Severe neural tube defects in the loop-tail mouse result from mutation of Lpp1, a novel gene involved in floor plate specification

Jennifer N. Murdoch 0 2 Kit Doudney 0 1 2 Caroline Paternotte 0 2 Andrew J. Copp 0 2 Philip Stanier 0 1 2 0 Imperial College School of Medicine , Hammersmith Campus, London W12 0NN , UK 1 Department of Maternal and Fetal Medicine, Institute of Reproductive and Developmental Biology 2 Neural Development Unit, Institute of Child Health, University College London , 30 Guilford Street, London WC1N 1EH Neural tube defects (NTD) are clinically important congenital malformations whose molecular mechanisms are poorly understood. The loop-tail (Lp) mutant mouse provides a model for the most severe NTD, craniorachischisis, in which the brain and spinal cord remain open. During a positional cloning approach, we have identified a mutation in a novel gene, Lpp1, in the Lp mouse, providing a strong candidate for the genetic causation of craniorachischisis in Lp. Lpp1 encodes a protein of 521 amino acids, with four transmembrane domains related to the Drosophila protein strabismus/van gogh (vang). The human orthologue, LPP1, shares 89% identity with the mouse gene at the nucleotide level and 99% identity at the amino acid level. Lpp1 is expressed in the ventral part of the developing neural tube, but is excluded from the floor plate where Sonic hedgehog (Shh) is expressed. Embryos lacking Shh express Lpp1 throughout the ventral neural tube, suggesting negative regulation of Lpp1 by Shh. Our findings suggest that the mutual interaction between Lpp1 and Shh may define the lateral boundary of floor plate differentiation. Loss of Lpp1 function disrupts neurulation by permitting more extensive floor plate induction by Shh, thereby inhibiting midline bending of the neural plate during initiation of neurulation. - Closure of the neural tube is essential for normal development of the brain and spinal cord. Failure of neural tube closure is among the commonest of human congenital malformations, with a prevalence of ~1 per 1000 pregnancies (1). In craniorachischisis, the most severe type of neural tube defect (NTD), almost the entire brain and spinal cord remain open (Fig. 1A and B), as a result of a failure to initiate closure at the start of neurulation (2,3). Craniorachischisis comprises 1020% of human NTD (46), and leads to death around the time of birth. Although the aetiology of human craniorachischisis is unknown, there is a close similarity between this defect and the phenotype of the loop-tail (Lp) mouse mutant (7,8). This resemblance has prompted a series of studies, over a 50 year period (710), aimed at determining the developmental basis of craniorachischisis and identifying the causative gene in the Lp mouse. Lp is one of only two known gene mutations that disrupt the onset of mouse neural tube closure, which occurs at the hindbraincervical boundary in embryos with six to seven somites. This initial neurulation event, so-called Closure 1, is essential for the subsequent closure of the entire spine and much of the brain: hence, the severe NTD phenotype resulting from failure of Closure 1 (2). The other gene known to be essential for Closure 1 is circletail (Crc), a recently described mutation, with a closely similar phenotype to Lp (11). The two mutations are not allelic, and yet they interact in Lp/Crc compound heterozygotes to produce craniorachischisis closely resembling the phenotype of the Lp and Crc single homozygotes (12). These findings suggest the existence of a developmental pathway, involving the Lp and Crc genes, that is critical in regulating the onset of neurulation. A clue to the underlying developmental defect in Lp is the finding of an enlarged presumptive floor plate region in the midline neural plate of Lp homozygous embryos (13). In normal circumstances, neurulation at the site of Closure 1 involves bending of the neural plate solely in the midline (14). Enlargement of the floor plate region in Lp/Lp embryos, prior to the onset of neural tube closure at embryonic day (E) 8.5, disrupts midline bending so that the neural folds are more widely spaced apart than normal. This defect appears to lead directly to the failure of neural tube closure (13). Subsequently, the floor plate differentiates as an abnormally broad structure in the posterior region of Lp/Lp embryos, with an abnormally extensive expression domain of the floor plate marker, Sonic hedgehog (Shh). Although the molecular mechanism responsible for enlargement of the presumptive floor plate region in Lp has not been determined, one possibility is a recruitment of cells into the floor plate from more lateral regions of the neural plate. According to this idea, the normal function of the Lp gene product might be to restrict the lateral extent of floor plate differentiation. In the present study, we report the outcome of a positional cloning project to identify candidate genes for Lp. We have identified a mutation in a novel gene, named Lp protein-1 (Lpp1), and demonstrate that its expression pattern is restricted The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors to the lateral boundary of the floor plate in the neurulation stage embryo. Independent studies confirm the mutation of Lpp1 in loop-tail mice (15), strongly indicating that this gene is indeed essential for the initiation of neurulation. Previous genetic studies have enabled Lp to be mapped within a 1.2 cM (600 kb) interval on distal mouse chromosome 1, between the markers D1Mit113 and Tagln2 (10,1619). This region has extensive homology to human chromosome 1q22q23 (20). In order to identify candidate genes, we obtained genomic sequence across the entire region in mouse and human. The mouse sequence led to the identification of new informative microsatellite markers which permitted refinement of the critical region to 450 kb (Fig. 1C). Lpp1: a candidate gene for Lp A complete transcript map over the refined Lp critical region was developed using a combination of computational-based gene prediction analysis, exon amplification, comparative sequence analysis and cross-species database searching (10,18). The region contains 12 candidate genes, many of which are expressed in the early neurulation stage embryo (10,21). Sequence analysis of the 128 coding exons and flanking intronic sequences constituting 10 of the 12 genes (Nhlh1, Ncstn, Copa, Pxf, Tim23, H326, Pea15, Casq1, Atp1a4 and Atp1a2; Fig. 1C) failed to identify any mutation in Lp mutant DNA compared with wild-type. Expression analysis of Nhlh1, Copa, Pxf and Pea15 also revealed normal expression of these genes in Lp homozygotes, providing no evidence for a disturbance of transcriptional regulation (10,21,22). An 11th gene, Cd84, is not expressed during neurulation (10), and was excluded from the analysis. In the one remaining candidate gene, we identified a mutation within the protein coding region. This gene was described previously as Kiaa1215, from its homology to a human cDNA clone (10), and we have now renam (...truncated)