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

Congenital Hydrocephalus and Abnormal Subcommissural Organ Development in Sox3 Transgenic Mice

Congenital hydrocephalus (CH) is a life-threatening medical condition in which excessive accumulation of CSF leads to ventricular expansion and increased intracranial pressure. Stenosis (blockage) of the Sylvian aqueduct (Aq; the narrow passageway that connects the third and fourth ventricles) is a common form of CH in humans, although the genetic basis of this condition is unknown. Mouse models of CH indicate that Aq stenosis is associated with abnormal development of the subcommmissural organ (SCO) a small secretory organ located at the dorsal midline of the caudal diencephalon. Glycoproteins secreted by the SCO generate Reissner's fibre (RF), a thread-like structure that descends into the Aq and is thought to maintain its patency. However, despite the importance of SCO function in CSF homeostasis, the genetic program that controls SCO development is poorly understood. Here, we show that the X-linked transcription factor SOX3 is expressed in the murine SCO throughout its development and in the mature organ. Importantly, overexpression of Sox3 in the dorsal diencephalic midline of transgenic mice induces CH via a dose-dependent mechanism. Histological, gene expression and cellular proliferation studies indicate that Sox3 overexpression disrupts the development of the SCO primordium through inhibition of diencephalic roof plate identity without inducing programmed cell death. This study provides further evidence that SCO function is essential for the prevention of hydrocephalus and indicates that overexpression of Sox3 in the dorsal midline alters progenitor cell differentiation in a dose-dependent manner

Deep sequencing analysis of the developing mouse brain reveals a novel microRNA

Extent: 15p.Background: MicroRNAs (miRNAs) are small non-coding RNAs that can exert multilevel inhibition/repression at a post-transcriptional or protein synthesis level during disease or development. Characterisation of miRNAs in adult mammalian brains by deep sequencing has been reported previously. However, to date, no small RNA profiling of the developing brain has been undertaken using this method. We have performed deep sequencing and small RNA analysis of a developing (E15.5) mouse brain. Results: We identified the expression of 294 known miRNAs in the E15.5 developing mouse brain, which were mostly represented by let-7 family and other brain-specific miRNAs such as miR-9 and miR-124. We also discovered 4 putative 22-23 nt miRNAs: mm_br_e15_1181, mm_br_e15_279920, mm_br_e15_96719 and mm_br_e15_294354 each with a 70-76 nt predicted pre-miRNA. We validated the 4 putative miRNAs and further characterised one of them, mm_br_e15_1181, throughout embryogenesis. Mm_br_e15_1181 biogenesis was Dicer1-dependent and was expressed in E3.5 blastocysts and E7 whole embryos. Embryo-wide expression patterns were observed at E9.5 and E11.5 followed by a near complete loss of expression by E13.5, with expression restricted to a specialised layer of cells within the developing and early postnatal brain. Mm_br_e15_1181 was upregulated during neurodifferentiation of P19 teratocarcinoma cells. This novel miRNA has been identified as miR-3099. Conclusions: We have generated and analysed the first deep sequencing dataset of small RNA sequences of the developing mouse brain. The analysis revealed a novel miRNA, miR-3099, with potential regulatory effects on early embryogenesis, and involvement in neuronal cell differentiation/function in the brain during late embryonic and early neonatal development.King-Hwa Ling, Peter J Brautigan, Christopher N Hahn, Tasman Daish, John R Rayner, Pike-See Cheah, Joy M Raison, Sandra Piltz Jeffrey R Mann, Deidre M Mattiske, Paul Q Thomas, David L Adelson and Hamish S Scot

<i>Sox3</i>-26ala cells cause pituitary defects indistinguishable from <i>Sox3</i>-null cells.

<p>WT, <i>Sox3</i>+/− or <i>Sox3</i>-26ala<->WT chimeras were cut sagittally at 11.5 dpc (A) or 13.5 dpc (B) and immunostained for SOX3 and NEO expression. Percentage chimerism for each embryo in (A) and (B) was determined by qPCR as outlined in the methods. ISH for <i>Neo</i> on adjacent sections at 11.5 dpc confirmed the identification of mutant cells within the ventral diencephalon (A). Examples at 11.5 dpc show the infundibulum (I) appears unaffected in a 5% chimera, shallow in a 20% chimera and absent in a 75% chimera that also displayed a Rathke's Pouch (*) that had failed to detach from the oral ectoderm. At 13.5 dpc, heterozygous and high percentage (65%) chimeric embryos displayed a distorted infundibulum (I) with a lobular edge (arrow heads) and a branched Rathke's Pouch (*). Low percentage chimeras (20%) look similar to WT (0%). C) Phase micrographs of 13.5 dpc coronal sections through the developing pituitary show a broadening at the base of the third ventricle in chimeras (arrows). Chimerism for embryos shown in (C) was determined based on immunoreactivity for NEO in adjacent sections (data not shown).</p

Generation of <i>Sox3</i>-26ala ES cells.

<p>Scale representation of the <i>Sox3</i> locus, targeting vector and recombinant alleles (A). Probing of <i>BglII</i> digested DNA from ES cell clones with the 5′ probe yielded an 8.8 kb fragment from the WT locus and a 5.9 kb fragment when the <i>Neo</i> cassette was recombined into the <i>Sox3</i> locus (<i>Sox3</i>-26ala or <i>Neo</i>). B) Representative Southern blot of 3 clones including a targeted clone (<i>Sox3</i>-26ala-3) is shown. C) PCR using primers spanning the alanine expansion (red arrows in A) was used to distinguish whether targeted clones carried the expansion and gave a 219 bp product instead of 186 bp as seen in WT.</p

Failure of <i>Sox3</i>-26ala targeted ES clones to transmit through the germline.

<p>Failure of <i>Sox3</i>-26ala targeted ES clones to transmit through the germline.</p

Residual nuclear SOX3-26ala protein rescues a gastrulation defect of <i>Sox3</i>-null <-> WT chimeric embryos.

<p><i>Sox3</i>-26ala <-> WT chimeras are normal at 7.5 dpc (gastrulation) unlike <i>Sox3</i>-null <-> WT chimeras. A total of 15 <i>Sox3</i>-flox<-> WT ES chimeras, 31 <i>Sox3</i>-null<-> WT chimeras and 21 <i>Sox3</i>-26ala<-> WT chimeras were blind scored by two independent operators as morphologically normal or abnormal. The average score for each embryo was used to plot the percentage of abnormal embryos for each condition and chi squared analysis was performed with <i>Sox3</i>-flox<-> WT embryos used to set expected outcomes. Significantly more <i>Sox3</i>-null<-> WT chimeras were abnormal (p = 0.0001) while <i>Sox3</i>-26ala<-> WT chimeras did not deviate from expected (p = 0.95). An example of normal morphology is shown for <i>Sox3-</i>flox<-> WT and <i>Sox3</i>-26ala<-> WT chimeras and an abnormal <i>Sox3</i>-null<-> WT chimera is also shown that exhibits distortion of the ectodermal layer and apparent expansion of cells at the primitive streak and the adjacent extra-embryonic region.</p

SOX3-26ala from mouse and human retains transactivation activity.

<p>A) COS-7 cells were transfected with pcDNA3.1 expression vector containing either mouse <i>Sox3</i>, human <i>SOX3</i>, mouse <i>Sox3</i>-26ala, human <i>SOX3</i>-26ala or an empty vector control. Values represent mean normalised luciferase values plus standard deviation of four independent experiments measured 48 hours after transfection. Student's T-tests (two tailed, unequal variance) of SOX3-26ala from human or mouse compared to empty vector control show a statistically significant increase in luciferase activity. B) Nuclear protein lysates prepared from duplicate plates 48 hours after transfection show that less SOX3 is detected in the nucleus of cells expressing both mouse and human SOX3-26ala. pcDNA3.1-EGFP transfected cells were used as a control and prepared for both nuclear protein and whole cell extracts (WCE). Blotting for Histone H3, indicates equal loading and blotting for α-Tubulin shows an absence of cytoplasmic contamination in nuclear preparations. Transfection efficiency was determined by co-transfecting EGFP and counting positive cells prior to harvesting and found to be equal for all plasmids.</p