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.

Control of vertebrate gastrulation: inducing signals and responding genes.

Recently, genes with similar expression patterns in the early gastrulae of several different vertebrate species have been identified. The remarkable conservation of these expression patterns suggests that fundamental similarities exist within the vertebrates at remarkably early stages. It has yet to be established exactly how these genes are activated in the correct spatial patterns and what their functions might be.

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.

Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions.

Non-isotopic whole-mount in situ hybridization of mRNA is a novel technique that has greatly facilitated the precise three-dimensional localization of transcripts from genes whose expression is important during development. This methodology has recently been applied to the study of the mouse embryo and offers particular advantages over conventional procedures.

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.

Cell fate and morphogenetic movement in the late mouse primitive streak.

A prospective fate map of the late gastrulation mouse primitive streak has been charted in 8.5 dpc mouse embryos developed in culture, using the lineage marker DiI to label groups of cells. As at earlier stages, the fate of cells in the 8.5 dpc primitive streak is regionalised such that successively more caudal regions of the streak give rise to more lateral mesoderm. While most labelled cells over a 24 or 48 h culture period exit from the primitive streak, some are consistently found to remain within it. The most conspicuous resident population is present in the node. To determine when ingression of ectoderm through the streak ceases, ectoderm cells of the streak and posterior neuropore of 8.5-10.0 dpc embryos were labelled. Involution of surface cells to form mesoderm continues until closure of the posterior neuropore but is not seen thereafter.

The role of the brachyury gene in heart development and left-right specification in the mouse.

The midline has a theoretical role in the development of left-right asymmetry, and this is supported by both genetic analyses and experimental manipulation of midline structures in vertebrates. The mouse brachyury (T) gene encodes a transcription factor which is expressed in the developing notochord and is required for its development. T/T mice lack a mature notochord and have a dorsalised neural tube. We have examined the hearts of T/T mice and have found consistent morphological abnormalities, resulting in ventrally displaced ventricular loops, and a 50% incidence of inverted heart situs. Three TGF-beta related genes, lefty-1, lefty-2 and nodal, are expressed asymmetrically in mouse embryos, and are implicated in the development of situs. We find that nodal, which is normally expressed around the node and in left lateral plate mesoderm in early somite embryos, is completely absent at this stage in T/T embryos. In contrast, lefty-1 and lefty-2, which are normally expressed in the left half of prospective floorplate and left lateral plate mesoderm, respectively, are both expressed in T/T embryos only in a broad patch of ventral cells in, and just rostral to, the node region. These results implicate the node as a source of instructive signals driving expression of nodal and lefty-2 in the left lateral plate mesoderm, and being required for normal looping and situs of the heart.

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.

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.

Alterations in gene expression during mesoderm formation and axial patterning in Brachyury (T) embryos.

The mouse T (Brachyury) deletion causes defective mesoderm formation and notochord morphogenesis, and abnormalities in the caudal neural tube and somites. To investigate the effect of the wild type T gene on concurrently expressed genes, we have compared expression of a panel of such genes in homozygous T mutants with that in wild type and heterozygous T/+ control embryos. Two classes of genes were used in this study: those implicated in primitive streak or mesoderm formation, and those which are differentially expressed in regions of the neural tube and somites. Results of wholemount in situ analysis show that the mRNA levels of Evx-1, Wnt-3a and Wnt-5a decrease in T/T embryos late in gastrulation, although earlier expression patterns are similar to control embryos. In contrast, BMP-4 and Msx-1 expression patterns remain similar throughout the period studied. Pax-3 and Pax-6, which are expressed in specific dorsoventral domains of the neural tube, both have ventrally extended expression domains in caudal T/T neural tube. This is consistent with a missing ventral signal provided by the notochord. However, the expression of Msx-1 in the most dorsal domain of the neural tube is unaltered in T/T embryos. Pax-1 and Pax-3, which are expressed in the sclerotome and dermamyotome respectively, are expressed correctly in anterior T/T somites, although the Pax-3 expression domain is widened ventromedially. This extension into ventromedial somite domains is more pronounced caudally, supporting a function for the notochord in ventralizing somites.

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.

An autoradiographic analysis of tissue potency in different regions of the embryonic ectoderm during gastrulation in the mouse.

In vitro chimaeras have been produced by injecting [3H]thymidine-labelled 8th day embryonic ectoderm, derived from the anterior, distal or posterior regions of the egg cylinder, into unlabelled synchronous embryos. Injected embryos were cultured for 36 h and the distribution of donor cells was analysed autoradiographically. One series of orthotopic injections was carried out and the results indicate that the developmental fate of embryonic ectoderm in the posterior part of the embryo is to form mesoderm, both embryonic and extraembryonic. Heterotopic injections of distal and posterior embryonic ectoderm demonstrate that these tissues readily conform to the colonisation patterns characteristic of their new location. In contrast, anterior embryonic ectoderm showed some preference for definitive ectoderm differentiation following heterotopic transplantation. However, there was no evidence that the normal fate of tissue from the three regions studied could be explained by pre-existing mosaicism in the embryonic ectoderm.

Induction of a second neural axis by the mouse node.

The anterior aspect of the mouse primitive streak resembles the organizer of Xenopus and chick in terms of its developmental fate, ability to alter pattern in the chick limb bud and with respect to the repertoire of genes that its constituent cells express. However, until now there has been no direct evidence that the mouse node organizes pattern during gastrulation, nor that the exceptionally small mouse embryonic egg cylinder can be induced to form a second axis. Grafts of transgenically marked midgastrulation mouse node, or node labelled with DiI, to a posterolateral location in a host embryo of the same developmental stage results in the induction of a second neural axis and the formation of ectopic somites. The graft gives rise predominantly to notochord and endoderm tissue whereas the neurectoderm and somites are mainly of host origin. The ectopic notochord formed is derived solely from the donor node which suggests that the node can serve as a 'stem cell' source of axial mesoderm. This is corroborated by the observation that labelling in situ the population of cells on the outer surface of the mid-gastrulation node with DiI results in continuous labelling of the notochord. DiI-labelled cells are present throughout the notochord from a rostral boundary in the cranial region to its most caudal extreme and the node itself always remains labelled.

The development of 12th to 14th day foetuses following reimplantation of pre- and early-primitive-streak-stage mouse embryos.

This paper describes a technique for transplanting early postimplantation mouse embryos from their own implantation site to a decidua in another pregnant mouse. Evidence is provided that this procedure is compatible with their continued development. At a low frequency both 6th and 7th day embryos can re-establish themselves and continue apparently normal development and placentation for at least 6-8 days.

Histogenetic and neoplastic potential of different regions of the mouse embryonic egg cylinder.

The histogenetic and neoplastic potentials of defined regions of the 8th day mouse embryonic egg cylinder were examined following ectopic transfer to beneath the testis capsule. No differences in histogenetic potential were detected between anterior and posterior slices of the embryo, either when composed of all three germ layers or of embryonic ectoderm alone. Small anterior and distal fragments of embryonic ectoderm also produced similar histogenetic profiles, although posterior fragments failed to grow in this ectopic site. The histogenetic potential of anterior and distal fragments exceeded the developmental fate ascribed to these two regions in the embryo (Beddington, 1981). There was some evidence for regionalization with respect to neoplastic potential, anterior slices of the embryo giving rise to a higher incidence of embryonal carcinoma cells than posterior slices.

BMP-7 influences pattern and growth of the developing hindbrain of mouse embryos.

The expression pattern of bone morphogenetic protein-7 (BMP-7) in the hindbrain region of the headfold and early somite stage developing mouse embryo suggests a role for BMP-7 in the patterning of this part of the cranial CNS. In chick embryos it is thought that BMP-7 is one of the secreted molecules which mediates the dorsalizing influence of surface ectoderm on the neural tube, and mouse surface ectoderm has been shown to have a similar dorsalizing effect. While we confirm that BMP-7 is expressed in the surface ectoderm of mouse embryos at the appropriate time to dorsalize the neural tube, we also show that at early stages of hindbrain development BMP-7 transcripts are present in paraxial and ventral tissues, within and surrounding the hindbrain neurectoderm, and only later does expression become restricted to a dorsal domain. To determine more directly the effect that BMP-7 may have on the developing hindbrain we have grafted COS cells expressing BMP-7 into the ventrolateral mesoderm abutting the neurectoderm in order to prolong BMP-7 expression in the vicinity of ventral hindbrain. Three distinct actvities of BMP-7 are apparent. Firstly, as expected from previous work in chick, BMP-7 can promote dorsal characteristics in the neural tube. Secondly, we show that it can also attenuate the expression of sonic hedgehog (Shh) in the floorplate without affecting Shh expression in the notochord. Finally, we find that ectopic BMP-7 appears to promote growth of the neurectoderm. These findings are discussed with respect to possible timing mechanisms necessary for the coordination of hindbrain dorsoventral patterning.

The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis.

Orthotopic grafts of [3H]thymidine-labelled cells have been used to demonstrate differences in the normal fate of tissue located adjacent to and in different regions of the primitive streak of 8th day mouse embryos developing in vitro. The posterior streak produces predominantly extraembryonic mesoderm, while the middle portion gives rise to lateral mesoderm and the anterior region generates mostly paraxial mesoderm, gut and notochord. Embryonic ectoderm adjacent to the anterior part of the streak contributes mainly to paraxial mesoderm and neurectoderm. This pattern of colonization is similar to the fate map constructed in primitive-streak-stage chick embryos. Similar grafts between early-somite-stage (9th day) embryos have established that the older primitive streak continues to generate embryonic mesoderm and endoderm, but ceases to make a substantial contribution to extraembryonic mesoderm. Orthotopic grafts and specific labelling of ectodermal cells with wheat germ agglutinin conjugated to colloidal gold (WGA-Au) have been used to analyse the recruitment of cells into the paraxial mesoderm of 8th and 9th day embryos. The continuous addition of primitive-streak-derived cells to the paraxial mesoderm is confirmed and the distribution of labelled cells along the craniocaudal sequence of somites is consistent with some cell mixing occurring within the presomitic mesoderm.

Multi-lineage 'stem' cells in the mammalian embryo.

The term 'stem' cell has acquired a rather more restricted meaning in cell biology than in embryology as a result of studies on the growth kinetics of renewing tissues in mature organisms. It is normally used in an embryological context as a synonym for 'progenitor' cell. Methods of establishing the existence of multi-lineage progenitor cells in mammals are examined briefly before the occurrence and properties of such cells in both embryonic and extra-embryonic tissues of the mouse conceptus are reviewed. Various attributes of 'stem' cells that can be obtained from outgrowths of blastocysts in vitro are also discussed.

An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo.

Embryonic stem cells (ES) cells were injected into host blastocysts either in groups of 10-15 cells or as single cells in order to test their developmental potential in the developing embryo. The analysis of midgestation chimaeras, by electrophoretic separation of glucose phosphate isomerase (GPI) isozymes, showed that ES cells were capable of colonizing trophectoderm and primitive endoderm derivatives at a low frequency, as well as producing a high rate of chimaerism in tissues of the fetus and extraembryonic mesoderm.