Disruption of scribble (Scrb1) causes severe neural tube defects in the circletail mouse

Jennifer N. Murdoch 2 Deborah J. Henderson 1 Kit Doudney 0 Carles Gaston-Massuet 2 Helen M. Phillips 1 Caroline Paternotte 2 Ruth Arkell 3 Philip Stanier 0 Andrew J. Copp 2 0 Department of Obstetrics and Gynaecology, Institute of Reproductive and Developmental Biology, Imperial College School of Medicine , London W12 0NN , UK 1 Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life , Newcastle upon Tyne NE1 3BZ , UK 2 Neural Development Unit, Institute of Child Health, University College London , 30 Guilford Street, London WC1N 1EH , UK 3 MRC Mammalian Genetics Unit , Harwell, Oxon OX11 0RD , UK Circletail is one of only two mouse mutants that exhibit the most severe form of neural tube defect (NTD), termed craniorachischisis. In this disorder, almost the entire brain and spinal cord is affected, owing to a failure to initiate neural tube closure. Craniorachischisis is a significant cause of lethality in humans, yet the molecular mechanisms involved remain poorly understood. Here, we report the identification of the gene mutated in circletail (Crc), using a positional cloning approach. This gene, Scrb1, encodes a member of the LAP protein family related to Drosophila scribble, with 16 leucine rich repeats and four PDZ domains. The Crc mutant contains a single base insertion that creates a frame shift and leads to premature termination of the Scrb1 protein. We report the expression pattern of Scrb1 during embryonic and fetal development, and show that Scrb1 expression closely mirrors the phenotypic defects observed in Crc/Crc mutants. In addition, circletail genetically interacts with the loop-tail mutant, and we reveal overlapping expression of Scrb1 with Vangl2, the gene mutated in loop-tail. The identification of the Crc gene further defines the nature of the genetic pathway required for the initiation of neural tube closure and provides an important new candidate that may be implicated in the aetiology of human NTDs. - INTRODUCTION The neural tube is the embryonic precursor of the brain and spinal cord, and is formed by the rolling up of a flat layer of ectodermal cells (the neural plate) to create a tube. In the mammalian embryo, initiation of neural tube closure occurs at three sites along the body axis (1). The first site of de novo closure (so-called Closure 1) occurs in the future cervical region, while the second and third sites of de novo closure (Closures 2 and 3) occur at about the forebrainmidbrain boundary, and at the most rostral extent of the forebrain, respectively (1). Closure between these three sites is responsible for the formation of the cranial neural tube, while continuation of closure caudally from the site of Closure 1 is necessary for the formation of the spinal cord. Disruption of neural tube closure leads to a group of disorders termed neural tube defects (NTDs), which are one of the commonest causes of congenital malformation. NTDs affect around 1 in 1000 pregnancies and are either severely disabling or lethal (2). Neural tube defects are classified according to the region of the body axis that is affected (3). Anencephaly is the consequence of a failure to complete neural tube closure in the brain, whereas spina bifida results from disruption of neural tube closure in the lower spine. The most severe form of NTD is craniorachischisis, in which almost the entire brain and spinal cord remain open. Craniorachischisis is caused by a failure to initiate neural tube formation at Closure 1, at the start of neurulation (3). Craniorachischisis comprises 1020% of human NTDs (46) and is invariably lethal, yet the molecular mechanisms involved are poorly understood. The estimated recurrence risk of NTD with one affected sibling is 25% and this increases to 16% with two affected siblings, revealing a strong genetic predisposition for NTD in humans (7). However, direct analysis of the molecular basis of human NTDs is difficult, owing to the practical and ethical constraints of studying human embryos early in development, and the lack of suitable families available for linkage analysis. In contrast, the mouse provides an excellent model system with which to identify the molecular mechanisms involved in neural tube closure. Indeed, there are over 60 existing mouse mutants that exhibit defects in neurulation resulting in, predominantly, spina bifida or anencephaly (8). Identification of the molecular mechanisms involved in craniorachischisis currently relies solely on two mutants that exhibit a failure of Closure 1: looptail (Lp) and circletail (Crc). These mutants are essential to our understanding of the molecular mechanisms involved in the initiation of neural tube closure. Homozygous Lp/Lp embryos and homozygous Crc/Crc mutants both exhibit failure of initiation of neural tube closure in the future cervical region of the embryo (Closure 1), at E8.5. Consequently, these mutants exhibit a neural tube that remains open from the midbrain/hindbrain boundary throughout the spine (Fig. 1AC) (911), closely modelling the human condition of craniorachischisis. We, and others, recently identified the gene mutated in loop-tail as Vangl2 (formerly known as Lpp1 or Ltap) (12,13). Vangl2 encodes a protein related to Drosophila van gogh/strabismus, with four transmembrane domains and a putative carboxy terminal PDZbinding motif (12,13). Disruption of Drosophila strabismus reveals an essential role for this gene in the establishment of planar cell polarity (PCP), also known as epithelial polarity or tissue polarity (1416). PCP is evident in a number of tissues in Drosophila, such as the regular arrangement of ommatidia in the eye, the formation and directionality of hairs in the wing, and the arrangement of sensory bristles in the thorax. In the strabismus mutant, this regular arrangement is lost (1416). Other genes known to be involved in the establishment of PCP in these tissues include frizzled, dishevelled, prickle, flamingo, rhoA and the JNK cascade (1719). The involvement of frizzled and dishevelled reveals molecular overlap with the Wnt signalling pathway, for which frizzled is the receptor, and dishevelled a downstream cytoplasmic factor. However, PCP appears not to involve the other downstream components of the canonical Wnt pathway, such as armadillo (b-catenin). Rather, planar cell polarity is mediated by a non-canonical Wnt signalling pathway. Homologues of all the Drosophila PCP pathway genes exist in vertebrates, and much recent evidence implicates the vertebrate PCP pathway in the regulation of the convergent extension cell movements that occur during gastrulation and neurulation. For instance, disruption of the Xenopus or zebrafish homologues of van gogh/strabismus results in failure of convergent extension, and subsequently to failure of neural tube closure (2023), while mutation of the JNK genes also affects convergent extension (24). A defect in convergent extension may underlie the failure of neural tu (...truncated)