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The Paxfamily of transcription factors plays important roles in vertebrate organogenesis. Pax-2is a critical factor in the development of the mammalian urogenital system. Pax-2is expressed in the epithelia of the ureter, the Müllerian duct, and the Wolffian duct and in the nephrogenic mesenchyme. Gene targeting in the mouse as well as natural mutations in mouse and man have demonstrated the requirement of Pax-2in the development of these structures. Little is known about the molecular mechanisms regulating Pax-2expression in the developing urogenital system. As a first step to reveal these mechanisms and to search for the elements and factors controlling Pax-2expression we have characterized regulatory sequences of the Pax-2gene in an in vivoreporter assay in the mouse. An 8.5-kb genomic region upstream of the Pax-2transcription start site directed reporter gene activity in the epithelium of the pronephric duct at 8.25 days postcoitum (dpc) and in the Wolffian duct starting from 9.0 dpc. Expression in the Wolffian duct and its derivatives, the ureter, the collecting duct system, the seminal vesicles, the vas deferens, and the epididymis, was maintained at least until 18.5 dpc. Hence, an element(s) in the 8.5-kb upstream region is sufficient to initiate and maintain Pax-2expression in the Wolffian duct and its derivatives. In order to more precisely map the Wolffian duct regulatory sequences, a deletion analysis of the 8.5-kb upstream region was performed in a transient in vivoreporter assay. A 0.4-kb subfragment was required for marker gene expression in the Wolffian duct. Misexpression of fgf8under the control of the 8.5-kb upstream region resulted in polycystic kidneys, demonstrating the general usefulness of Pax-2regulatory sequences in misexpression of foreign genes in the ureter and collecting duct system of the kidney in transgenic approaches in mice.

The gastrointestinal tract develops from the embryonic gut, which is composed of an endodermally derived epithelium surrounded by cells of mesodermal origin. Cell signaling between these two tissue layers appears to play a critical role in coordinating patterning and organogenesis of the gut and its derivatives. We have assessed the function of Sonic hedgehog and Indian hedgehog genes, which encode members of the Hedgehog family of cell signals. Both are expressed in gut endoderm, whereas target genes are expressed in discrete layers in the mesenchyme. It was unclear whether functional redundancy between the two genes would preclude a genetic analysis of the roles of Hedgehog signaling in the mouse gut. We show here that the mouse gut has both common and separate requirements for Sonic hedgehog and Indian hedgehog. Both Sonic hedgehog and Indian hedgehog mutant mice show reduced smooth muscle, gut malrotation and annular pancreas. Sonic hedgehog mutants display intestinal transformation of the stomach, duodenal stenosis (obstruction), abnormal innervation of the gut and imperforate anus. Indian hedgehog mutants show reduced epithelial stem cell proliferation and differentiation, together with features typical of Hirschsprung’s disease (aganglionic colon). These results show that Hedgehog signals are essential for organogenesis of the mammalian gastrointestinal tract and suggest that mutations in members of this signaling pathway may be involved in human gastrointestinal malformations.

Neural crest cells are multipotential stem cells that contribute extensively to vertebrate development and give rise to various cell and tissue types. Determination of the fate of mammalian neural crest has been inhibited by the lack of appropriate markers. Here, we make use of a two-component genetic system for indelibly marking the progeny of the cranial neural crest during tooth and mandible development. In the first mouse line, Cre recombinase is expressed under the control of the Wnt1 promoter as a transgene. Significantly, Wnt1 transgene expression is limited to the migrating neural crest cells that are derived from the dorsal CNS. The second mouse line, the ROSA26 conditional reporter (R26R), serves as a substrate for the Cre-mediated recombination. Using this two-component genetic system, we have systematically followed the migration and differentiation of the cranial neural crest (CNC) cells from E9.5 to 6 weeks after birth. Our results demonstrate, for the first time, that CNC cells contribute to the formation of condensed dental mesenchyme, dental papilla, odontoblasts, dentine matrix, pulp, cementum, periodontal ligaments, chondrocytes in Meckel’s cartilage, mandible, the articulating disc of temporomandibular joint and branchial arch nerve ganglia. More importantly, there is a dynamic distribution of CNC- and non-CNC-derived cells during tooth and mandibular morphogenesis. These results are a first step towards a comprehensive understanding of neural crest cell migration and differentiation during mammalian craniofacial development. Furthermore, this transgenic model also provides a new tool for cell lineage analysis and genetic manipulation of neural-crest-derived components in normal and abnormal embryogenesis.

A subpopulation of neural crest termed the cardiac neural crest is required in avian embryos to initiate reorganization of the outflow tract of the developing cardiovascular system. In mammalian embryos, it has not been previously experimentally possible to study the long-term fate of this population, although there is strong inference that a similar population exists and is perturbed in a number of genetic and teratogenic contexts. We have employed a two-component genetic system based on Cre/lox recombination to label indelibly the entire mouse neural crest population at the time of its formation, and to detect it at any time thereafter. Labeled cells are detected throughout gestation and in postnatal stages in major tissues that are known or predicted to be derived from neural crest. Labeling is highly specific and highly efficient. In the region of the heart, neural-crest-derived cells surround the pharyngeal arch arteries from the time of their formation and undergo an altered distribution coincident with the reorganization of these vessels. Labeled cells populate the aorticopulmonary septum and conotruncal cushions prior to and during overt septation of the outflow tract, and surround the thymus and thyroid as these organs form. Neural-crest-derived mesenchymal cells are abundantly distributed in midgestation (E9.5-12.5), and adult derivatives of the third, fourth and sixth pharyngeal arch arteries retain a substantial contribution of labeled cells. However, the population of neural-crest-derived cells that infiltrates the conotruncus and which surrounds the noncardiac pharyngeal organs is either overgrown or selectively eliminated as development proceeds, resulting for these tissues in a modest to marginal contribution in late fetal and postnatal life.

Indian hedgehog (Ihh) and Parathyroid Hormone-related Protein (PTHrP) play a critical role in the morphogenesis of the vertebrate skeleton. Targeted deletion of Ihh results in short-limbed dwarfism, with decreased chondrocyte proliferation and extensive hypertrophy, features shared by mutants in PTHrP and its receptor. Activation of Ihh signaling upregulates PTHrP at the articular surface and prevents chondrocyte hypertrophy in wild-type but not PTHrP null explants, suggesting that Ihh acts through PTHrP. To investigate the relationship between these factors during development of the appendicular skeleton, mice were produced with various combinations of an Ihh null mutation (Ihh–/–), a PTHrP null mutation (PTHrP–/–), and a constitutively active PTHrP/Parathyroid hormone Receptor expressed under the control of the Collagen II promoter (PTHrPR*). PTHrPR* rescues PTHrP–/– embryos, demonstrating this construct can completely compensate for PTHrP signalling. At 18.5 dpc, limb skeletons of Ihh, PTHrP compound mutants were identical to Ihh single mutants suggesting Ihh is necessary for PTHrP function. Expression of PTHrPR* in chondrocytes of Ihh–/– mice prevented premature chondrocyte hypertrophy but did not rescue either the short-limbed dwarfism or decreased chondrocyte proliferation. These experiments demonstrate that the molecular mechanism that prevents chondrocyte hypertrophy is distinct from that which drives proliferation. Ihh positively regulates PTHrP, which is sufficient to prevent chondrocyte hypertrophy and maintain a normal domain of cells competent to undergo proliferation. In contrast, Ihh is necessary for normal chondrocyte proliferation in a pathway that can not be rescued by PTHrP signaling. This identifies Ihh as a coordinator of skeletal growth and morphogenesis, and refines the role of PTHrP in mediating a subset of Ihh’s actions.

The mechanisms that regulate patterning and growth of the developing cerebral cortex remain unclear. Suggesting a role for Wnt signaling in these processes, multiple Wnt genes are expressed in selective patterns in the embryonic cortex. We have examined the role of Wnt-3a signaling at the caudomedial margin of the developing cerebral cortex, the site of hippocampal development. We show that Wnt-3a acts locally to regulate the expansion of the caudomedial cortex, from which the hippocampus develops. In mice lacking Wnt-3a, caudomedial cortical progenitor cells appear to be specified normally, but then underproliferate. By mid-gestation, the hippocampus is missing or represented by tiny populations of residual hippocampal cells. Thus, Wnt-3a signaling is crucial for the normal growth of the hippocampus. We suggest that the coordination of growth with patterning may be a general role for Wnts during vertebrate development.

We show that Schwann cell–derived Desert hedgehog (Dhh) signals the formation of the connective tissue sheath around peripheral nerves. mRNAs for dhhand its receptor patched(ptc) are expressed in Schwann cells and perineural mesenchyme, respectively. In dhh−/−mice, epineurial collagen is reduced, while the perineurium is thin and disorganized, has patchy basal lamina, and fails to express connexin 43. Perineurial tight junctions are abnormal and allow the passage of proteins and neutrophils. In nerve fibroblasts, Dhh upregulates ptcand hedgehog-interacting protein(hip). These experiments reveal a novel developmental signaling pathway between glia and mesenchymal connective tissue and demonstrate its molecular identity in peripheral nerve. They also show that Schwann cell–derived signals can act as important regulators of nerve development.

In WEXPZ-En-1 transgenic mice, Engrailed-1, a homeodomain-containing transcription factor, is ectopically expressed in the developing brain under control of the Wnt-1 enhancer. En-1 is a developmental regulatory control gene which has an essential role in the formation of the midbrain and cerebellum. Approximately 28% of WEXPZ-En-1⁺ mice develop cystic malformations of the posterior lobe of the cerebellar vermis, fourth ventricular dilatation, and postnatal hydrocephalus. These anatomic features are also found among the spectrum of posterior fossa malformations in humans. Expression characteristics of the WEXP transgene suggest that the neuropathology observed in WEXPZ-En-1⁺ mice stems from overexpression of En-1 during fetal and neonatal phases of cerebellar development. These observations raise the possibility that abnormal regulation of Engrailed genes, or targets of Engrailed, may be involved in the pathogenesis of cystic central nervous system malformations of the posterior fossa in humans. Teratology 60:22–28, 1999. © 1999 Wiley-Liss, Inc.

Sonic hedgehog (Shh) is a secreted glycoprotein expressed by the notochord and floor plate that is involved in the induction and specification of ventral phenotypes in the vertebrate neural tube. Recently, Shh has also been shown to promote the survival of cultured rat embryo ventral brain and spinal cord cells. We have examined whether Shh can promote the survival of chick embryo neurons in vivoor in vitro. In the chick, Shh is expressed in notochord, floor plate, and ventral neural tube/spinal cord at several stages at which programmed cell death (PCD) occurs. However, the administration of exogenous Shh to embryos in vivoor to motoneuron cultures at these stages failed to promote the survival of several different neuronal populations, including spinal motoneurons, spinal interneurons, sympathetic preganglionic neurons, sensory neurons, and neuronal precursor cells. Rather, at the earliest stage of PCD examined here (embryonic day 3) Shh selectively induced the death of ventral neuronal precursors and floor-plate cells, resulting in a net loss of cells in the neural tube. Altered concentrations of Shh induce aberrant phenotypes that are removed by PCD. Accordingly, normal PCD in the early neural tube may play a role in dorsal-ventral patterning.

Morphogenesis depends on the precise control of basic cellular processes such as cell proliferation and differentiation. Wnt5a may regulate these processes since it is expressed in a gradient at the caudal end of the growing embryo during gastrulation, and later in the distal-most aspect of several structures that extend from the body. A loss-of-function mutation of Wnt5a leads to an inability to extend the A-P axis due to a progressive reduction in the size of caudal structures. In the limbs, truncation of the proximal skeleton and absence of distal digits correlates with reduced proliferation of putative progenitor cells within the progress zone. However, expression of progress zone markers, and several genes implicated in distal outgrowth and patterning including Distalless, Hoxd and Fgf family members was not altered. Taken together with the outgrowth defects observed in the developing face, ears and genitals, our data indicates that Wnt5a regulates a pathway common to many structures whose development requires extension from the primary body axis. The reduced number of proliferating cells in both the progress zone and the primitive streak mesoderm suggests that one function of Wnt5a is to regulate the proliferation of progenitor cells.

Fibroblast growth factor (FGF) signaling is required prior to gastrulation in the mouse embryo. To test for the spatial and temporal requirements of FGF signaling, a dominant negative FGF receptor (dnFGFR) was used to make transgenic mouse embryos. In mosaic embryos, cell division ceased at the fifth cell division in all cells that expressed the mutant receptor, but cell death did not increase. After the fifth cell division, the progeny of unaltered cells and cells expressing lacZ continued to accumulate at the same rate, suggesting that the FGF requirement is cell autonomous. In mosaic embryos, lacZ, but not dnFGFR expression was detected in mitotic trophoblasts adjacent to the ICM. Conversely, dnFGFR-expressing extraembryonic ectoderm cells were detected at the abembryonic pole in postmitotic cells. In blastocysts expressing the dnFGFR in all cells, the morphology appeared normal and inner cell masses (ICMs) formed, but resultant embryos had only one-third the number of cells as control embryos. In these blastocysts, cell division had also ceased at the fifth cell division, but cavitation, a concurrent morphogenetic event, initiated and progressed normally. To test for the continuing requirement of FGF, FGFR-3 was overexpressed in all cells and resulted in an increase in cell numbers after the fifth cell cycle. In a model for postimplantation development, addition of FGF-4 to blastocyst outgrowths increased the number of extraembryonic ectoderm cells, suggesting a continuing role for FGF. Thus, FGF signaling induces the cell division of embryonic and extraembryonic cells in the preimplantation mouse embryo starting at the fifth cell division. The signal requirement for FGF is cell autonomous, but is not required to prevent cell death. This provides the first evidence for the necessity of a growth factor before implantation.

Abstract: In Victoria, Australia, both dryland salinity and salinity in irrigation regions are serious agricultural problems. One option to control the latter is to pump groundwater to maintain it below the surface. However, this leaves a saline wastewater for disposal, probably into local streams or wetlands. This review of the salt sensitivity of the biota of Australian streams and wetlands gives information of interest to those responsible for developing controls on these discharges. The review addresses the lethal and sub-lethal effects of salinity on microbes (mainly bacteria), macrophytes and micro-algae, riparian vegetation, invertebrates, fish, amphibians, reptiles, mammals, and birds. Data suggest that direct adverse biological effects are likely to occur in Australian river, stream and wetland ecosystems if salinity is increased to around 1 000 mg L−1. The review highlights a general lack of data on the sensitivity of freshwater plants and animals to salinity increases.

Several lines of evidence suggest that Wntgenes play a critical role in regulating development of the vertebrate embryo. To address the role that this family may play in the development of the chicken central nervous system (CNS), we have used a PCR based strategy to clone partial sequences for Wntgenes. At least six different Wntgenes are expressed in the developing CNS of the chick embryo. The domains of expression overlap either partially or completely, and are expressed in spatial domains that prefigure morphological subunits of the embryonic neural tube. Wnt-1and Wnt-4are first expressed in the open neural plate in the region of the presumptive mesencephalon. Wnt-3aexpression is first observed in the rhombencephalic regions of the open neural plate. After neural tube closure, when the embryonic subdivisions of the neural tube became apparent, Wnt-1, Wnt-3aand Wnt-4are all broadly expressed in partially overlapping domains in the mesencephalon and caudal diencephalon, as well as in the rhombencephalon and spinal cord. The mesencephalic expression patterns are subsequently modified such that Wnt-1and Wnt-4are expressed in a characteristic ring just rostral to the isthmus, at the mesencephalic/metencephalic junction; and Wnt-1and Wnt-3aexpression become restricted to the dorsal midline. Wnt-1, Wnt-3a, Wnt-4, Wnt-5aand Wnt-8bare expressed in one or two caudal subdivisions of the developing diencephalon, the synencephalon and posterior parencephalon, but do not extend ventral to the zona limitans interparencephalica. In contrast, Wnt-7bis expressed in the anterior parencephalon. Both Wnt-7band Wnt-8bare expressed in telencephalic portions of the secondary prosencephalon. The timing and spatial distribution of Wnt-gene expression in the chick embryo further support the general hypothesis that Wntgenes play key roles in patterning the developing vertebrate nervous system.

In the Drosophilaembryo, activation of winglessand engrailedin the parasegment requires paired, a member of the Paxfamily of transcription factors. We have explored the possible conservation of this regulatory hierarchy in the developing mouse brain. We find that Pax-2is expressed prior to somite formation in the presumptive mid/hindbrain region. Shortly thereafter, Wnt-1(the winglessorthologue) and Engrailed-1are expressed in overlapping regions within the Pax-2domain. Pax-5expression commences later, at the 3-somite stage. Thus, the spatial and temporal expression of Pax-2is consistent with a possible regulatory role in the activation of Wnt-1and En-1.

The secreted signaling molecule GDNF is expressed in the metanephric mesenchyme and has recently been implicated as a factor necessary for development of the metanephric kidney. We have examined the effects of GDNF on mouse kidney explants. We show that GDNF increases cell proliferation in ureter tips. There is an increase in the number of ureter tips and expansion and fusion of adjacent tips and some tips appear to grow toward the source of GDNF. These events are accompanied by transcriptional upregulation of several genes localized to the tips, including its own receptor,c-ret,the transcription factorSox9,and the signalWnt-11.These results support a model in which GDNF supplied by the mesenchyme regulates growth and branching in the metanephric kidney through the local regulation of ureter tip-specific factors.

Studies on the mouseSplotch(Sp) mutation, a deletion in the transcription factor Pax-3, have revealed that Pax-3 is essential for normal development of the neural crest. We have investigated the defect in neural crest development using a Wnt-l::LacZ reporter construct to mark neural crest cells. Staining embryos for β-galactosidase activity at different developmental stages revealed a severe reduction in the number of neural crest cells which emigrated from the neural tube at the vagal and rostral trunk levels. At the caudal thoracic, lumbar, and sacral levels there was a complete loss of neural crest cell emigration. In contrast to previous work in culture, we saw no evidence for any delay in the onset of neural crest cell migration at anterior levels. Pax-3 is expressed in the dorsal neural tube, where the neural crest cells originate, in migrating neural crest cells, and in somitic cells along the migratory pathway. Hence, it is not clear which aspect of the Pax-3 expression accounts for the observed phenotype. We addressed this problem by transplanting neural tissue between mouse and chick embryos. Our studies indicate that the defect in theSplotchmutation is not intrinsic to the neural crest cells themselves, but appears to reflect inappropriate cell interactions either within the neural tube or between the neural tube and the somite.

Development of the metanephric kidney requires the concerted interaction of two tissues, the epithelium of the ureteric duct and the metanephric mesenchyme. Signals from the ureter induce the metanephric mesenchyme to condense and proliferate around the ureter tip, reciprocal signals from the mesenchyme induce the ureter tip to grow and to branch. Wnt genes encode secreted glycoproteins, which are candidate mediators of these signaling events. We have identified three Wnt genes with specific, non-over-lapping expression patterns in the metanephric kidney, Wnt-4, Wnt-7b and Wnt-11. Wnt-4 is expressed in the condensing mesenchyme and the comma- and S-shaped bodies. Wnt-7b is expressed in the collecting duct epithelium from 13.5 days post coitum onward. Wnt-11 is first expressed in the nephric duct adjacent to the metanephric blastema prior to the outgrowth of the ureteric bud. Wnt-11 expression in Danforth’s short-tail mice suggests that signaling from the mesenchyme may regulate Wnt-11 activation. During metanephric development, Wnt-11 expression is confined to the tips of the branching ureter. Maintenance of this expression is independent of Wnt-4 signaling and mature mesenchymal elements in the kidney. Moreover, Wnt-11 expression is maintained in recombinants between ureter and lung mesenchyme suggesting that branching morphogenesis and maintenance of Wnt-11 expression are independent of metanephric mesenchyme-specific factors. Interference with proteoglycan synthesis leads to loss of Wnt-11 expression in the ureter tip. We suggest that Wnt-11 acts as an autocrine factor within the ureter epithelium and that its expression is regulated at least in part by proteoglycans.

cDNA clones encoding the murine int-1 -related protein (m-irp) were isolated from an 8.5-day mouse embryo library, m-irp and its human counterpart, h-irp, share extensive nucleotide homology in coding (92 %) and 3’ untranslated (69 %) regions. At the amino acid level, m-irp and h-irp share 97 % of amino acids including all 24 cysteine residues, which are highly conserved among members of the int-1 family. However, in contrast to h-irp and int-1, the predicted m-irp protein sequence did not contain a signal peptide sequence. Analysis of polymerase chain reaction, amplified cDNA, and genomic sequences strongly suggests that a single-base substitution has created a new S’ splice site 17 bp 5’ of a highly conserved splice site. Splicing at this new site generates a mRNA-encoding an amino-terminal truncated protein. Splicing at the conserved splice site generates a mRNA species encoding a protein with a signal peptide sequence similar to h-irp. Close linkage between m-irp and the met oncogene maps m-irp sequences to proximal mouse chromosome 6. Adult and fetal expression of m-irp was examined by RNA blot analysis. Adult expression of m-irp is restricted to lungs and heart, and fetal expression, to placental tissue and to all stages of fetal development examined. In situ hybridization localized early fetal m-irp expression to the pericardium of the heart, to the umbilicus and associated allantoic mesoderm, and to the ventral lateral mesenchyme tissue surrounding the umbilical vein in the fetus. These results suggest a role for m-irp in the development of fetal allantoic communication.

Pax1 expression in vertebrate limb buds is confined to cells in a discrete anterior proximal domain (Timmons et al. [1994] Development 120:2773–2785; Ebensperger et al. [1995] Anat. Embryol. 191:297–310). In dorsoventral patterning of Drosophila, expression of pox meso, an insect gene with high sequence similarity to Pax1, is repressed by decapentaplegic (dpp) in dorsal mesoderm and, thus, is restricted to a discrete ventral domain (Staehling-Hampton et al. [1994] Nature 372:783–786). In the chick wing, cells expressing a vertebrate homolog of dpp, bone morphogenetic protein 4 (Bmp4), abut the Pax1 domain, suggesting a similar relationship between homologous genes in both vertebrates and invertebrates. Here, we show that two BMPs (BMP4, and BMP2, also highly related to dpp) can repress Pax1 in the developing chick wing. Chick wing bud cells expressing Pax1 give rise to the shoulder girdle. Cells in an equivalent position in the mouse forelimb also express Pax1, and Pax1 mutant mice display shoulder girdle defects. Similarly in chick embryos, girdle defects are produced by treatments with signalling molecules that lead to expression of BMPs, which subsequently reduce Pax1 expression in the limb bud. Recently, BMP4 has been shown to inhibit Pax1 expression in the developing trunk (Monsoro-Burq et al. [1996] Development 122:3607–3616) and Pax9 expression in developing teeth (Neubüser et al. [1997] Cell 90:247–255). Thus, a property of BMPs appears to be to regulate pox meso homologs negatively and, thus, limit their expression domains. Dev. Dyn. 1998;213:199–206. © 1998 Wiley-Liss, Inc.

Development of the mammalian kidney is initiated by ingrowth of the ureteric bud into the metanephric blastema. In response to signal(s) from the ureter, mesenchymal cells condense, aggregate into pretubular clusters, and undergo epithelialisation to form simple epithelial tubules. Subsequent morphogenesis and differentiation of the tubular epithelium lead to the establishment of a functional nephron. Here we demonstrate that Wnt-4, a secreted glycoprotein which is required for tubule formation, is sufficient to trigger tubulogenesis in isolated metanephric mesenchyme, whereas Wnt-11 which is expressed in the tip of the growing ureter is not. Wnt-4 signaling depends on cell contact and sulphated glycosaminoglycans and is only required for triggering tubulogenesis but not for later events. The Wnt-4 signal can be replaced by other members of the Wnt gene family including Wnt-1, Wnt-3a, Wnt-7a and Wnt-7b. Further, dorsal spinal cord, which has been thought to mimic ureteric signaling in tubule induction induces Wnt-4 mutant as well as wild-type mesenchyme suggesting that spinal cord derived signal(s) most likely act by mimicking the normal mesenchymal action of Wnt-4. These results lend additional support to the notion that Wnt-4 is a key auto-regulator of the mesenchymal to epithelial transformation that underpins nephrogenesis adding another level of complexity in the hierarchy of molecular events mediating tubulogenesis.

Recent results have indicated that cAMP-dependent protein kinase (PKA) acts as a negative regulator of Hedgehog signaling in target cells of the vertebrate embryo. Consequently, suppression of PKA activity is sufficient to mimic the effect of receiving a Hedgehog signal. We have explored whether PKA-inhibiting Gi-proteins (GiPs) may also be involved in the regulation of Hedgehog signaling. Zebrafish embryos were injected with RNA encoding pertussis toxin (Ptx), a specific inhibitor of GiPs. These embryos developed phenotypic traits opposite to embryos expressing a dominant negative form of the PKA regulatory subunit (dnPKA), including a fusion of the eyes, a lack of ventral specification in the forebrain, and an expansion of the sclerotome at the expense of adaxial fates in the posterior somites. These effects can be partially rescued by coexpression of dnPKA, but not by coexpression of Indian Hedgehog, suggesting that GiPs act upstream of PKA and downstream of Hedgehogs. Other Hedgehog- and PKA-dependent processes, sclerotomal specification and adaxial specification in the first five somites, are not negatively affected by Ptx. Thus, GiPs may be involved in Hedgehog signaling in some, but not all target cells.

Epithelial–mesenchymal interactions govern the development of epidermal organs such as teeth. During the early stages of tooth development, a local ectodermal thickening which expresses several signaling molecules appears. It is believed that these in turn signal to the underlying mesenchyme triggering mesenchymal condensation and tooth development. For example, epithelially expressed Bmp4 inducesMsx1andLef1as well as itself in the underlying mesenchyme. In this paper we have investigated the role of four epithelial signaling molecules, Bmp2, Shh, Wnt10a, and Wnt10b, in the early inductive cascades that govern tooth development. We show that all four genes are specifically expressed in the epithelium between E11.0 and E12.0 when tooth morphogenesis is first apparent. Although Shh, Bmp2, and Wnt10b have similar, if not identical, expression patterns, each signal has a distinct molecular action on the jaw mesenchyme. Whereas Shh and Wnt10b can induce general Hedgehog and Wnt targets,PtcandGlifor Shh andLef1for Wnt10b, only Bmp2 is able to induce tooth-specific expression ofMsx1. Thus, there are distinct targets for all three pathways. Interestingly, both Bmp and Wnt signaling activateLef1, making it a candidate for integrating the two distinct signaling pathways.

The Notch gene of Drosophila encodes a large transmembrane protein involved in cell-cell interactions and cell fate decisions in the Drosophila embryo. To determine if a gene homologous to Drosophila Notch plays a role in early mouse development, we screened a mouse embryo cDNA library with probes from the Xenopus Notch homolog, Xotch. A partial cDNA clone encoding the mouse Notch homolog, which we have termed Motch, was used to analyze expression of the Motch gene. Motch transcripts were detected in a wide variety of adult tissues, which included derivatives of all three germ layers. Differentiation of P19 embryonal carcinoma cells into neuronal cell types resulted in increased expression of Motch RNA. In the postimplantation mouse embryo Motch transcripts were first detected in mesoderm at 7.5 days post coitum (dpc). By 8.5 dpc, transcript levels were highest in presomitic mesoderm, mesenchyme and endothelial cells, while much lower levels were detected in neuroepithelium. In contrast, at 9.5 dpc, neuroepithelium was a major site of Motch expression. Transcripts were also abundant in cell types derived from neural crest. These data suggest that the Motch gene plays multiple roles in patterning and differentiation of the early postimplantation mouse embryo.

Patterning of the vertebrate limb along the anterior-posterior axis is controlled by the zone of polarizing activity (ZPA) located at the posterior limb margin. One of the vertebrate Hh family members, Shh, has been shown to be able to mediate the function of the ZPA. Several naturally occurring mouse mutations with the phenotype of preaxial polydactyly exhibit ectopic Shh expression at the anterior limb margin. In this study, we report the molecular characterization of a spontaneous mouse mutation, Doublefoot (Dbf). Dbf is a dominant mutation which maps to chromosome 1. Heterozygous and homozygous embryos display a severe polydactyly with 6 to 8 digits on each limb. We show here that Shh is expressed normally in Dbf mutants. In contrast, a second Hh family member, Indian hedgehog (Ihh) which maps close to Dbf, is ectopically expressed in the distal limb bud. Ectopic Ihh expression in the distal and anterior limb bud results in the ectopic activation of several genes associated with anterior-posterior and proximal-distal patterning (Fgf4, Hoxd13, Bmp2). In addition, specific components in the Hedgehog pathway are either ectopically activated (Ptc, Ptc-2, Gli1) or repressed (Gli2). We propose that misexpression of Ihh, and not a novel Smoothened ligand as recently suggested (Hayes et al., 1998), is responsible for the Dbf phenotype. We consider that Ihh has a similar activity to Shh when expressed in the early Shh-responsive limb bud. To determine whether Dbf maps to the Ihh locus, which is also on chromosome 1, we performed an interspecific backcross. These results demonstrate that Dbf and Ihh are genetically separated by approximately 1.3 centimorgans, suggesting that Dbf mutation may cause an exceptionally long-range disruption of Ihh regulation. Although this leads to ectopic activation of Ihh, normal expression of Ihh in the cartilaginous elements is retained.

The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at early somite stages. Wnt-1 encodes a signal that is expressed in the developing midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located downstream of the Wnt-1 coding sequence which is necessary and sufficient for Wnt-1 expression in vivo. Using a transgenic mouse reporter assay, we have now identified a 110 base pair regulatory sequence within the 5.5 kilobase enhancer, which is sufficient for expression of a lacZ reporter in the approximate Wnt-1 pattern at neural plate stages. Multimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1−/− embryos. The possibility that this region represents an evolutionarily conserved regulatory module is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes). These sequences are capable of appropriate temporal and spatial activation of a reporter gene in the embryonic mouse midbrain; although, later aspects of the Wnt-1 expression pattern are absent. Genetic evidence has implicated Pax transcription factors in the regulation of Wnt-1. Although Pax-2 binds to the 110 base pair murine regulatory element in vitro, the location of the binding sites could not be precisely established and mutation of two putative low affinity sites did not abolish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unlikely that Pax proteins regulate Wnt-1 by direct interactions with this cis-acting regulatory region. Our analysis of the 110 base pair minimal regulatory element suggests that Wnt-1 regulation is complex, involving different regulatory interactions for activation and the later maintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction.

Mutation and expression studies have implicated the Wnt gene family in early developmental decision making in vertebrates and flies. In a detailed comparative analysis, we have used in situ hybridization of 8.0- to 9.5-day mouse embryos to characterize expression of all ten published Wnt genes in the central nervous system (CNS) and limb buds. Seven of the family members show restricted expression patterns in the brain. At least three genes (Wnt-3, Wnt-3a, and Wnt-7b) exhibit sharp bound-aries of expression in the forebrain that may predict sub-divisions of the region later in development. In the spinal cord, Wnt-1, Wnt-3, and Wnt-3a are expressed dorsally, Wnt-5a, Wnt-7a, and Wnt-7b more ventrally, and Wnt-4 both dorsally and in the floor plate. In the forelimb primordia, Wnt-3, Wnt-4, Wnt-6 and Wnt-7b are expressed fairly uniformly throughout the limb ectoderm. Wnt-5a RNA is distributed in a proximal to distal gradient through the limb mesenchyme and ectoderm. Along the limb’s dorsal-ventral axis, Wnt-5a is expressed in the ventral ectoderm and Wnt-7a in the dorsal ectoderm. We discuss the significance of these patterns of restricted and partially overlapping domains of expression with respect to the putative function of Wnt signalling in early CNS and limb development.

The analysis of mutant alleles at the Wnt-1 locus has demonstrated that Wnt-1-mediated cell signalling plays a critical role in development of distinct regions of the embryonic central nervous system (CNS). To determine how these signals participate in the formation of the CNS, we have ectopically expressed this factor in the spinal cord under the control of the Hoxb-4 Region A enhancer. Ectopic Wnt-1 expression causes a dramatic increase in the number of cells undergoing mitosis in the ventricular region and a concomitant ventricular expansion. Although this leads to consistent changes in the relative proportions of dorsal and ventral regions, Wnt-1 does not appear to act as a primary patterning signal. Rather, our experiments indicate that Wnt-1 can act as a mitogen in the developing CNS.

The pattern of protein synthesis in 8-, 9- and 10-day post coitum (p.c.) mouse embryos was examined by 2-D gel electrophoresis of [35S]methionine-labelled proteins. Of the 600-800 polypeptides detected only one, a 14×103Mr (14 K) protein, was found to accumulate over this period. To isolate cDNA clones that potentially encode this protein, 32P-labelled cDNA was synthesized from 9 and 10 days p.c. embryo poly(A)+RNA, and used for the differential screening of an 8·5-day p.c. mouse embryo cDNA library cloned in λgt10. Six clones that hybridized strongly to the 10-day probe were purified and their inserts subcloned into plasmid vectors. Cross hybridization and restriction mapping of these inserts indicate that they fall into four distinct groups. Each of these hybridize with transcripts of approximately 600 nucleotides, which accumulate in the embryo from 9 to 10 days p.c. Expression was barely detectable in adult tissues and restricted to liver and spleen. Expression of one of these clones, 10.1, was examined by in situ hybridization of 35S-labelled RNA probes to 8·5-12·5 day p.c. embryo sections. Strong hybridization was observed in yolk sac blood islands, fetal liver and embryonic erythrocytes, suggesting that 10.1A encodes an erythrocyte-specific protein. DNA sequence analysis indicates that the four classes of cDNA were derived from transcripts of the α 1, ζ, βh1 and ϵ globin genes. Labelling of 10-day p.c. erythrocyte proteins with [35S]methionine, followed by 2-D gel electrophoresis, clearly demonstrates that the most abundant polypeptide migrates to the same position as the 14 K protein which accumulates from 8 to 10 days p.c. Thus the only abundant transcripts and corresponding proteins that change over a period of profound morphogenetic change correspond to globins of the newly established blood system.

Sonic hedgehog (Shh) encodes a signal that is implicated in both short-and long-range interactions that pattern the vertebrate central nervous system (CNS), somite and limb. Studies in vitro indicate that Shh protein undergoes an internal cleavage to generate two secreted peptides. We have investigated the distribution of Shh peptides with respect to these patterning events using peptide-specific antibodies. Immunostaining of chick and mouse embryos indicates that Shh peptides are expressed in the notochord, floor plate and posterior mesenchyme of the limb at the appropriate times for their postulated patterning functions. The amino peptide that is implicated in intercellular signaling is secreted but remains tightly associated with expressing cells. The distribution of peptides in the ventral CNS is polarized with the highest levels of protein accumulating towards the luminal surface. Interestingly, Shh expression extends beyond the floor plate, into ventrolateral regions from which some motor neuron precursors are emerging. In the limb bud, peptides are restricted to a small region of posterior-distal mesenchyme in close association with the apical ectodermal ridge; a region that extends 50-75 μm along the anterior-posterior axis. Temporal expression of Shh peptides is consistent with induction of sclerotome in somites and floor plate and motor neurons in the CNS, as well as the regulation of anterior-posterior polarity in the limb. However, we can find no direct evidence for long-range diffusion of the 19×103Mr peptide which is thought to mediate both short-and long-range cell interactions. Thus, either long-range signaling is mediated indirectly by the activation of other signals, or alternatively the low levels of diffusing peptide are undetectable using available techniques.

Dishevelled (Dsh/Dvl) proteins are known to mediate Wnt signaling by up-regulating β-catenin levels and stimulating T cell factor (TCF)/LEF-1-dependent transcription. We have identified a new Dvl-mediated signaling pathway in that mouse Dvl proteins, when expressed in COS-7 cells, stimulate c-Jun-dependent transcription activity and the kinase activity of the c-Jun N-terminal kinase (JNK). The DEP domain of Dvl1 is essential for JNK activation. By contrast, all three conserved domains of Dvl, including DIX, PDZ, and DEP, are required for up-regulation of β-catenin and for stimulation of LEF-1-mediated transcription in mammalian cells. Thus, Dvl can lead to two different signaling pathways. Furthermore, the small G proteins of Cdc42 or Rac1, which are involved in JNK activation by many stimuli, do not appear to play a major role in Dvl-mediated JNK activation, because the dominant negative mutants of Cdc42 and Rac1 could not inhibit Dvl-induced JNK activation. This suggests that Dvl may activate JNK via novel pathways.

Hedgehoggenes in Drosophila and vertebrates control patterning of a number of different structures during embryogenesis. They code for secreted signaling proteins that are cleaved into an active aminopeptide and a carboxypeptide. The aminopeptide can mediate local and long range events and can act as a morphogen, inducing differentiation of distinct cell types in a concentration-dependent manner. We demonstrate here that the expression ofIndian hedgehogmRNA and protein is upregulated dramatically as F9 cells differentiate in response to retinoic acid, into either parietal endoderm or embryoid bodies, containing an outer visceral endoderm layer. The ES cell line D3 forms embryoid bodies in suspension culture without addition of retinoic acid and also upregulatesIndian hedgehogexpression. RT-PCR analysis of blastocyst outgrowth cultures demonstrates that whereas little or noIndian hedgehogmessage is present in blastocysts, significant levels appear upon subsequent days of culture, coincident with the emergence of parietal endoderm cells.In situhybridization analysis forIndian hedgehogmRNA expression demonstrates the presence of elevated levels of message in the outer visceral endoderm cells relative to the core cells in mature embryoid bodies and in the visceral endoderm of Day 6.5 embryos. Whole-mountin situhybridization analysis of Day 7.5 and 8.5 embryos indicates thatIndian hedgehogexpression is highest in the visceral yolk sac at this stage. F9 cell lines expressing a full lengthIndian hedgehogcDNA express a number of characteristics of differentiated cells, in the absence of retinoic acid. Taken together, these data suggest thatIndian hedgehogis involved in mediating differentiation of extraembryonic endoderm during early mouse embryogenesis.

We have investigated developmental expression of the gene Egr-1, which encodes a protein containing three zinc fingers. Egr-1 like c-fos is a serum inducible, early response gene, which is co-induced with c-fos in a variety of quite different situations. A single 3.7-kb RNA was detected throughout fetal mouse development, which increased in absolute levels in total fetal RNA from 9.5 to 12.5 days post coitum (p.c.). In situ hybridization to 14.5- and 17.5-day p.c. fetal tissues demonstrated Egr-1 accumulation at several specific sites. These included mesenchymal components of the developing tooth germs and salivary and nasal glands; an ectodermally derived component of the whisker pad and developing muscle, cartilage, and bone. Expression of Egr-1 in cartilage and bone showed a strikingly similar expression to previously published reports of c-fos in these tissues. High levels of Egr-1 RNA was observed at the perichondrial interface of opposing cartilaginous elements and in interstitial cells that lie in between. Bone expression was observed in membranous bone of the head, alveolar bone around the tooth germs, and at periosteal and endochondral ossification sites in the limb bones. Our data support the idea that Egr-1 and c-fos may be coregulated in vivo and together may regulate normal development of the skeleton.

The int-1 gene was originally identified as a locus activated by mouse mammary tumor virus insertion. Cloning and sequencing of the mouse gene indicates that int-1 encodes a 41K, 370 amino acid, cysteine-rich protein with a potential hydrophobic signal peptide sequence. Expression studies clearly indicate that int-1 enters the secretory pathway and is probably secreted, although definitive evidence is lacking. Drosophila int-1 encodes the wingless gene, wingless, a segment-polarity gene, is required for the establishment of normal pattern in each segment. Genetic studies indicate that the wingless protein is probably secreted since it is required for the maintenance of stable gene expression in neighboring cells. int-1 is also expressed during early neural stages of frog and mouse development. In the mouse, where expression is well characterized, int-1 RNA is restricted to the dorsal midline of the neural tube. By analogy with Drosophila, int-1 may operate to specify position within this structure. To test this idea, we have interfered with normal int-1 expression by injection of int-1 RNA into frog embryos. This results in a striking and specific aberration, bifurcation of the anterior neural tube. Thus, it seems possible that in vertebrates int-1 is able to influence patterning events.