Functional conservation of Pax6 regulatory elements in humans and mice demonstrated with a novel transgenic reporter mouse

<p>Abstract</p> <p>Background</p> <p>The Pax6 transcription factor is expressed during development in the eyes and in specific CNS regions, where it is essential for normal cell proliferation and differentiation. Mice lacking one or both copies of the <it>Pax6 </it>gene model closely humans with loss-of-function mutations in the <it>PAX6 </it>locus. The sequence of the Pax6/PAX6 protein is identical in mice and humans and previous studies have shown <it>structural </it>conservation of the gene's regulatory regions.</p> <p>Results</p> <p>We generated a transgenic mouse expressing green fluorescent protein (GFP) and neomycin resistance under the control of the entire complement of human <it>PAX6 </it>regulatory elements using a modified yeast artificial chromosome (YAC). Expression of GFP was studied in embryos from 9.5 days on and was confined to cells known to express Pax6. GFP expression was sufficiently strong that expressing cells could be distinguished from non-expressing cells using flow cytometry.</p> <p>Conclusion</p> <p>This work demonstrates the <it>functional </it>conservation of the regulatory elements controlling <it>Pax6/PAX6 </it>expression in mice and humans. The transgene provides an excellent tool for studying the functions of different <it>Pax6/PAX6 </it>regulatory elements in controlling Pax6 expression in animals that are otherwise normal. It will allow the analysis and isolation of cells in which <it>Pax6 </it>is activated, irrespective of the status of the endogenous locus.</p

Controlled overexpression of Pax6 in vivo negatively autoregulates the Pax6 locus, causing cell-autonomous defects of late cortical progenitor proliferation with little effect on cortical arealization

Levels of expression of the transcription factor Pax6 vary throughout corticogenesis in a rostro-lateral(high) to caudo-medial(low) gradient across the cortical proliferative zone. Previous loss-of-function studies have indicated that Pax6 is required for normal cortical progenitor proliferation, neuronal differentiation, cortical lamination and cortical arealization, but whether and how its level of expression affects its function is unclear. We studied the developing cortex of PAX77 YAC transgenic mice carrying several copies of the human PAX6 locus with its full complement of regulatory regions. We found that PAX77 embryos express Pax6 in a normal spatial pattern, with levels up to three times higher than wild type. By crossing PAX77 mice with a new YAC transgenic line that reports Pax6 expression (DTy54), we showed that increased expression is limited by negative autoregulation. Increased expression reduces proliferation of late cortical progenitors specifically, and analysis of PAX77↔wild-type chimeras indicates that the defect is cell autonomous. We analyzed cortical arealization in PAX77 mice and found that, whereas the loss of Pax6 shifts caudal cortical areas rostrally, Pax6 overexpression at levels predicted to shift rostral areas caudally has very little effect. These findings indicate that Pax6 levels are stabilized by autoregulation, that the proliferation of cortical progenitors is sensitive to altered Pax6 levels and that cortical arealization is not

Childhood socioeconomic position and objectively measured physical capability levels in adulthood: a systematic review and meta-analysis

&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; Grip strength, walking speed, chair rising and standing balance time are objective measures of physical capability that characterise current health and predict survival in older populations. Socioeconomic position (SEP) in childhood may influence the peak level of physical capability achieved in early adulthood, thereby affecting levels in later adulthood. We have undertaken a systematic review with meta-analyses to test the hypothesis that adverse childhood SEP is associated with lower levels of objectively measured physical capability in adulthood.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methods and Findings:&lt;/b&gt; Relevant studies published by May 2010 were identified through literature searches using EMBASE and MEDLINE. Unpublished results were obtained from study investigators. Results were provided by all study investigators in a standard format and pooled using random-effects meta-analyses. 19 studies were included in the review. Total sample sizes in meta-analyses ranged from N = 17,215 for chair rise time to N = 1,061,855 for grip strength. Although heterogeneity was detected, there was consistent evidence in age adjusted models that lower childhood SEP was associated with modest reductions in physical capability levels in adulthood: comparing the lowest with the highest childhood SEP there was a reduction in grip strength of 0.13 standard deviations (95% CI: 0.06, 0.21), a reduction in mean walking speed of 0.07 m/s (0.05, 0.10), an increase in mean chair rise time of 6% (4%, 8%) and an odds ratio of an inability to balance for 5s of 1.26 (1.02, 1.55). Adjustment for the potential mediating factors, adult SEP and body size attenuated associations greatly. However, despite this attenuation, for walking speed and chair rise time, there was still evidence of moderate associations.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Policies targeting socioeconomic inequalities in childhood may have additional benefits in promoting the maintenance of independence in later life.&lt;/p&gt

Diabetes Is Associated with Cerebrovascular but not Alzheimer\u27s Disease Neuropathology

INTRODUCTION: The relationship of diabetes to specific neuropathologic causes of dementia is incompletely understood. METHODS: We used logistic regression to evaluate the association between diabetes and infarcts, Braak neurofibrillary tangle stage, and neuritic plaque score in 2365 autopsied persons. In a subset of \u3e1300 persons with available cognitive data, we examined the association between diabetes and cognition using Poisson regression. RESULTS: Diabetes increased odds of brain infarcts (odds ratio [OR] = 1.57, P \u3c .0001), specifically lacunes (OR = 1.71, P \u3c .0001), but not Alzheimer\u27s disease neuropathology. Diabetes plus infarcts was associated with lower cognitive scores at end of life than infarcts or diabetes alone, and diabetes plus high level of Alzheimer\u27s neuropathologic changes was associated with lower mini-mental state examination scores than the pathology alone. DISCUSSION: This study supports the conclusions that diabetes increases the risk of cerebrovascular but not Alzheimer\u27s disease pathology, and at least some of diabetes\u27 relationship to cognitive impairment may be modified by neuropathology

A review of survival analysis methods used in NICE technology appraisals of cancer treatments: consistency, limitations and areas for improvement

Objectives: In June 2011, the National Institute for Health and Care Excellence (NICE) Decision Support Unit published a Technical Support Document (TSD) providing recommendations on survival analysis for NICE technology appraisals (TAs). Survival analysis outputs are influential inputs into economic models estimating the cost-effectiveness of new cancer treatments. Hence, it is important that systematic and justifiable model selection approaches are used. This study investigates the extent to which the TSD recommendations have been followed since its publication. Methods: We reviewed NICE cancer TAs completed between July 2011 and July 2017. Information on survival analyses undertaken and associated critiques for overall survival (OS) and progressionfree survival were extracted from the company submissions, Evidence Review Group (ERG) reports and final appraisal determination documents. Results: Information was extracted from 58 TAs. Only four (7%) followed all TSD recommendations for OS outcomes. The vast majority (91%) compared a range of common parametric models and assessed their fit to the data (86%). Only a minority of TAs included an assessment of the shape of the hazard function (38%) or proportional hazards assumption (40%). Validation of the extrapolated portion of the survival function using external data was attempted in a minority of TAs (40%). Extrapolated survival functions were frequently criticised by ERGs (71%). Conclusions: Survival analysis within NICE TAs remains sub-optimal, despite publication of the TSD. Model selection is not undertaken in a systematic way resulting in inconsistencies between TAs. More attention needs to be given to assessing hazard functions and validation of extrapolated survival functions. Novel methods not described in the TSD have been used, particularly in the context of immuno-oncology, suggesting that an updated TSD may be of value

Long-range downstream enhancers are essential for Pax6 expression

AbstractPax6 is a developmental control gene with an essential role in development of the eye, brain and pancreas. Pax6, as many other developmental regulators, depends on a substantial number of cis-regulatory elements in addition to its promoters for correct spatiotemporal and quantitative expression. Here we report on our analysis of a set of mice transgenic for a modified yeast artificial chromosome carrying the human PAX6 locus. In this 420 kb YAC a tauGFP-IRES-Neomycin reporter cassette has been inserted into the PAX6 translational start site in exon 4. The YAC has been further engineered to insert LoxP sites flanking a 35 kb long, distant downstream regulatory region (DRR) containing previously described DNaseI hypersensitive sites, to allow direct comparison between the presence or absence of this region in the same genomic context. Five independent transgenic lines were obtained that vary in the extent of downstream PAX6 locus that has integrated. Analysis of transgenic embryos carrying full-length and truncated versions of the YAC indicates the location and putative function of several novel tissue-specific enhancers. Absence of these distal regulatory elements abolishes expression in specific tissues despite the presence of more proximal enhancers with overlapping specificity, strongly suggesting interaction between these control elements. Using plasmid-based reporter transgenic analysis we provide detailed characterization of one of these enhancers in isolation. Furthermore, we show that overexpression of a short PAX6 isoform derived from an internal promoter in a multicopy YAC transgenic line results in a microphthalmia phenotype. Finally, direct comparison of a single-copy line with the floxed DRR before and after Cre-mediated deletion demonstrates unequivocally the essential role of these long-range control elements for PAX6 expression

DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of migratory defects in both pathways in Pax6(Sey/Sey) mice

Comparison of two related lines of tauGFP transgenic mice designed for lineage tracing

Abstract Background The tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3 Tg/Tg homozygotes are not viable and TgTP6.3 Tg/− hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3. Results Although TgTP6.4 Tg/Tg homozygotes died before weaning, TgTP6.4 Tg/− hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4 Tg/− hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex. Conclusions Mosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing

Regulatory role of Pax6 on cell division cycle associated 7 and cortical progenitor cell proliferation

Forebrain development is controlled by a set of transcription factors which are expressed in dynamic spatiotemporal patterns in the embryonic forebrain and are known to regulate complex gene networks. Pax6 is a transcription factor that regulates corticogenesis and mutations affecting Pax6 protein levels cause neurodevelopmental defects in the eyes and forebrain in both humans and mice. In previous studies, it was shown that the graded expression pattern of Pax6 protein, which is high rostro-laterally to low caudo-medially in the cerebral cortex, is critical for its control of cell cycle progression and proliferation of cortical progenitors. However the underlying mechanisms are still unclear. Based on a microarray analysis carried out in our laboratory, a number of cell cycle-related candidate genes that may be affected by Pax6 have been identified. One such gene, Cell division cycle associated 7 (Cdca7) is expressed in a counter-gradient against that of Pax6. In my current study, I found that Cdca7 mRNA expression in the telencephalon is upregulated in Pax6 null (Small eye) mutants and downregulated in mice that overexpress PAX6 (PAX77) across developing time points from E12.5 to E15.5. There are several potential Pax6 binding motifs located in the genomic locus upstream of Cdca7. However, by chromatin immunoprecipitation, it is showed that none of the predicted binding sites are physically bound by Pax6. Promoter luciferase assays using fragments combining five suspected binding motifs show that Pax6 is functionally critical. Cdca7 is also identified as a Myc and E2F1 direct target and is upregulated in some tumours but its biological role is not fully understood. Current work using in utero electroporation to overexpress Cdca7 around the lateral telencephalon, where Cdca7 expression levels are normally low, tested the effects on the proliferation and differentiation of cortical progenitor cells in this region. In E12.5 mice embryos, overexpression of Cdca7 protein causes fewer intermediate progenitor cells and post-mitotic neurons to be produced but these effects were not found in E14.5 embryos. This result implies that Cdca7 may affect cell fate decision during cortical development