α-Tanycytes of the adult hypothalamic third ventricle include distinct populations of FGF-responsive neural progenitors.

Emerging evidence suggests that new cells, including neurons, can be generated within the adult hypothalamus, suggesting the existence of a local neural stem/progenitor cell niche. Here, we identify α-tanycytes as key components of a hypothalamic niche in the adult mouse. Long-term lineage tracing in vivo using a GLAST::CreER(T2) conditional driver indicates that α-tanycytes are self-renewing cells that constitutively give rise to new tanycytes, astrocytes and sparse numbers of neurons. In vitro studies demonstrate that α-tanycytes, but not ß-tanycytes or parenchymal cells, are neurospherogenic. Distinct subpopulations of α-tanycytes exist, amongst which only GFAP-positive dorsal α2-tanycytes possess stem-like neurospherogenic activity. Fgf-10 and Fgf-18 are expressed specifically within ventral tanycyte subpopulations; α-tanycytes require fibroblast growth factor signalling to maintain their proliferation ex vivo and elevated fibroblast growth factor levels lead to enhanced proliferation of α-tanycytes in vivo. Our results suggest that α-tanycytes form the critical component of a hypothalamic stem cell niche, and that local fibroblast growth factor signalling governs their proliferation.

Molecular effects of novel mutations in Hesx1/HESX1 associated with human pituitary disorders.

The homeobox gene Hesx1/HESX1 has been implicated in the establishment of anterior pattern in the central nervous system (CNS) in a number of vertebrate species. Its role in pituitary development has been documented through loss-of-function studies in the mouse. A homozygous missense point mutation resulting in a single amino acid substitution, Arg160Cys (R160C), is associated with a heritable form of the human condition of septo-optic dysplasia (SOD). We have examined the phenotype of affected members in this pedigree in more detail and demonstrate for the first time a genetic basis for midline defects associated with an undescended or ectopic posterior pituitary. A similar structural pituitary abnormality was observed in a second patient heterozygous for another mutation in HESX1, Ser170Leu (S170L). Association of S170L with a pituitary phenotype may be a direct consequence of the HESX1 mutation since S170L is also associated with a dominant familial form of pituitary disease. However, a third mutation in HESX1, Asn125Ser (N125S), occurs at a high frequency in the Afro-Caribbean population and may therefore reflect a population-specific polymorphism. To investigate the molecular basis for these clinical phenotypes, we have examined the impact of these mutations on the regulatory functions of HESX1. We show that Hesx1 is a promoter-specific transcriptional repressor with a minimal 36 amino acid repression domain which can mediate promoter-specific repression by suppressing the activity of homeodomain-containing activator proteins. Mutations in HESX1 associated with pituitary disease appear to modulate the DNA-binding affinity of HESX1 rather than its transcriptional activity. Wild-type HESX1 binds a dimeric homeodomain site with high affinity (K(d) 31 nM) whilst HESX1(S170L) binds with a 5-fold lower activity (K(d) 150 nM) and HESX1(R160C) does not bind at all. Although HESX1(R160C) has only been shown to be associated with the SOD phenotype in children homozygous for the mutation, HESX1(R160C) can inhibit DNA binding by wild-type HESX1 both in vitro and in vivo in cell culture. This dominant negative activity of HESX1(R160C) is mediated by the Hesx1 repression domain, supporting the idea that the repression domain is implicated in interactions between homeodomain proteins. Our data suggest a possible molecular paradigm for the dominant inheritance observed in some pituitary disorders.

Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility.

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

Heterozygous HESX1 mutations associated with isolated congenital pituitary hypoplasia and septo-optic dysplasia.

We have previously shown that familial septo-optic dysplasia (SOD), a syndromic form of congenital hypopituitarism involving optic nerve hypoplasia and agenesis of midline brain structures, is associated with homozygosity for an inactivating mutation in the homeobox gene HESX1/Hesx1 in man and mouse. However, as most SOD/congenital hypopituitarism occurs sporadically, the possible contribution of HESX1 mutations to the aetiology of these cases is presently unclear. Interestingly, a small proportion of mice heterozygous for the Hesx1 null allele show a milder SOD phenocopy, implying that heterozygous mutations in human HESX1 could underlie some cases of congenital pituitary hypoplasia with or without midline defects. Accordingly, we have now scanned for HESX1 mutations in 228 patients with a broad spectrum of congenital pituitary defects, ranging in severity from isolated growth hormone deficiency to SOD with panhypopituitarism. Three different heterozygous missense mutations were detected in individuals with relatively mild pituitary hypoplasia or SOD, which display incomplete penetrance and variable phenotype amongst heterozygous family members. Gel shift analysis of the HESX1-S170L mutant protein, which is encoded by the C509T mutated allele, indicated that a significant reduction in relative DNA binding activity results from this mutation. Segregation analysis of a haplotype spanning 6.1 cM, which contains the HESX1 locus, indicated that only one HESX1 mutation was present in the families containing the C509T and A541G mutations. These results demonstrate that some sporadic cases of the more common mild forms of pituitary hypoplasia have a genetic basis, resulting from heterozygous mutation of the HESX1 gene.

Multiple source localization using genetic algorithms.

We present a new procedure for localizing simultaneously active multiple brain sources that overlap in both space and time on EEG recordings. The source localization technique was based on a spatio-temporal model and a genetic algorithm search routine. The method was successfully applied to the localization of two dipole sources from several sets of simulated potentials with various signal-to-noise ratios (SNR). The different SNR values resembled evoked responses and epileptic spikes as commonly seen in the laboratory. Results of the simulation studies yielded localization accuracy ranging from 0.01 to 0.07 cm with an SNR of 10; from 0.02 to 0.26 cm with an SNR of 5; and from 0.06 to 0.73 cm when the SNR was equal to 2. Additionally, two sets of simulations were based on the dipole arrangements and time activities of data obtained during electrical stimulation of the median nerve in human subjects. These studies yielded localization accuracy within 0.1 cm. We also studied the localization accuracy of the algorithm using a physical model incorporating potential measurements of two current dipoles embedded in a sphere. In this situation the algorithm was successful in localizing the two simultaneously active sources to within 0.07-0.15 cm.

Somatosensory evoked response source localization using actual cortical surface as the spatial constraint.

We localized right median nerve somatosensory evoked responses in a normal human subject using an equivalent dipole method applied to magnetic field recordings. High resolution, 3-dimensional MRI data were used to confine source locations to the cortical surface. Results localized in Brodmann area 3b corresponding to location of hand somatosensory cortex derived from direct brain stimulation studies. The solution was unique and total computational time for an exhaustive, brute-force search was small and the results realistic due to applied anatomical constraints. This study demonstrates feasibility of accurate, non-invasive, realistic localization of dynamic human cortical function using spatial constraints provided by MRI images.