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.

The molecular phenotype of heparan sulfate in the Hs2st-/- mutant mouse.

Heparan sulfate (HS) is a co-receptor for a number of growth factors, morphogens, and adhesion proteins. HS biosynthetic modifications may determine the strength and outcome of HS-ligand interactions. We previously described the phenotype of mice with a gene-trap mutation in Hs2st, encoding the key HS 2-O-sulfotransferase enzyme in HS polymer modification. In contrast to the early developmental failure of embryos lacking HS, the onset of abnormalities in the Hs2st(-/-) mice occurs only after midgestation, the most dramatic being the complete failure of kidney development. Uronate 2-O-sulfates were not detected in the mutant HS, indicating a complete loss of function of Hs2st. However, the domain structure of the mutant HS is conserved, and compensatory increases in N- and 6-O-sulfation maintain the overall charge density. The apparent affinities of the mutant HS for hepatocyte growth factor/scatter factor and fibronectin were unchanged but were reduced for fibroblast growth factor-1 and -2. Surprisingly, the Hs2st(-/-) cells were able to mount an apparently normal signaling response to fibroblast growth factor-1 and -2 as well as to hepatocyte growth factor/scatter factor.

Nodal signalling in the epiblast patterns the early mouse embryo.

Shortly after implantation the mouse embryo comprises three tissue layers. The founder tissue of the embryo proper, the epiblast, forms a radially symmetric cup of epithelial cells that grows in close apposition to the extra-embryonic ectoderm and the visceral endoderm. This simple cylindrical structure exhibits a distinct molecular pattern along its proximal-distal axis. The anterior-posterior axis of the embryo is positioned later by coordinated cell movements that rotate the pre-existing proximal-distal axis. The transforming growth factor-beta family member Nodal is known to be required for formation of the anterior-posterior axis. Here we show that signals from the epiblast are responsible for the initiation of proximal-distal polarity. Nodal acts to promote posterior cell fates in the epiblast and to maintain molecular pattern in the adjacent extra-embryonic ectoderm. Both of these functions are independent of Smad2. Moreover, Nodal signals from the epiblast also pattern the visceral endoderm by activating the Smad2-dependent pathway required for specification of anterior identity in overlying epiblast cells. Our experiments show that proximal-distal and subsequent anterior-posterior polarity of the pregastrulation embryo result from reciprocal cell-cell interactions between the epiblast and the two extra-embryonic tissues.

Distinct enhancer elements control Hex expression during gastrulation and early organogenesis.

In the mouse, embryological and genetic studies have indicated that two spatially distinct signalling centres, the anterior visceral endoderm and the node and its derivatives, are required for the correct patterning of the anterior neural ectoderm. The divergent homeobox gene Hex is expressed in the anterior visceral endoderm, in the node (transiently), and in the anterior definitive endoderm. Other sites of Hex expression include the liver and thyroid primordia and the endothelial cell precursors. We have used transgenic analysis to map the cis-acting regulatory elements controlling Hex expression during early mouse development. A 4.2-kb upstream region is important for Hex expression in the endothelial cell precursors, liver, and thyroid, and a 633-bp intronic fragment is both necessary and sufficient for Hex expression in the anterior visceral endoderm and the anterior definitive endoderm. These same regions drive expression in homologous structures in Xenopus laevis, indicating conservation of these regulatory regions in vertebrates. Analysis of the anterior visceral endoderm/anterior definitive endoderm enhancer identifies a repressor region that is required to downregulate Hex expression in the node once the anterior definitive endoderm has formed. This analysis also reveals that the initiation of Hex expression in the anterior visceral endoderm and axial mesendoderm requires common elements, but maintenance of expression is regulated independently in these tissues.

Getting your head around Hex and Hesx1: forebrain formation in mouse.

An increasing amount of evidence suggests that in mouse there are two signalling centres required for the formation of a complete neural axis: the anterior visceral endoderm (AVE), and the node and its derivatives. Embryological and genetic studies suggest that the AVE has a head-inducing activity. In contrast, the node appears to act first as a head inducer in synergy with the AVE initiating anterior neural patterning at early stages of mouse development, and later, node derivatives are necessary for maintenance and embellishment of anterior neural character. Hex and Hesx1 are homeobox genes that are expressed in relevant tissues involved in anterior patterning. The analysis of the Hex and Hesx1 mutant mice has revealed that the lack of these genes has little or no effect on the early steps of anterior neural induction. However, both genes are required subsequently for the proper expansion of the forebrain region. We suggest that disturbance in the specification of an Fgf8 signalling centre in the anterior neural ridge may account for the anterior defects observed in these mutants.

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.

Sp5, a new member of the Sp1 family, is dynamically expressed during development and genetically interacts with Brachyury.

We describe the identification, biochemical characterisation, and mutation of a novel mouse gene: Sp5. Sp5 encodes a protein having a C-terminal C(2)H(2) zinc finger domain closely related to that of the transcription factor Sp1. In vitro, DNA binding studies show that it binds to the GC box, a DNA motif present in the promoter of a very large number of genes, including Brachyury, and recognised by members of the Sp1 family. However, outside of its DNA binding domain, Sp5 has little homology with any other member of the Sp1 family. In contrast to the ubiquitous expression of Sp1, Sp5 exhibits a remarkably dynamic pattern of expression throughout early development. This is suggestive of a role in numerous tissue patterning events, including gastrulation and axial elongation; differentiation and patterning of the neural tube, pharyngeal region, and somites; and formation of skeletal muscle in the body and limbs. Mice homozygous for a targeted mutation in Sp5 show no overt phenotype. However, the enhancement of the T/+ phenotype in compound mutant mice (Sp5(lacZ)/Sp5(lacZ), T/+) indicates a genetic interaction between Sp5 and Brachyury. These observations are consistent with a role for Sp5 in the coordination of changes in transcription required to generate pattern in the developing embryo.

Molecular genetics of septo-optic dysplasia.

Septo-optic dysplasia (SOD) is a highly variable condition characterized by midline neurological abnormalities associated with pituitary hypoplasia and optic nerve hypoplasia. The aetiology is unknown. Mutant mice, in which a novel homeobox gene, Hesx1, has been disrupted, exhibit a phenotype that resembles the phenotype of SOD. We therefore wished to explore the possibility that this gene is implicated in SOD. We cloned and sequenced the human homologue HESX1 and screened for mutations in affected individuals using single-stranded conformational polymorphism analysis, followed by cloning and sequencing of any exons which showed a band shift. Two siblings with SOD were homozygous for an Arg53Cys missense mutation within the HESX1 homeodomain, leading to a loss of in vitro DNA binding. Subsequently, we have identified heterozygous mutations in HESX1 that are associated with milder pituitary phenotypes. Our studies indicate a vital role for Hesx1/HESX1 in forebrain and pituitary development in mouse and man, and hence in some cases of SOD.

The homeobox gene Hesx1 is required in the anterior neural ectoderm for normal forebrain formation.

The homeobox gene Hesx1 is expressed in the anterior visceral endoderm (AVE), anterior axial mesendoderm (AME), and anterior neural ectoderm (ANE) during early mouse embryogenesis. Previous studies have shown that Hesx1 is essential for normal murine forebrain development. Hesx1 homozygous mutants showed variable forebrain truncations ranging from mild to severe lack of forebrain tissue. Here, we have investigated the requirement of Hesx1 in the AVE, AME, and ANE using chimeric and in situ hybridization analyses to understand better the nature of the forebrain defects. Chimeric embryos composed predominantly of Hesx1(+/+) cells developing within Hesx1(-/-) visceral endoderm showed no evident forebrain abnormalities. In contrast, injection of Hesx1(-/-) ES cells into wild-type blastocysts gave rise to chimeras with forebrain defects similar to those observed in the Hesx1(-/-) mutants. RNA in situ hybridization analysis showed that the AVE and AME markers Cerrl, Lim1, and Shh were normally expressed in 6.5- and 7.5-dpc Hesx1(-/-) mutants. Expression of the ANE markers Six3 and Rax/Rx was also unperturbed in the Hesx1(-/-) mutants from late gastrula to late headfold stages. However, transcripts for both genes were markedly reduced by the early somite stage, about 24 h after Hesx1 is first expressed in the ANE. Therefore, Hesx1 seems to be required autonomously in the ANE for normal forebrain formation.

Hex is a transcriptional repressor that contributes to anterior identity and suppresses Spemann organiser function.

One of the earliest markers of anterior asymmetry in vertebrate embryos is the transcription factor Hex. We find that Hex is a transcriptional repressor that can be converted to an activator by fusing full length Hex to two copies of the minimal transcriptional activation domain of VP16 together with the flexible hinge region of the (lambda) repressor (Hex-(lambda)VP2). Retention of the entire Hex open reading frame allows one to examine Hex function without disrupting potential protein-protein interactions. Expression of Hex-(lambda)VP2 in Xenopus inhibits expression of the anterior marker Cerberus and results in anterior truncations. Such embryos have multiple notochords and disorganised muscle tissue. These effects can occur in a cell non-autonomous manner, suggesting that one role of wild-type Hex is to specify anterior structures by suppressing signals that promote dorsal mesoderm formation. In support of this idea, over-expression of wild-type Hex causes cell non-autonomous dorso-anteriorization, as well as cell autonomous suppression of dorsal mesoderm. Suppression of dorsal mesoderm by Hex is accompanied by the down-regulation of Goosecoid and Chordin, while induction of dorsal mesoderm by Hex-(lambda)VP2 results in activation of these genes. Transient transfection experiments in ES cells suggest that Goosecoid is a direct target of Hex. Together, our results support a model in which Hex suppresses organiser activity and defines anterior identity.

The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation.

The homeobox gene Hex is expressed in the anterior visceral endoderm (AVE) and rostral definitive endoderm of early mouse embryos. Later, Hex transcripts are detected in liver, thyroid and endothelial precursor cells. A null mutation was introduced into the Hex locus by homologous recombination in embryonic stem cells. Hex mutant embryos exhibit varying degrees of anterior truncation as well as liver and thyroid dysplasia. The liver diverticulum is formed but migration of hepatocytes into the septum transversum fails to occur. Development of the thyroid is arrested at the thyroid bud stage at 9.5 dpc. Brain defects are restricted to the rostral forebrain and have a caudal limit at the zona limitans intrathalamica, the boundary between dorsal and ventral thalamus. Analysis of Hex(-/-) mutants at early stages shows that the prospective forebrain ectoderm is correctly induced and patterned at 7.5 days post coitum (dpc), but subsequently fails to develop. AVE markers are expressed and correctly positioned but development of rostral definitive endoderm is greatly disturbed in Hex(-/-) embryos. Chimeric embryos composed of Hex(-/-) cells developing within a wild-type visceral endoderm show forebrain defects indicating that Hex is required in the definitive endoderm. All together, these results demonstrate that Hex function is essential in definitive endoderm for normal development of the forebrain, liver and thyroid gland.

Wnt signaling in Xenopus embryos inhibits bmp4 expression and activates neural development.

We report a new role for Wnt signaling in the vertebrate embryo: the induction of neural tissue from ectoderm. Early expression of mouse wnt8, Xwnt8, beta-catenin, or dominant-negative GSK3 induces the expression of neural-specific markers and inhibits the expression of Bmp4 in Xenopus ectoderm. We show that Wnt8, but not the BMP antagonist Noggin, can inhibit Bmp4 expression at early gastrula stages. Furthermore, inhibition of beta-catenin activity in the neural ectoderm of whole embryos by a truncated TCF results in a decrease in neural development. Therefore, we suggest that a cleavage-stage Wnt signal normally contributes to an early repression of Bmp4 on the dorsal side of the embryo and sensitizes the ectoderm to respond to neural inducing signals from the organizer. The Wnt targets Xnr3 and siamois have been shown previously to have neuralizing activity when overexpressed. However, antagonists of Wnt signaling, dnXwnt8 and Nxfrz8, inhibit Wnt-mediated Xnr3 and siamois induction, but not neural induction, suggesting an alternative mechanism for Bmp repression and neuralization. Conversely, dnTCF blocks both Wnt-mediated Xnr3 and neural induction, suggesting that both pathways require this transcription factor.

An anterior signalling centre in Xenopus revealed by the homeobox gene XHex.

BACKGROUND: Signals from anterior endodermal cells that express the homeobox gene Hex initiate development of the most rostral tissues of the mouse embryo. The dorsal/anterior endoderm of the Xenopus gastrula, which expresses Hex and the putative head-inducing gene cerberus, is proposed to be equivalent to the mouse anterior endoderm. Here, we report the origin and signalling properties of this population of cells in the early Xenopus embryo. RESULTS: Xenopus anterior endoderm was found to derive in part from cells at the centre of the blastocoel floor that express XHex, the Xenopus cognate of Hex. Like their counterparts in the mouse embryo, these Hex-expressing blastomeres moved to the dorsal side of the Xenopus embryo as gastrulation commenced, and populated deep endodermal adjacent to Spemann's organiser. Experiments involving the induction of secondary axes confirmed that XHex expression was associated with anterior development. Ventral misexpression of XHex induced ectopic cerberus expression and conferred anterior signalling properties to the endoderm. Unlike the effect of misexpressing cerberus, these signals could not neuralise overlying ectoderm. CONCLUSIONS: XHex expression reveals the unexpected origin of an anterior signalling centre in Xenopus, which arises in part from the centre of the blastula and localises to the deep endoderm adjacent to Spemann's organiser. Signals originating from these endodermal cells impart an anterior identity to the overlying ectoderm, but are insufficient for neural induction. The anterior movement of Hex-expressing cells in both Xenopus and mouse embryos suggests that this process is a conserved feature of vertebrate development.

HESX1: a novel gene implicated in a familial form of septo-optic dysplasia.

The homeobox gene Hesx1, which encodes a pituitary transcription factor, is first expressed at gastrulation in the mouse embryo. Hesx1 expression begins in prospective forebrain tissue but later becomes restricted to Rathke's pouch, the primordium of the anterior pituitary gland. Transgenic mice lacking Hesx1 exhibit a phenotype comprising variable anterior CNS defects, such as a reduced prosencephalon, abnormalities in the corpus callosum and septum pellucidum, anophthalmia or microphthalmia, defective olfactory development and bifurcations in Rathke's pouch with pituitary dysplasia. A comparable and highly variable phenotype in humans is septo-optic dysplasia. We have cloned and sequenced the human homologue HESX1 and screened for mutations in affected individuals using single-stranded conformational polymorphism analysis. Two siblings with septo-optic dysplasia were homozygous for a missense mutation within the HESX1 homeobox. This mutation resulted in the substitution of a highly conserved arginine residue (Arg53) by cysteine and led to a loss of in vitro DNA binding. Hence, a vital role for Hesx1/HESX1 in forebrain and pituitary development in mice and humans is suggested.

Isolation of developmentally regulated genes by differential display screening of cDNA libraries.

mRNA differential display RT-PCR has been extensively used for the isolation of genes differentially expressed between RNA populations. We have assessed its utility for the identification of developmentally regulated genes in plasmid cDNA libraries derived from individual tissues dissected from early mouse embryos. Using plasmid Southern blot hybridisation as a secondary screen, we are able to identify such genes and show by whole-mount in situ hybridisation that their expression pattern is that expected from the differential display profile.

Anterior patterning in mouse.

The anteroposterior axis of the vertebrate embryo becomes explicit during gastrulation, the process that converts a relatively featureless embryonic precursor population into new tissues assembled into a recognisable body pattern. Vertebrate embryos arrive at gastrulation in very different states in terms of their size, cell number and reliance on factors inherited from the unfertilized egg. However, they emerge from gastrulation looking very similar, and there is now extensive molecular genetic evidence to indicate that the bare essentials of the gastrulation process have been well conserved during evolution. Here, we review recent findings in the mouse that suggest that anterior identity is, in fact, established before gastrulation starts. They suggest that it is first manifest in extraembryonic tissue and that this tissue is essential for the embryo to develop normal anterior structures, such as the forebrain. We also argue that this precocious anterior pattern could have a counterpart in other non-mammalian vertebrates.

Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse.

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.

Renal agenesis in mice homozygous for a gene trap mutation in the gene encoding heparan sulfate 2-sulfotransferase.

Heparan sulfate proteoglycans have been implicated in the presentation of a number of secreted signaling molecules to their signal-transducing receptors. We have characterized a gene trap mutation in the gene encoding a heparan sulfate biosynthetic enzyme, heparan sulfate 2-sulfotransferase (HS2ST). Transgenic mice were generated from embryonic stem cells harboring this insertion. lacZ reporter gene activity in heterozygous embryos demonstrates that the gene is expressed differentially during embryogenesis, presumably directing dynamic changes in heparan sulfate structure. Moreover, mice homozygous for the Hs2st gene trap allele die in the neonatal period, exhibiting bilateral renal agenesis and defects of the eye and the skeleton. Analysis of kidney development in Hs2st mutants reveals that the gene is not required for two early events-ureteric bud outgrowth from the Wolffian duct and initial induction of Pax-2 expression in the metanephric mesenchyme. It is required, however, for mesenchymal condensation around the ureteric bud and initiation of branching morphogenesis. Because 2-O-sulfation has been shown to influence the functional interactions of ligands with heparan sulfate in vitro, we discuss the possibility that the Hs2st mutant phenotype is a consequence of compromised interactions between growth factors and their signal-transducing receptors. These data provide the first genetic evidence that the regulated synthesis of differentially glycosylated proteoglycans can affect morphogenesis during vertebrate development.

Msg1 and Mrg1, founding members of a gene family, show distinct patterns of gene expression during mouse embryogenesis.

Msg1 and Mrg1 are founding members of a gene family which exhibit distinct patterns of gene expression during mouse embryogenesis. Sequence analysis reveals that these genes are unlike any other gene identified to date, but they share two near-identical sequence domains. The Msg1 and Mrg1 expression profiles during early development are distinct from each other. Msg1 is predominantly expressed in nascent mesoderm, the heart tube, limb bud and sclerotome. Intriguingly, Msg1 expression is restricted, within these developing mesodermal sites, to posterior domains. Mrg1 is expressed prior to gastrulation in the anterior visceral endoderm. Expression is maintained in the endoderm once gastrulation has begun and commences in the rostralmost embryonic mesoderm which underlies the anterior visceral endoderm. Mrg1 expression persists in this rostral mesoderm as it is translocated caudalwards during the invagination of the foregut and the formation of the heart. Later Mrg1 expression predominates in the septum transversum caudal to the heart. This expression pattern suggests that the septum transversum originates from the rostralmost embryonic mesoderm which first expressed Mrg1 at the late primitive streak stage.