Ectopic expression of Msx2 in chick retinal pigmented epithelium cultures suggests a role in patterning the optic vesicle.

During the initial stages of vertebrate retinogenesis, cells of the optic vesicle adopt one of two alternate cell fates. Cells in the distal-most part of the vesicle, immediately beneath the surface ectoderm, undergo neural differentiation; cells in the proximal part differentiate into retinal pigmented epithelial cells. The mechanisms that establish this pattern of differentiation are poorly understood. In the mouse embryo, Msx2, a homeobox-containing transcription factor, is expressed in cells of the optic vesicle that will form the neural retina, whilst the developing retinal pigmented epithelium (RPE) does not express this gene. Msx2 could therefore be involved in patterning the optic vesicle into neural and pigmented domains. To explore this possibility we ectopically expressed mouse Msx2 in cultures of chick RPE cells. Compared with cultures transfected with a control construct, Msx2-transfected cultures contained fewer cells expressing the RPE marker, Mitf, and more cells expressing class III beta-tubulin, a neuronal marker. In addition a small proportion of Msx2-transfected cells acquired a neural-like morphology. These results show that Msx2 can suppress the differentiated state of RPE cells and promote their differentiation into neural cell types. We suggest that Msx2 may pattern the optic vesicle into neural and pigmented domains by affecting the balance between RPE and neural retina differentiation.

An internet-accessible database of mouse developmental anatomy based on a systematic nomenclature.

This paper reports an internet-accessible database of mouse developmental anatomy (DMDA) that currently holds a hierarchy of the names and synonyms of the tissues in the first 22 Theiler stages of development (E1-E13.5), together with other appropriate information. The purposes of the database are to provide, first, a nomenclature for analyzing normal and mutant mouse anatomy, and second a language for inputting, storing and querying gene-expression and other spatially organized data. DMDA currently contains some 6900 named and staged tissues (e.g. 360 and 1161 tissues in Theiler stage (TS) 14 (E9) and TS22 (E13.5) embryos). DMDA will be extended to include further lineage and other data when it becomes available. The database can be interactively accessed over the internet using either a Java or a non-Java WWW browser at http://genex.hgu.mrc.ac.uk/.

Antagonistic effects of FGF4 on BMP induction of apoptosis and chondrogenesis in the chick limb bud.

In an effort to define the roles of bone morphogenic proteins (BMPs) and fibroblast growth factors (FGFs) during chick limb development more closely, we have implanted beads impregnated with these growth factors into chick limb buds between stages 20 and 26. Embryos were sacrificed at the time the bone chondrocyte condensations first appear (stages 27-28). Implantation of beads containing BMPs at the earlier stages (20-22) caused apoptosis to occur, in the most severe cases leading to complete limb degeneration. Application of FGF4, either in the same, or in a different bead, prevented the BMP-induced apoptosis. We argue that the apoptosis observed on removal of the AER prior to stage 23 of development could be brought about by BMPs. The action of epithelial FGF in preventing BMP-mediated apoptosis in the mesenchyme would define a novel aspect of epithelial-mesenchymal interactions. Implanting the BMP4 beads into the core of the limb bud a day later (stages 25-26) caused intense chondrogenesis rather than apoptosis. FGF4 could again nullify this effect and by itself caused a reduction in bone size. This is the reverse of the functional relationship these growth factors have in mouse tooth specification (where it is BMP4 that inhibits the FGF8 function), and suggests that the balance between the effects of FGFs and BMPs could control the size of the chondrocyte precursor cell pool. In this way members of these two growth factor families could control the size of appendages when they are initially formed.

Computer-generated three-dimensional reconstructions of serially sectioned mouse embryos.

We have been involved with a group of computer scientists and anatomists in the development of computer-based methodologies that not only combine the advantages of scanning electron microscopy and conventional histology, but provide the additional dimension of tissue recognition. The latter is achieved by the appropriate labelling of tissues and structures by delineation or 'painting'. Individually segmented anatomically defined tissues can be highlighted in a particular colour and viewed either in isolation or in combination with other appropriately labelled tissues and organs. Tissues can be shown in any orientation either as a transparent overlay on computer-generated histological sections or as 3-D images without the histological background. An additional feature of the system is that computer graphics technology combined with 3-D glasses now also allows the viewer to see the object under analysis in stereo. This facility has been found to be particularly helpful in drawing attention to topological relationships that had not previously been readily noted. As the mouse is now the mammalian model of choice in many areas of developmental research, it is of critical importance that a basic level of skill is available in the research community in the interpretation of serially sectioned material, for example, for the rapidly expanding field in which gene expression studies play a significant role. It is equally important that there is an understanding of the dynamic changes that occur in relation to the differentiation of the various organ systems seen in these early stages of development. What we emphasise here is the additional information that it is possible to gain from the use of this tool which, in our view, could not readily have been gained from the analysis of scanning electron micrographs or by studying conventional serial histological sections of similar stages of mouse embryonic development. The methodology has been developed as part of a large project to prepare a database of mouse developmental anatomy covering all stages from fertilisation to birth in order to allow the accurate spatial mapping of gene expression and cell lineage data onto the digital Atlas of normal mouse development. In this paper we show how this digital anatomical Atlas also represents a valuable teaching aid and research tool in anatomy.

The role of the nurse executive: in the corporatization of health care.

This article describes one of the changing roles of the executive nurse. It attempts to sketch the environment, the changing politics, possible role expectations, and perceived barriers of the system role. A brief list of additional skills that may be helpful in assuring greater success is also detailed. This article is an account of "a" perspective and experience.

The role of Pax-6 in eye and nasal development.

Small eye (Sey) mice homozygous for mutations in the Pax-6 gene have no lenses and no nasal cavities. We have examined the ontogeny of eye and nasal defects in Sey/Sey embryos and have related the defects seen to the pattern of Pax-6 mRNA expression in the mouse during normal eye and nasal development. There are two principal components of the early eye, the neural ectoderm of the optic vesicle, which forms the retina, and the overlying surface ectoderm, which forms the lens and cornea. By studying these interacting tissues in normal and Sey/Sey embryos, we have identified processes for which Pax-6 is important and can thus suggest possible roles for the Pax-6 gene. Pax-6 is essential for the formation of lens placodes from surface ectoderm. In normal development, early Pax-6 mRNA expression in a broad domain of surface ectoderm is downregulated, but expression is specifically maintained in the developing lens placode. Moreover, other Pax-6-expressing tissues are frequently those that have can transdifferentiate into lens. Thus, phenotype and expression together suggest a role for Pax-6 in lens determination. At least some functions of Pax-6 can be separated from the influence of other tissues. Early Sey/Sey optic vesicles are abnormally broad and fail to constrict proximally. These defects occur prior to the time of lens placode formation and probably reflect a requirement for Pax-6 in neural ectoderm. In surface ectoderm domains, where Pax-6 expression is known to be independent of the presence of an optic vesicle, Pax-6 function is required for the maintenance of its own transcription. The mutual dependency of lens and optic vesicle development can also be studied using the Small eye mutation. Using region-specific markers we find that, in the morphologically abnormal Sey/Sey optic vesicles, aspects of normal proximo-distal specification nevertheless persist, despite the complete absence of lens. Like the lens, the nasal cavities develop from ectodermal placodes that normally express Pax-6 mRNA, fail to form in Sey/Sey mice and show Pax-6-dependent Pax-6 mRNA regulation. Analysis of patterns of programmed cell death and absence of nasal region expression from an Msx-1 transgene in Sey/Sey embryos suggest a requirement for Pax-6 in the transition from presumptive nasal ectoderm to placode, and that Msx-1, or genes regulating it, are possible targets for Pax-6.

Comparative development. Seeing eye to eye.

Comparative studies of homologous developmental genes in mouse and Drosophila are suggesting that organs in these species may have closer evolutionary relationships than was hitherto suspected.

Experimental analysis of the control of expression of the homeobox-gene Msx-1 in the developing limb and face.

Mouse mesenchyme was grafted into chick embryos to investigate the control of mesenchymal expression of Msx-1 in the developing limb and face. In situ hybridization, using species-specific probes, allows a comparison between Msx-1 expression in the graft and the host tissue. The results show that Msx-1 expression in both limb-to-limb and face-to-face grafts corresponds closely with the level of Msx-1 expression in the surrounding chick mesenchyme. Cells in grafts that end up within the host domain of Msx-1 express the gene irrespective of whether they were from normally expressing, or non-expressing, regions. Therefore Msx-1 expression in both the developing limb and the developing face appears to be position-dependent. Mesenchyme from each of the three major facial primordia behaved in the same way when grafted to the chick maxillary primordium. Reciprocal grafts between face and limb gave a different result: Msx-1 expression was activated when facial mesenchyme was grafted to the limb but not when limb mesenchyme was grafted to the face. This suggests either that there are quantitative or qualitative differences in two local signalling systems or that additional factors determine the responsiveness of the mesenchyme cells.

TRP-2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor.

We have used a probe derived from TRP-2/DT to detect migratory melanoblasts shortly after they emerge from the neural crest, as early as 10 days post coitum (dpc). TRP-2/DT expression is otherwise restricted to the presumptive pigmented retinal epithelium, the developing telencephalon and the endolymphatic duct. The pattern of steel and c-kit hybridisation in the developing brain differed from that of TRP-2. TRP-1 and tyrosinase probes also detected melanoblasts but were both expressed later in development than TRP-2. We used the TRP-2/DT probe to investigate the way that the Steel-dickie (Sld) mutation interferes with melanocyte development, and found that the membrane-bound steel growth factor which is missing in Sld/Sld mutants is necessary for the survival of melanoblasts but not for their early migration and initial differentiation.

Effects of retinoic acid excess on expression of Hox-2.9 and Krox-20 and on morphological segmentation in the hindbrain of mouse embryos.

Mouse embryos were exposed to maternally administered RA on day 8.0 or day 7 3/4 of development, i.e. at or just before the differentiation of the cranial neural plate, and before the start of segmentation. On day 9.0, the RA-treated embryos had a shorter preotic hindbrain than the controls and clear rhombomeric segmentation was absent. These morphological effects were correlated with alterations in the spatiotemporal distribution patterns of two genes, Hox-2.9 and Krox-20, which are expressed in the otic and preotic hindbrain and in specific neural crest cell populations. Hox-2.9 was expressed throughout the preotic hindbrain region, instead of being confined to rhombomere 4. Krox-20 was not expressed rostral to the Hox-2.9 domain, i.e. its normal rhombomere 3 domain was absent. The Hox-2.9/Krox-20 boundary was ill-defined, with patches of alternating expression of the two genes. In migrating neural crest cells, Hox-2.9 expression was both abnormally extensive and abnormally prolonged. Neural crest cells expressing Krox-20 remained close to the neural tube. Embryos exposed to RA on day 8 1/4 appeared to be morphologically normal. We suggest that early events leading to rhombomeric segmentation and rhombomere-specific gene expression are specifically vulnerable to raised RA levels, and may require RA levels lower than those in the region of somitic segmentation.

Isolation of two cDNAs encoding novel alpha 1-antichymotrypsin-like proteins in a murine chondrocytic cell line.

We have isolated two novel serpin-encoding sequences from EB22, a chondrocytic cell line derived from a mouse teratocarcinoma. Both sequences fall within the Spi-2 sub-family, and are related to the gene encoding human alpha 1-antichymotrypsin (ACT), a major acute-phase reactant. Considerable amplification of the Spi-2 gene family in the mouse has occurred, hindering the identification of a functional equivalent of the human gene. However, one of the sequences described here, EB22/4, exhibits several features which indicate that it may represent the physiological rodent equivalent of ACT. The sequence is expressed in the liver, as expected, and is induced several-fold during the acute-phase response. The P1 amino acid residue, which is primarily responsible for inhibitor specificity, is Met rather than the human Leu, most probably a functionally conservative substitution. Analysis of the orthologous sequence in related rodents demonstrates conservation of the predicted reactive centre-encoded specificity. The second isolated cDNA, EB22/3, encodes an unexpected Cys residue at the P1 position in the reactive centre, and represents a novel sub-class of the Spi-2 serine proteinase inhibitor (serpin)-encoding gene family. At least one of the sequences appears to be expressed at sites of skeletal deposition during the later stages of mouse foetal development, indicating a role for serpins during development.

The Msh-like homeobox genes define domains in the developing vertebrate eye.

The mouse Hox-7.1 gene has previously been shown to be related to the Drosophila Msh homeobox-containing gene. Here we report the isolation of a new member of this family which resides at an unlinked chromosomal location and has been designated Hox-8.1. Both Hox-7.1 and Hox-8.1 are expressed in the mouse embryo during the early stages of eye development in a distinct spatial and temporal relationship. Hox-8.1 is expressed in the surface ectoderm and in the optic vesicle before invagination occurs in regions corresponding to the prospective corneal epithelium and neural retina, respectively. Hox-7.1 is expressed after formation of the optic cup, marking the domain that will give rise to the ciliary body. The activity of these genes indicates that the inner layer of the optic cup is differentiated into three distinct compartments before overt cellular differentiation occurs. Our results suggest that these genes are involved in defining the region that gives rise to the inner layer of the optic cup and in patterning this tissue to define the iris, ciliary body and retina.

Position-dependent expression of two related homeobox genes in developing vertebrate limbs.

Many genes that control pattern formation in insects contain a conserved homeobox region which encodes a domain involved in DNA binding. One approach to understanding pattern formation in vertebrates is to examine the role of homeobox-containing genes in the developing limb. Two such genes, Hox-7.1 and Hox-8.1, are expressed in distal mesoderm, but not in the proximal core, of mouse forelimb (refs 3, 4, and D.R.D., manuscript in preparation). The proximodistal cartilage pattern in the chick wing is progressively determined in the distal mesoderm, which is maintained as a 'progress zone' by the overlying apical ectodermal ridge. Indeed, proximal cells are reprogrammed to form distal structures when placed in the progress zone and we therefore expect that genes involved in controlling limb pattern should be activated in such grafts. We tested this requirement for Hox-7.1 and Hox-8.1 in mouse limb mesoderm placed in chick wing buds. Our results reported here indicate that both genes are rapidly activated by a signal from the apical ectoderm. These properties, taken with the DNA-binding properties of the homeodomain, strongly suggest that Hox-7.1 and Hox-8.1 have fundamental roles in limb-pattern formation.

Expression of the chicken homeobox-containing gene GHox-8 during embryonic chick limb development.

Homeobox-containing genes are thought to be involved in the regulation of pattern formation and specification of positional information during vertebrate limb development. Because of its accessibility to microsurgical manipulation, the developing chick limb bud provides a powerful system for investigating the role of homeobox-containing genes in patterning events. We report the isolation from a chick limb bud cDNA library of a chicken homeobox-containing cDNA, which on the basis of its nucleotide and deduced amino acid sequences has been identified as the chicken cognate of mouse Hox-8. The gene encoding this chicken (Gallus) homeobox-containing cDNA has been designated GHox-8, and is a member of a family of vertebrate homeobox-containing genes that are highly similar in sequence to the Drosophila msh gene. GHox-8 encodes an mRNA transcript of about 3 kb that is expressed at several early stages of chick limb development. In situ and dot-blot hybridization analyses have revealed that GHox-8 is expressed in limb bud mesoderm in a temporal and spatial fashion consistent with its involvement in specifying anterior positional identity. At early stages (stages 20-21) of chick limb development when positional values along the anterior-posterior (A-P) axis are being specified, GHox-8 is expressed in high amounts in the anterior mesoderm of the wing bud. Little expression of the gene is detectable in the middle region of the wing bud mesoderm or in the posterior mesoderm that contains the zone of polarizing activity, which is thought to be the source of a diffusible morphogen, possibly retinoic acid, that specifies the A-P positional values of the skeletal elements of the limb according to its local concentration. Similarly, at later stages of development (stages 23-25), high expression of GHox-8 is localized to the proximal anterior periphery of the wing bud, with no detectable expression in the proximal dorsal and ventral (myogenic) regions, or in the chondrogenic central core. In the proximal posterior periphery of the wing bud at these later stages of development, expression of GHox-8 is limited to a small region in the mid-proximal periphery corresponding to the posterior necrotic zone in which programmed cell death is occurring. The possible involvement of GHox-8 in programmed cell death during limb development is also suggested by the fact that it is expressed in the necrotic interdigital mesenchyme in 6-7 day (stage 31-32) wing buds.(ABSTRACT TRUNCATED AT 400 WORDS)

Segment-specific expression of a homoeobox-containing gene in the mouse hindbrain.

The process of segmentation, in which the developing embryo is divided into repetitive structures along its antero-posterior (A-P) axis, as a means of organizing and coordinating the body plan is found in a wide range of organisms. In Drosophila, homoeotic genes are involved in all levels of segmental organization and in determining segment identity. The roles of these genes in segmentation have been found mainly by mutational studies, but also by in situ hybridization, which has shown their domains of expression. In contrast to Drosophila, however, embryonic expression of homoeobox-containing genes in vertebrate organisms has not been found to follow a segmental pattern. Vertebrate segmentation can be clearly seen in the mesodermal somites, but repetitive morphological structures in the central nervous system (neuromeres) have only recently been shown to have developmental significance. Neuromeres in the hindbrain (rhombomeres) have been defined as segmental units by their pattern of nerve formation in the developing chick and by the alternating expression of Krox-20, a gene encoding a zinc-finger DNA-binding protein, in the 9.5-day-old mouse. Here we report that a mouse homoeobox-containing gene, Hox-2.9, is expressed in a segment-specific manner in the developing mouse hindbrain. This expression is in a region which is flanked by the regions of expression of Krox-20, and is precisely contained within a single neuromere, rhombomere 4.

A new family of mouse homeo box-containing genes: molecular structure, chromosomal location, and developmental expression of Hox-7.1.

Two families of homeo box-containing genes have been identified in mammals to date, the Antennapedia- and engrailed-like homeo boxes, based on the sequence similarity to those from Drosophila. Here, we report the isolation of a homeo box-containing gene that belongs to a new family of which there are at least three related genes in the mouse genome. The homeo box of this new gene shows remarkable similarity to the Drosophila Msh homeo box that we designate as the prototype for this family. The gene maps to the proximal end of mouse chromosome 5 and does not cosegregate with any known homeo box-containing gene. We designate this locus Hox-7.1. In situ hybridizations to mouse embryos at different stages show a unique pattern of expression, as compared to other homeo box-containing genes described thus far. Hox-7.1 transcripts are detected in 9.5-day-old embryos in the neural crest, developing limb bud, and visceral arches. Later, this gene is expressed in regions of the face that are derived from neural crest and in the interdigital mesenchymal tissues in both the fore- and hindlimbs.