Deciphering the Transcriptional Mechanisms and Function of SOX3 in the Developing Embryonic Mouse Brain and Postnatal Testes

SOX3 is a transcription factor found within neural progenitor cells (NPC) of the developing and adult vertebrate central nervous system. SOX3 is also found in other tissues, most notably the spermatogonial progenitor/stem cell populations in the testes. Normal brain development in both humans and mice, and sperm production in mice, is reliant on the correct expression and dosage of SOX3. The function of SOX3 has been explored through a number of different cell and mouse model based techniques, however, the mechanisms through which SOX3 acts remain largely unknown. This thesis explores the genome wide DNA binding profile of SOX3 in both NPCs and postnatal testes, two very different sources of SOX3 expressing cells. We identified 8064 binding sites within NPCs derived from cultured mouse embryonic stem cells, linking SOX3 to a number of different neural development pathways. Additionally, we identified 778 SOX3 binding sites within postnatal day 7 mouse testes, linking SOX3 to the control of histones and histone variants, most of which was also true for NPCs. We utilised our Sox3 null mouse model and a number of different marker genes of spermatogenesis to identify that SOX3 is found within the committed progenitor fraction of the undifferentiated spermatogonial pool. We identified that SOX3 is required for the transition from a GFRα1+ state to a NGN3+ committed progenitor state, and in the absence of SOX3 GFRα1+ cells accumulate and spermatogonia fail to differentiate, leading to empty testes with no mature sperm. We provide further evidence that Ngn3 is a direct target of SOX3 in both NPCs and the testes albeit thought different regulatory regions. We have generated two invaluable genome wide ChIP-seq datasets that will deepen our understanding of mechanisms by which SOX3 controls context-specific differentiation. Taken together, the data presented in this thesis expand our knowledge of the genomic regions bound by SOX3 and its role in neurogenesis and spermatogenesis.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202

Bulge formed cooling channels with a variable lead helix on a hollow body of revolution

A method of constructing a nozzle having cooling channels comprises a shell and a liner which are formed into a body of revolution having an axis of revolution. Helical welds are formed to hold the liner and shell to each other with a channel position being defined between each pair of helical welds. Pressurized fluid which may be a gas or a liquid, is introduced between the weld pairs to outwardly bulge the material of at least one of the liner and shell to define the channels

SOX3 promotes generation of committed spermatogonia in postnatal mouse testes

SOX3 is a transcription factor expressed within the developing and adult nervous system where it mostly functions to help maintain neural precursors. Sox3 is also expressed in other locations, notably within the spermatogonial stem/progenitor cell population in postnatal testis. Independent studies have shown that Sox3 null mice exhibit a spermatogenic block as young adults, the mechanism of which remains poorly understood. Using a panel of spermatogonial cell marker genes, we demonstrate that Sox3 is expressed within the committed progenitor fraction of the undifferentiated spermatogonial pool. Additionally, we use a Sox3 null mouse model to define a potential role for this factor in progenitor cell function. We demonstrate that Sox3 expression is required for transition of undifferentiated cells from a GFRα1+ self-renewing state to the NGN3 + transit-amplifying compartment. Critically, using chromatin immunoprecipitation, we demonstrate that SOX3 binds to a highly conserved region in the Ngn3 promoter region in vivo, indicating that Ngn3 is a direct target of SOX3. Together these studies indicate that SOX3 functions as a pro-commitment factor in spermatogonial stem/progenitor cells.</p

Mechanistic insight into the pathology of polyalanine expansion disorders revealed by a mouse model for x linked hypopituitarism

Extent: 9 p.Polyalanine expansions in transcription factors have been associated with eight distinct congenital human diseases. It is thought that in each case the polyalanine expansion causes misfolding of the protein that abrogates protein function. Misfolded proteins form aggregates when expressed in vitro; however, it is less clear whether aggregation is of relevance to these diseases in vivo. To investigate this issue, we used targeted mutagenesis of embryonic stem (ES) cells to generate mice with a polyalanine expansion mutation in Sox3 (Sox3-26ala) that is associated with X-linked Hypopituitarism (XH) in humans. By investigating both ES cells and chimeric mice, we show that endogenous polyalanine expanded SOX3 does not form protein aggregates in vivo but rather is present at dramatically reduced levels within the nucleus of mutant cells. Importantly, the residual mutant protein of chimeric embryos is able to rescue a block in gastrulation but is not sufficient for normal development of the hypothalamus, a region that is functionally compromised in Sox3 null embryos and individuals with XH. Together, these data provide the first definitive example of a disease-relevant PA mutant protein that is both nuclear and functional, thereby manifesting as a partial loss-of-function allele.James Hughes Sandra Piltz, Nicholas Rogers, Dale McAninch, Lynn Rowley and Paul Thoma

SOX3 binding sites at enhancer regions.

<p>The overlap of SOX3 peaks with P300 binding sites identified from 11.5 dpc mouse; forebrain (A), midbrain (B), and limb (C), showing a high degree of overlap in the developing brain and not within the limb. Average phastCons score of the common peaks between SOX3 and P300 forebrain (D) and midbrain (E) binding sites.</p

Differentially expressed genes from <i>Sox3</i> null NPCs with nearby SOX3 ChIP binding sites.

<p>Differentially expressed genes from <i>Sox3</i> null NPCs with nearby SOX3 ChIP binding sites.</p

Evolutionary conservation of SOX3 bound regions.

<p>(A) The average phastCons score of each SOX3 bound peak showing 20% of peaks are highly conserved across 30 placental mammals. (B) A highly conserved peak within the second intron of <i>Dbx1</i> giving the highest conservation score of 0.97 (compared to all peaks). (C) A peak within the second intron of the neural gene <i>Nestin</i>, with an average phastCons score of 0.51.</p

Overview of SOX3 ChIP-Seq data from mouse neural progenitor cells.

<p>(A) Genomic classification of SOX3 binding sites relative to nearest transcriptional start sites. (B) Validation of SOX3 ChIP by qPCR. Fold change is relative to both input DNA and IgG control values for the same genomic location. Error bars correspond to standard deviation of three independent sample replicates, P-values indicated as <0.05 (ns), >0.05 (*) and >0.001 (**). (C) Highest enriched DNA motifs identified by MEME-ChIP as i. a SOX motif and ii. a SOX-POU motif. (D) Enriched Gene Ontology terms associated with subsets of SOX3 ChIP peaks.</p

Identification of common SOXB1 regulatory regions.

<p>(A) Overlap of this SOX3 ChIP-seq dataset with previously published SOX2 and SOX3 datasets from similar NPCs (2). (B) SOX motif identified with MEME-ChIP present in (i) all 648 SOXB1 common peaks and (ii) 295 peaks. (C) Average phastCons scores for the 648 SOXB1 peaks, showing more than 80% of peaks are either moderately (32%) or highly (42%) conserved. (D) Genomic localisation of the common SOXB1 peaks. Enriched Gene Ontology terms for (E) all 648 SOXB1 peaks and (F) both intronic and intergenic peaks.</p

Mechanistic Insight into Long Noncoding RNAs and the Placenta

Long non-coding RNAs (lncRNAs) are classified as RNAs greater than 200 nucleotides in length that do not produce a protein product. lncRNAs are expressed with cellular and temporal specificity and have been shown to play a role in many cellular events, including the regulation of gene expression, post-transcriptional modifications and epigenetic modifications. Since lncRNAs were first discovered, there has been increasing evidence that they play important roles in the development and function of most organs, including the placenta. The placenta is an essential transient organ that facilitates communication and nutrient exchange between the mother and foetus. The placenta is of foetal origin and begins to form shortly after the embryo implants into the uterine wall. The placenta relies heavily on the successful differentiation and function of trophoblast cells, including invasion as well as the formation of the maternal/foetal interface. Here, we review the current literature surrounding the involvement of lncRNAs in the development and function of trophoblasts and the human placenta