Combined genome-wide linkage and targeted association analysis of head circumference in autism spectrum disorder families.

BACKGROUND: It has long been recognized that there is an association between enlarged head circumference (HC) and autism spectrum disorder (ASD), but the genetics of HC in ASD is not well understood. In order to investigate the genetic underpinning of HC in ASD, we undertook a genome-wide linkage study of HC followed by linkage signal targeted association among a sample of 67 extended pedigrees with ASD. METHODS: HC measurements on members of 67 multiplex ASD extended pedigrees were used as a quantitative trait in a genome-wide linkage analysis. The Illumina 6K SNP linkage panel was used, and analyses were carried out using the SOLAR implemented variance components model. Loci identified in this way formed the target for subsequent association analysis using the Illumina OmniExpress chip and imputed genotypes. A modification of the qTDT was used as implemented in SOLAR. RESULTS: We identified a linkage signal spanning 6p21.31 to 6p22.2 (maximum LOD = 3.4). Although targeted association did not find evidence of association with any SNP overall, in one family with the strongest evidence of linkage, there was evidence for association (rs17586672, p = 1.72E-07). CONCLUSIONS: Although this region does not overlap with ASD linkage signals in these same samples, it has been associated with other psychiatric risk, including ADHD, developmental dyslexia, schizophrenia, specific language impairment, and juvenile bipolar disorder. The genome-wide significant linkage signal represents the first reported observation of a potential quantitative trait locus for HC in ASD and may be relevant in the context of complex multivariate risk likely leading to ASD.

Psychiatric symptoms and disorders in phenylketonuria.

Psychological and psychiatric problems are well documented across the lifespan of individuals with early-treated phenylketonuria (PKU). Early-treated children and adolescents tend to display attentional problems, school problems, lower achievement motivation, decreased social competence, decreased autonomy, and low-self-esteem. As they enter adulthood, early-treated individuals may carry forward low self-esteem and lack of autonomy but also tend to develop depressed mood, generalized anxiety, phobias, decreased positive emotions, social maturity deficits, and social isolation. The correlation between level of metabolic control and severity of symptoms suggests a biological basis of psychiatric dysfunction. Additionally, psychosocial factors such as the burden of living with a chronic illness may contribute to psychological and psychiatric outcomes in PKU. The lack of a PKU-specific psychiatric phenotype combined with the observation that not everyone with PKU is affected highlights the complexity of the problem. More research on psychiatric and psychological outcomes in PKU is required. Of particular importance is the routine monitoring of emotional, behavioral, and psychosocial symptoms in individuals with this metabolic disorder. Longitudinal studies are required to evaluate the impact of new and emerging therapies on psychiatric and psychosocial functioning in PKU. Unidentified or untreated emotional and behavioral symptoms may have a significant, lifelong impact on the quality of life and social status of patients.

Quantitative analysis of epithelial morphogenesis in Drosophila oogenesis: New insights based on morphometric analysis and mechanical modeling.

The process of epithelial morphogenesis is ubiquitous in animal development, but much remains to be learned about the mechanisms that shape epithelial tissues. The follicle cell (FC) epithelium encapsulating the growing germline of Drosophila is an excellent system to study fundamental elements of epithelial development. During stages 8 to 10 of oogenesis, the FC epithelium transitions between simple geometries-cuboidal, columnar and squamous-and redistributes cell populations in processes described as posterior migration, squamous cell flattening and main body cell columnarization. Here we have carried out a quantitative morphometric analysis of these poorly understood events in order to establish the parameters of and delimit the potential processes that regulate the transitions. Our results compel a striking revision of accepted views of these phenomena, by showing that posterior migration does not involve FC movements, that there is no role for columnar cell apical constriction in FC morphogenesis, and that squamous cell flattening may be a compliant response to germline growth. We utilize mechanical modeling involving finite element computational technologies to demonstrate that time-varying viscoelastic properties and growth are sufficient to account for the bulk of the FC morphogenetic changes.

PDZ proteins and polarity: functions from the fly.

Proteins that contain PDZ domains have been implicated in the localization of interacting partners to specific regions of the cell membrane. Although PDZ proteins that bind to a number of important mammalian proteins have been isolated, the significance of these interactions is unclear. In recent years, the fruit fly has emerged as a rich system for genetic analysis of PDZ protein function. Here, I discuss what the Drosophila data tell us about the roles and mechanisms of PDZ protein activity, and how this informs our understanding of PDZ function in other organisms.

Cell polarity: squaring the circle.

Recent studies have found that Drosophila gene products required for zonula adherens formation in the ectoderm are also involved in the asymmetric cell division of the neuroblast. The results illustrate the reiterated use of groups of proteins to dictate cell polarity in epithelial and non-epithelial cells.

Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors.

Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. We provide evidence from Drosophila that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Scribble (Scrib) is a cell junction-localized protein required for polarization of embryonic and, as demonstrated here, imaginal disc and follicular epithelia. We show that the tumor suppressors lethal giant larvae (lgl) and discs-large (dlg) have identical effects on all three epithelia, and that scrib also acts as a tumor suppressor. Scrib and Dlg colocalize and overlap with Lgl in epithelia; activity of all three genes is required for cortical localization of Lgl and junctional localization of Scrib and Dlg. scrib, dlg, and lgl show strong genetic interactions. Our data indicate that the three tumor suppressors act together in a common pathway to regulate cell polarity and growth control.

Distinct hox protein sequences determine specificity in different tissues.

Hox genes encode evolutionarily conserved transcription factors that control the morphological diversification along the anteroposterior (A/P) body axis. Expressed in precise locations in the ectoderm, mesoderm, and endoderm, Hox proteins have distinct regulatory activities in different tissues. How Hox proteins achieve tissue-specific functions and why cells lying at equivalent A/P positions but in different germ layers have distinctive responses to the same Hox protein remains to be determined. Here, we examine this question by identifying parts of Hox proteins necessary for Hox function in different tissues. Available genetic markers allow the regulatory effects of two Hox proteins, Abdominal-A (AbdA) and Ultrabithorax (Ubx), to be distinguished in the Drosophila embryonic epidermis and visceral mesoderm (VM). Chimeric Ubx/AbdA proteins were tested in both tissues and used to identify protein sequences that endow AbdA with a different target gene specificity from Ubx. We found that distinct protein sequences define AbdA, as opposed to Ubx, function in the epidermis vs. the VM. These sequences lie mostly outside the homeodomain (HD), emphasizing the importance of non-HD residues for specific Hox activities. Hox tissue specificity is therefore achieved by sensing distinct Hox protein structures in different tissues.

Localization of apical epithelial determinants by the basolateral PDZ protein Scribble.

The generation of membrane domains with distinct protein constituents is a hallmark of cell polarization. In epithelia, segregation of membrane proteins into apical and basolateral compartments is critical for cell morphology, tissue physiology and cell signalling. Drosophila proteins that confer apical membrane identity have been found, but the mechanisms that restrict these determinants to the apical cell surface are unknown. Here we show that a laterally localized protein is required for the apical confinement of polarity determinants. Mutations in Drosophila scribble (scrib), which encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein, cause aberrant cell shapes and loss of the monolayer organization of embryonic epithelia. Scrib is localized to the epithelial septate junction, the analogue of the vertebrate tight junction, at the boundary of the apical and basolateral cell surfaces. Loss of scrib function results in the misdistribution of apical proteins and adherens junctions to the basolateral cell surface, but basolateral protein localization remains intact. These phenotypes can be accounted for by mislocalization of the apical determinant Crumbs. Our results show that the lateral domain of epithelia, particularly the septate junction, functions in restricting apical membrane identity and correctly placing adherens junctions.

Hedgehog and wingless induce metameric pattern in the Drosophila visceral mesoderm.

The Drosophila visceral mesoderm (VM) is a favorite system for studying the regulation of target genes by Hox proteins. The VM is formed by cells from only the anterior subdivision of each mesodermal parasegment (PS). We show here that the VM itself acquires modular anterior-posterior subdivisions similar to those found in the ectoderm. As VM progenitors merge to form a continuous band running anterior to posterior along the embryo, expression of connectin (con) in 11 metameric patches within the VM reveals VM subdivisions analagous to ectodermal compartments. The VM subdivisions form in response to ectodermal production of secreted signals encoded by the segment polarity genes hedgehog (hh) and wingless (wg) and are independent of Hox gene activity. A cascade of induction from ectoderm to mesoderm to endoderm thus subdivides the gut tissues along the A-P axis. Induction of VM subdivisions may converge with Hox-mediated information to refine spatial patterning in the VM. Con patches align with ectodermal engrailed stripes, so the VM subdivisions correspond to PS 2-12 boundaries in the VM. The PS boundaries demarcated by Con in the VM can be used to map expression domains of Hox genes and their targets with high resolution. The resultant map suggests a model for the origins of VM-specific Hox expression in which Hox domains clonally inherited from blastoderm ancestors are modified by diffusible signals acting on VM-specific enhancers.

Wnt and TGFbeta signals subdivide the AbdA Hox domain during Drosophila mesoderm patterning.

Hox genes have large expression domains yet control the formation of fine pattern elements at specific locations. We have examined the mechanism underlying subdivision of the abdominal-A (abdA) Hox domain in the visceral mesoderm. AbdA directs formation of an embryonic midgut constriction at a precise location within the broad and uniform abdA expression domain. The constriction divides the abdA domain of the midgut into two chambers, the anterior one producing the Pointed (Pnt) ETS transcription factors and the posterior one the Odd-paired (Opa) zinc finger protein. Transcription of both pnt and opa is activated by abdA but the adjacent non-overlapping patterns are not due to mutual opa-pnt regulation. Near the anterior limit of the abdA domain, two signals, Dpp (a TGFbeta) and Wg (a Wnt), are produced, in adjacent non-overlapping patterns, under Hox control in mesoderm cells. The two signals are known to regulate local mesodermal cell fates and to signal to the endoderm. We find that, in addition, they precisely subdivide the abdA domain: Wg acts upon anterior abdA domain cells to activate pnt transcription, while Dpp is essential in the same region to prevent abdA from activating opa transcription. pnt activation is required to determine the appropriate numbers of mesodermal cells in the third midgut chamber.

Genomic regions required for morphogenesis of the Drosophila embryonic midgut.

The Drosophila midgut is an excellent system for studying the cell migration, cell-cell communication, and morphogenetic events that occur in organ formation. Genes representative of regulatory gene families common to all animals, including homeotic, TGF beta, and Wnt genes, play roles in midgut development. To find additional regulators of midgut morphogenesis, we screened a set of genomic deficiencies for midgut phenotypes. Fifteen genomic intervals necessary for proper midgut morphogenesis were identified, three contain genes already known to act in the midgut. Three other genomic regions are required for formation of the endoderm or visceral mesoderm components of the midgut. Nine regions are required for proper formation of the midgut constrictions. The E75 ecdysone-induced gene, which encodes a nuclear receptor superfamily member, is the relevant gene in one region and is essential for proper formation of midgut constrictions. E75 acts downstream of the previously known constriction regulators or in parallel. Temporal hormonal control may therefore work in conjunction with spatial regulation by the homeotic genes in midgut development. Another genomic region is required to activate transcription of the homeotic genes Antp and Scr specifically in visceral mesoderm. The genomic regions identified by this screen provide a map to novel midgut development regulators.

Spatial domains in the developing forebrain: developmental regulation of a restricted cell surface protein.

We have isolated a monoclonal antibody, mAb 52G9, that recognizes a 55-kDa cell surface protein restricted to the early embryonic rat forebrain and to placode-derived structures. In the central nervous system (CNS), 52G9 immunoreactivity appears at Embryonic Day 11 (E11) in the rostral-most area of the telencephalon. It then spreads to the neuroepithelium of the telencephalon and basal diencephalon. Most strikingly, it appears at E14 in a distinct zone at the caudal end of the ventral diencephalic neuroepithelium. This area is sharply defined by strong 52G9 immunoreactivity bounded by unlabeled neuroepithelium. The pattern revealed by 52G9 is the first biochemical demonstration of spatial domains in the forebrain at a time prior to neuronal differentiation. By E18, 52G9 immunoreactivity has progressively disappeared from the forebrain; the glomerular layer of the olfactory bulb is the only 52G9-positive area in the CNS. The olfactory, otic, and hypophyseal placodes, which can be identified as early as E10, are also 52G9 positive as are their derivatives, the sensory epithelial of the nasal passage and inner ear, and also Rathke's pouch. The distribution and regulation of the 52G9 protein suggests that this novel cell surface molecule may be involved in the formation of spatial domains in the developing forebrain.

The B30 ganglioside is a cell surface marker for neural crest-derived neurons in the developing mouse.

We have previously reported the isolation of a monoclonal antibody, mAb B30, that recognizes two minor gangliosides specifically expressed in a small subset of neurons in the developing mouse central nervous system (Stainier and Gilbert, 1989). B30 labels mesencephalic trigeminal neurons shortly after differentiation until about 2 weeks after birth. Postnatally, it also labels two specific monolayers of cerebellar neurons. In this study, we have characterized the B30 immunoreactivity in the developing peripheral nervous system of the mouse. We report that B30 is a marker for neural crest-derived neurons and have used it to follow the neuronal differentiation of neural crest cells in a serum-free chemically defined culture system. Within hours after plating, neural crest cells migrate away from the neural tube explant on a fibronectin or laminin substrate and by 24 hr, up to 15% of them have differentiated into morphologically identifable neurons. In vitro as in vivo, undifferentiated mouse neural crest cells express the GD3 ganglioside which is recognized by mAb B33, and neural crest-derived neurons can be labeled by mAbs B33, B30, and also E1.9, a specific neuronal cytoskeletal marker. We also show the unique biochemical specificity of mAb B30 and provide experimental evidence for the role of the B30 ganglioside in the cellular adhesion process.