During early mouse development the homeobox gene Hesx1 is expressed in prospective forebrain tissue, but later becomes restricted to Rathke's pouch, the primordium of the anterior pituitary gland. Mice lacking Hesx1 exhibit variable anterior CNS defects and pituitary dysplasia. Mutants have a reduced prosencephalon, anopthalmia or micropthalmia, defective olfactory development and bifurcations in Rathke's pouch. Neonates exhibit abnormalities in the corpus callosum, the anterior and hippocampal commissures, and the septum pellucidum. A comparable and equally variable phenotype in humans is septo-optic dysplasia (SOD). We have cloned human HESX1 and screened for mutations in affected individuals. Two siblings with SOD were homozygous for an Arg53Cys missense mutation within the HESX1 homeodomain which destroyed its ability to bind target DNA. These data suggest an important role for Hesx1/HESX1 in forebrain, midline and pituitary development in mouse and human.

Epilepsy and mental retardation limited to females (EFMR) is a disorder with an X-linked mode of inheritance and an unusual expression pattern. Disorders arising from mutations on the X chromosome are typically characterized by affected males and unaffected carrier females. In contrast, EFMR spares transmitting males and affects only carrier females. Aided by systematic resequencing of 737 X chromosome genes, we identified different protocadherin 19 (PCDH19) gene mutations in seven families with EFMR. Five mutations resulted in the introduction of a premature termination codon. Study of two of these demonstrated nonsense-mediated decay of PCDH19 mRNA. The two missense mutations were predicted to affect adhesiveness of PCDH19 through impaired calcium binding. PCDH19 is expressed in developing brains of human and mouse and is the first member of the cadherin superfamily to be directly implicated in epilepsy or mental retardation.

Pierre Robin sequence (PRS) is an important subgroup of cleft palate. We report several lines of evidence for the existence of a 17q24 locus underlying PRS, including linkage analysis results, a clustering of translocation breakpoints 1.06-1.23 Mb upstream of SOX9, and microdeletions both approximately 1.5 Mb centromeric and approximately 1.5 Mb telomeric of SOX9. We have also identified a heterozygous point mutation in an evolutionarily conserved region of DNA with in vitro and in vivo features of a developmental enhancer. This enhancer is centromeric to the breakpoint cluster and maps within one of the microdeletion regions. The mutation abrogates the in vitro enhancer function and alters binding of the transcription factor MSX1 as compared to the wild-type sequence. In the developing mouse mandible, the 3-Mb region bounded by the microdeletions shows a regionally specific chromatin decompaction in cells expressing Sox9. Some cases of PRS may thus result from developmental misexpression of SOX9 due to disruption of very-long-range cis-regulatory elements.

As overt anterior pattern is present in the visceral embryonic endoderm prior to formation of any axial mesendoderm, a mechanism for bestowing anterior pattern must exist which is independent of primitive streak descendants. Furthermore, correct molecular patterning of the most rostral neurectoderm appears to depend on the presence of this anterior visceral embryonic endoderm during the early stages of gastrulation. We propose that primitive endoderm is responsible for the initial induction of rostral identity in the embryo, and in particular for the correct definition of the future prosencephalic neurectoderm. Subsequently, this identity will be reinforced and maintained by axial mesendoderm when it displaces the visceral embryonic endoderm during the course of gastrulation.

The majority of epilepsies are focal in origin, with seizures emanating from one brain region. Although focal epilepsies often arise from structural brain lesions, many affected individuals have normal brain imaging. The etiology is unknown in the majority of individuals, although genetic factors are increasingly recognized. Autosomal dominant familial focal epilepsy with variable foci (FFEVF) is notable because family members have seizures originating from different cortical regions. Using exome sequencing, we detected DEPDC5 mutations in two affected families. We subsequently identified mutations in five of six additional published large families with FFEVF. Study of families with focal epilepsy that were too small for conventional clinical diagnosis with FFEVF identified DEPDC5 mutations in approximately 12% of families (10/82). This high frequency establishes DEPDC5 mutations as a common cause of familial focal epilepsies. Shared homology with G protein signaling molecules and localization in human neurons suggest a role of DEPDC5 in neuronal signal transduction.

Nonsense-mediated mRNA decay (NMD) is of universal biological significance1-3. It has emerged as an important global RNA, DNA and translation regulatory pathway4. By systematically sequencing 737 genes (annotated in the Vertebrate Genome Annotation database) on the human X chromosome in 250 families with X-linked mental retardation, we identified mutations in the UPF3 regulator of nonsense transcripts homolog B (yeast) (UPF3B) leading to protein truncations in three families: two with the Lujan-Fryns phenotype5,6 and one with the FG phenotype7. We also identified a missense mutation in another family with nonsyndromic mental retardation. Three mutations lead to the introduction of a premature termination codon and subsequent NMD of mutant UPF3B mRNA. Protein blot analysis using lymphoblastoid cell lines from affected individuals showed an absence of the UPF3B protein in two families. The UPF3B protein is an important component of the NMD surveillance machinery8,9. Our results directly implicate abnormalities of NMD in human disease and suggest at least partial redundancy of NMD pathways.

The development of complex organs composed of different cell types frequently depends on reciprocal induction events occurring between distinct tissue layers that lie adjacent to one another in the embryo. The pituitary is a well studied case in point. It originates from two ectoderm-derived tissues, with the posterior lobe developing from an evagination of the ventral diencephalon, the infundibulum, and the intermediate and anterior lobes deriving from Rathke's pouch, an invaginating domain in the roof of the oral ectoderm 1 . In the mature gland, the posterior lobe contains axon terminal projections from two populations of hypothalamic neuroendocrine neurons. The anterior and intermediate lobes contain several different endocrine cell types whose proliferation, hormone synthesis and secretion are regulated by factors secreted from hypothalamic neuroendocrine neurons.During early development the infundibulum has an inductive role in the formation of the pituitary. For example, the genetic ablation of this domain in Titf1-null mice results in the complete absence of the pituitary gland 2 . The essential signaling molecules made by the infundibulum are thought to be FGF8 and BMP4, as both are necessary to induce early development of Rathke's pouch. FGF and BMP signals are also required to control the pattern of differentiation of cell types derived from Rathke's pouch 3,4 .Even subtle mutations that affect signaling pathways during early organogenesis can have profound effects on subsequent development and specification of mature cell types. These could arise in genes encoding the signaling molecules or their receptors, in transcription factors responsible for their expression or in genes required to specify the interacting tissues. In humans, SOX3, an HMG box protein (for review see ref. 5), is implicated in a syndrome of X-linked hypopituitarism and mental retardation 6 . In a single family whose males were deficient in growth hormone, a mutation in SOX3 was identified. The consequences of this mutation on the function of the protein are not known. X-chromosome deletions encompassing SOX3 are linked to several syndromes in humans, including mental retardation, but defects in pituitary function have not been reported 7,8 .SOX3 is a single-exon gene on the X chromosome in all mammals and is thought to be the gene from which SRY, the Y-linked testis determining gene, evolved 9,10 . Based on sequence homology, however, SOX3 is more closely related to SOX1 and SOX2 (refs. 5,11). Together they comprise the SOXB1 subfamily and are coexpressed throughout the developing CNS [11][12][13] . To study the role of Sox3, we targeted null mutations in the gene into XY embryonic stem (ES) cells, but injection of these cells into blastocysts resulted in early lethality of the chimeras due to a gastrulation defect (M. Parsons, C. Wise, S.B., M. CohenTannoudji, K.R., L. Pevny & R.L.-B., unpublished data).Therefore, to access later functions of SOX3, we initiated experiments using a conditional targeting strategy. In contrast to th...

Benign familial infantile epilepsy (BFIE) is a self-limited seizure disorder that occurs in infancy and has autosomal-dominant inheritance. We have identified heterozygous mutations in PRRT2, which encodes proline-rich transmembrane protein 2, in 14 of 17 families (82%) affected by BFIE, indicating that PRRT2 mutations are the most frequent cause of this disorder. We also report PRRT2 mutations in five of six (83%) families affected by infantile convulsions and choreoathetosis (ICCA) syndrome, a familial syndrome in which infantile seizures and an adolescent-onset movement disorder, paroxysmal kinesigenic choreoathetosis (PKC), co-occur. These findings show that mutations in PRRT2 cause both epilepsy and a movement disorder. Furthermore, PRRT2 mutations elicit pleiotropy in terms of both age of expression (infancy versus later childhood) and anatomical substrate (cortex versus basal ganglia).