulating Hox gene expression.
Finally, I end this dissertation with five appendices describing separate and related studies. In the first appendix we examine how the number of skeletal elements in the chicken forelimb zeugopod (radius/ulna) is determined and implicate SOX6 as having an Avian-specific role in activating chondrogenesis in the anterior zeugopod. The subsequent four appendices are publications I contributed to as a co-author over the course of my PhD. In the second appendix our chicken lncRNA data was used as part of a larger analysis of the principles of lncRNA evolution derived from a comparison of 17 transcriptomes. In the third appendix we shared our chicken lncRNA data with the Avian RNA-Seq Consortium as part of the third report on chicken genes and chromosomes. In the fourth appendix we examine the roles of the miR-196 family of microRNA in independently regulating vertebral number and identity in the mouse axial skeleton. Finally, in appendix five we identify a Gremlin1 positive osteochondroreticular stem cell population in the mouse bone marrow with the potential to self-renew and to generate osteoblasts, chondrocytes, and reticular marrow stromal cells.
Taken together, this dissertation provides insight into the many diverse gene regulatory mechanisms required for patterning the vertebrate embryo. / Medical Sciences
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/33493379 |
| Date | 25 July 2017 |
| Creators | Schwartz, Matthew Gabriel |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation, text |
| Format | application/pdf |
| Rights | open |
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