Allyson Ross

Influence of PAX6 Gene Dosage on Development: Overexpression Causes Severe Eye Abnormalities

Aniridia in man and Small eye in mice are semidominant developmental disorders caused by mutations within the paired box gene PAX6. Whereas heterozygotes suffer from iris hypoplasia, homozygous mice lack eyes and nasal cavities and exhibit brain abnormalities. To investigate the role of gene dosage in more detail, we have generated yeast artificial chromosome transgenic mice carrying the human PAX6 locus. When crossed onto the Small eye background, the transgene rescues the mutant phenotype. Strikingly, mice carrying multiple copies on a wild-type background show specific developmental abnormalities of the eye, but not of other tissues expressing the gene. Thus, at least five different eye phenotypes are associated with changes in PAX6 expression. We provide evidence that not only reduced, but also increased levels of transcriptional regulators can cause developmental defects.

EMAP and EMAGE: a framework for understanding spatially organized data.

The Edinburgh Mouse Atlas Project (EMAP) is a time-series of mouse-embryo volumetric models. The models provide a context-free spatial framework onto which structural interpretations and experimental data can be mapped. This enables collation, comparison, and query of complex spatial patterns with respect to each other and with respect to known or hypothesized structure. The atlas also includes a time-dependent anatomical ontology and mapping between the ontology and the spatial models in the form of delineated anatomical regions or tissues. The models provide a natural, graphical context for browsing and visualizing complex data.The Edinburgh Mouse Atlas Gene-Expression Database (EMAGE) is one of the first applications of the EMAP framework and provides a spatially mapped gene-expression database with associated tools for data mapping, submission, and query. In this article, we describe the underlying principles of the Atlas and the gene-expression database, and provide a practical introduction to the use of the EMAP and EMAGE tools, including use of new techniques for whole body gene-expression data capture and mapping.

Genetic heterogeneity in Cornelia de Lange syndrome (CdLS) and CdLS-like phenotypes with observed and predicted levels of mosaicism

Background Cornelia de Lange syndrome (CdLS) is a multisystem disorder with distinctive facial appearance, intellectual disability and growth failure as prominent features. Most individuals with typical CdLS have de novo heterozygous loss-of-function mutations in NIPBL with mosaic individuals representing a significant proportion. Mutations in other cohesin components, SMC1A, SMC3, HDAC8 and RAD21 cause less typical CdLS. Methods We screened 163 affected individuals for coding region mutations in the known genes, 90 for genomic rearrangements, 19 for deep intronic variants in NIPBL and 5 had whole-exome sequencing. Results Pathogenic mutations [including mosaic changes] were identified in: NIPBL 46 [3] (28.2%); SMC1A 5 [1] (3.1%); SMC3 5 [1] (3.1%); HDAC8 6 [0] (3.6%) and RAD21 1 [0] (0.6%). One individual had a de novo 1.3 Mb deletion of 1p36.3. Another had a 520 kb duplication of 12q13.13 encompassing ESPL1, encoding separase, an enzyme that cleaves the cohesin ring. Three de novo mutations were identified in ANKRD11 demonstrating a phenotypic overlap with KBG syndrome. To estimate the number of undetected mosaic cases we used recursive partitioning to identify discriminating features in the NIPBL-positive subgroup. Filtering of the mutation-negative group on these features classified at least 18% as ‘NIPBL-like’. A computer composition of the average face of this NIPBL-like subgroup was also more typical in appearance than that of all others in the mutation-negative group supporting the existence of undetected mosaic cases. Conclusions Future diagnostic testing in ‘mutation-negative’ CdLS thus merits deeper sequencing of multiple DNA samples derived from different tissues.

The morphogenesis of the ciliary body of the avian eye: I. Lateral cell detachment facilitates epithelial folding

Abstract The ciliary body of the avian eye comprises about 90 radial folds in the anterior retina that form over a period of about 2 days (stages 29–32). They extend from close to the lens circumference outwards for about 1 mm and within each there is a small blood vessel. The tip of the retina itself does not fold, but adheres to the anterior surface of the lens and eventually forms the iris. TEM studies show that the area of the retina that folds is a domain displaying a most unusual phenomenon: its inner cells, the anterior region of the monolayer neural retinal epithelium (NRE) detach themselves from one another over most of their lateral surfaces some 2 days before folding occurs and reattach about 3 days afterwards. Gap junctions between the cells of the pigmented retinal epithelium (PRE) and the neutral retinal epithelium maintain the integrity of the bilayer while junctional complexes between the basal regions of the cells in each layer reinforce the structure. The well-defined basement membrane on the vitreous-facing surface of the NRE also, presumably, helps maintain its integrity. A simple physical analysis shows that lateral detachment within the NRE facilitates tissue bending in that, for a given input of energy, it permits the amount of folding to increase by a factor of 20 or more. The retinal tip itself does not fold, presumably because the NRE cells there do not detach; indeed, the region seems particularly rigid. The strength of this tip region may be enhanced by the fiber adhesions (Zonules of Zinn) that it forms with the lens just before folding occurs. Study of the developing blood system of the eye shows that small radial vessels from the ophthalmic cerebral artery surrounding the eye are regularly spaced around the retinal tip some 6 hr before the first folds appear. These results suggest that, before morphogenesis occurs, several earlier morphological events ensure that the tissue has been made ready to fold. The observations do not, however, give insight into the dynamic forces that are responsible for folding; these are investigated in the following paper.

The wt1-heterozygous mouse; a model to study the development of glomerular sclerosis

In the present study, it is shown that mice heterozygous for wt1 develop glomerular sclerosis and the nature and time course of events leading to the glomerular scarring are determined. Wt1‐heterozygous (wt1het) mice and their wild‐type littermates were closely monitored from birth and plasma levels of urea, creatinine, and albumin were compared with histological data and clinical features. One of the first indications of nephropathy in the wt1het mouse was the development of proteinuria, accompanied by progressive elevation of the plasma levels of urea and creatinine. Subsequently, the mice developed albuminuria, which correlated with thickening of the glomerular basement membrane and fusion of the podocyte foot processes. Glomerulosclerosis was a relatively late event, accompanied by severe albuminuria and loss of WT1, nephrin, CD2AP, and α‐actinin‐4. Copyright

Genetic background influences embryonic lethality and the occurrence of neural tube defects in Men1 null mice: relevance to genetic modifiers

Germline mutations of the multiple endocrine neoplasia type 1 (MEN1) gene cause parathyroid, pancreatic and pituitary tumours in man. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP) and the same MEN1 mutations, in different families, can cause either FIHP or MEN1. This suggests a role for genetic background and modifier genes in altering the expression of a mutation. We investigated the effects of genetic background on the phenotype of embryonic lethality that occurs in a mouse model for MEN1. Men1(+/-) mice were backcrossed to generate C57BL/6 and 129S6/SvEv incipient congenic strains, and used to obtain homozygous Men1(-/-) mice. No viable Men1(-/-) mice were obtained. The analysis of 411 live embryos obtained at 9.5-16.5 days post-coitum (dpc) revealed that significant deviations from the expected Mendelian 1:2:1 genotype ratio were first observed at 12.5 and 14.5 dpc in the 129S6/SvEv and C57BL/6 strains respectively (P<0.05). Moreover, live Men1(-/-) embryos were absent by 13.5 and 15.5 dpc in the 129S6/SvEv and C57BL/6 strains respectively thereby indicating an earlier lethality by 2 days in the 129S6/SvEv strain (P<0.01). Men1(-/-) embryos had macroscopic haemorrhages, and histology and optical projection tomography revealed them to have internal haemorrhages, myocardial hypotrophy, pericardial effusion, hepatic abnormalities and neural tube defects. The neural tube defects occurred exclusively in 129S6/SvEv embryos (21 vs 0%, P<0.01). Thus, our findings demonstrate the importance of genetic background in influencing the phenotypes of embryonic lethality and neural tube defects in Men1(-/-) mice, and implicate a role for genetic modifiers.

The morphogenesis of the ciliary body of the avian eye: II. Differential enlargement causes an epithelium to form radial folds

Abstract The morphogenetic mechanism responsible for the radial folding of the anterior retina of the chick eye as it forms the ciliary body has been investigated in two ways. First, eye growth and cell division have been assayed to find out the origins of the extra fold material, and second, possible mechanisms have been tested, and in some cases excluded, by artificially increasing the size of the embryonic eye in vitro under a range of restrictive conditions. Growth studies show that, while on average the eyeball increases linearly in area by a factor of about 14 over the period 4–8 days or stages 24–33, there is a slowing down in growth at stage 28 which is followed by a rapid catch up as the surface area increases by about two-thirds in the 12 hr between stages 29 and 30, just as the ciliary body forms. Thymidine incorporation studies show that cell division is roughly uniform over the eye at this stage. The sudden increase in the overall size of the eye is not, however, matched by growth in the region of the pupil at the retinal tip; this ring of tissue grows slowly and its diameter remains virtually constant over the period of ciliary body morphogenesis. These observations suggest a simple morphogenetic mechanism. Tissue near the retinal tip, unlike such tissue in the rest of the eye, is unable to swell uniformly under intraocular pressure as it is constrained by the rigid pupillary ring; the resulting complex tensions cause radial folding. This process is facilitated by lateral cell detachment in the neural retinal epithelium (NRE) and nucleated by existing radial capillaries superficial to the retina. It is a prediction of the mechanism that any differential growth of the eyeball should cause stage 29 eyes to fold. Such growth has been induced in vitro by immersing early stage 29 eyes in 50% ethanol and water, a solution which causes an 8% or so increase in eye diameter in a few minutes, equivalent to about several hours growth in vivo. Pupillary expansion, however, lags some 15 min behind that of the rest of the eye. In most cases, immersed eyes generate large numbers of radial folds over about half their circumference in about 10 min after immersion, the degree of folding expected for similar growth in vivo. This result argues against morphogenetic mechanisms based on localised growth or on other slow developmental events. Such radial fold formation in vitro after treatment of eyes in vivo with colchicine and cytochalasin B has also been observed, results which argue against any morphogenetic mechanism based on microtubules or microfilaments. Some folds also form with ethanol swelling after the cornea and peripheral mesenchyme have been removed from the eye, a result which excludes any major directive role for this part of the tissue. The observations therefore support the simple differential growth stimulus for ciliary body morphogenesis.

eMouseAtlas, EMAGE, and the spatial dimension of the transcriptome

AbstracteMouseAtlas (www.emouseatlas.org) is a comprehensive online resource to visualise mouse development and investigate gene expression in the mouse embryo. We have recently deployed a completely redesigned Mouse Anatomy Atlas website (www.emouseatlas.org/emap/ema) that allows users to view 3D embryo reconstructions, delineated anatomy, and high-resolution histological sections. A new feature of the website is the IIP3D web tool that allows a user to view arbitrary sections of 3D embryo reconstructions using a web browser. This feature provides interactive access to very high-volume 3D images via a tiled pan-and-zoom style interface and circumvents the need to download large image files for visualisation. eMouseAtlas additionally includes EMAGE (Edinburgh Mouse Atlas of Gene Expression) (www.emouseatlas.org/emage), a freely available, curated online database of in situ gene expression patterns, where gene expression domains extracted from raw data images are spatially mapped into atlas embryo models. In this way, EMAGE introduces a spatial dimension to transcriptome data and allows exploration of the spatial similarity between gene expression patterns. New features of the EMAGE interface allow complex queries to be built, and users can view and compare multiple gene expression patterns. EMAGE now includes mapping of 3D gene expression domains captured using the imaging technique optical projection tomography. 3D mapping uses WlzWarp, an open-source software tool developed by eMouseAtlas.

A Restricted Repertoire of De Novo Mutations in ITPR1 Cause Gillespie Syndrome with Evidence for Dominant-Negative Effect

Gillespie syndrome (GS) is characterized by bilateral iris hypoplasia, congenital hypotonia, non-progressive ataxia, and progressive cerebellar atrophy. Trio-based exome sequencing identified de novo mutations in ITPR1 in three unrelated individuals with GS recruited to the Deciphering Developmental Disorders study. Whole-exome or targeted sequence analysis identified plausible disease-causing ITPR1 mutations in 10/10 additional GS-affected individuals. These ultra-rare protein-altering variants affected only three residues in ITPR1: Glu2094 missense (one de novo, one co-segregating), Gly2539 missense (five de novo, one inheritance uncertain), and Lys2596 in-frame deletion (four de novo). No clinical or radiological differences were evident between individuals with different mutations. ITPR1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. The homo-tetrameric structure has been solved by cryoelectron microscopy. Using estimations of the degree of structural change induced by known recessive- and dominant-negative mutations in other disease-associated multimeric channels, we developed a generalizable computational approach to indicate the likely mutational mechanism. This analysis supports a dominant-negative mechanism for GS variants in ITPR1. In GS-derived lymphoblastoid cell lines (LCLs), the proportion of ITPR1-positive cells using immunofluorescence was significantly higher in mutant than control LCLs, consistent with an abnormality of nuclear calcium signaling feedback control. Super-resolution imaging supports the existence of an ITPR1-lined nucleoplasmic reticulum. Mice with Itpr1 heterozygous null mutations showed no major iris defects. Purkinje cells of the cerebellum appear to be the most sensitive to impaired ITPR1 function in humans. Iris hypoplasia is likely to result from either complete loss of ITPR1 activity or structure-specific disruption of multimeric interactions.

Partial retinal dysplasia and subsequent degeneration in a mutant strain of domestic fowl (rdd).

An inherited recessive form of retinopathy has been discovered in the domestic fowl (rdd) which is characterized by progressive deterioration of the retina, culminating in blindness by sexual maturity. Morphologically, the condition is recognizable by abnormalities in both the retinal pigment epithelium and the neural retina. Gaps in the pigment epithelium which are first detected macroscopically at nine days of incubation become larger and more numerous until the time of hatching, then disappear during the subsequent week. Undulations in the outer nuclear, outer plexiform, and inner nuclear layers are obvious by 11 days of incubation. There is a marked reduction of photoreceptors at 18 days of incubation as compared to normal controls. After hatching, the thickness of the retina decreases with age, primarily due to cell loss from the photoreceptor region and inner nuclear layer. Detachment of the atrophic retinas generally occurs in adults, and is followed in some adults by granulation and ossification of the vitreous. Problems concerning the site of the lesion are discussed.