The secreted protein sonic hedgehog (Shh) plays an integral role in forming the ventral midline of the vertebrate central nervous system (CNS). In the absence of Shh function, ventral midline development is perturbed resulting in holoprosencephaly (HPE), a structural malformation of the brain, as well as in neuronal patterning and path finding defects along the length of the anteroposterior neuraxis. Central to the understanding of ventral neural tube development is how Shh transcription is regulated in the CNS. To address this issue, we devised an enhancer trap assay to systematically screen 1 Mb of DNA surrounding the Shh locus for the ability to target reporter gene expression to sites of Shh transcription in transgenic mouse embryos. This analysis uncovered six enhancers distributed over 400 kb, the combined activity of which covered all sites of Shh expression in the mouse embryonic CNS from the ventral forebrain to the posterior extent of the spinal cord. To evaluate the relative contribution of these enhancers to the overall pattern of Shh expression, individual elements were deleted in the context of a transgenic Bac reporter assay. Redundant mechanisms were found to control Shh-like reporter activity in the ventral spinal cord, hindbrain and regions of the telencephalon, whereas unique elements regulated Shh-like expression in the ventral midbrain, the majority of the ventral diencephalon and parts of the telencephalon. Three ventral forebrain enhancers locate on the distal side of translocation breakpoints that occurred upstream of Shh in human cases of HPE, suggesting that displacement of these regulatory elements from the Shh promoter is a likely cause of HPE in these individuals.

The secreted morphogen, Sonic hedgehog (Shh) is a significant determinant of brain size and craniofacial morphology1-4. In humans, SHH haploinsufficiency results in holoprosencephaly (HPE)5, a defect in anterior midline formation. Despite the importance of maintaining SHH transcript levels above a critical threshold, we know little about the upstream regulators of SHH expression in the forebrain. Here we describe a combination of genetic and biochemical experiments to uncover a critical pair of cis and trans acting determinants of Shh forebrain expression. A rare nucleotide variant located 460kb upstream of SHH was discovered in an individual with HPE that resulted in the loss of Shh brain enhancer-2 (SBE2) activity in the hypothalamus of transgenic mouse embryos. Using a DNA affinity capture assay we screened SBE2 sequence for DNA binding proteins and identified members of the Six3/Six6 homeodomain family as candidate regulators of Shh transcription. Six3 and Six6 showed reduced binding affinity for the mutant compared to wild type SBE2 sequence. Moreover, HPE causing mutations in Six3 failed to bind and activate SBE2, whereas, Shh forebrain expression was unaltered in Six6 −/− embryos. These data provide a direct link between Six3 and Shh regulation during normal forebrain development and in the pathogenesis of HPE. Previous efforts to address this issue focused on determining the genomic location of functional Shh regulatory elements13. These experiments identified six enhancers distributed over a 500 kb interval surrounding the Shh gene that directed reporter activity to most areas of Shh expression in the mouse CNS, including the ventral forebrain ( Fig. 1). In particular, the highly conserved Shh brain enhancer-2 (SBE2), located 460 kb upstream of the SHH coding sequence, was identified as unique in its ability to regulate Shh-like expression throughout the hypothalamus. KeywordsTo identify functionally relevant nucleotides in SBE2, we screened the 1.1 kb sequence mediating its activity for mutations in humans with HPE. We reasoned that HPE causing variants in SBE2 could aid in identifying critical cis and trans determinants of SHH expression in the forebrain. Similar resequencing approaches have been successful in identifying common and rare coding sequence variants in genes associated with common diseases, but have not been routinely applied to the study of remote noncoding regions in rare diseases such as HPE (1:16,000 livebirths)12,14.From 474 HPE patients, we identified one individual who was heterozygous for a C to T base change at nucleotide position 444 of the enhancer sequence. The C/T variant is situated within a block of 10 nucleotides that have been maintained in human, mouse, chicken and frog for over 350 million years ( Fig. 1). This C/T nucleotide variant was not observed in DNA samples from 450 unrelated control individuals. The affected female exhibited features of semilobar HPE including microcephaly, midfacial hypoplasia, cleft-lip and palate, diabetes insipidus, and moderate fus...

Background Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates. Objective To characterise genetic and clinical findings in patients with SIX3 mutations. Methods Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish. Results In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%. Conclusions Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype–phenotype correlation, as shown by functional studies using animal models.