Noggin mRNA Research Articles

Molecular signaling in intervertebral disk development

The purpose of this investigation is to identify and study the expression pattern of pertinent molecular factors involved in the differentiation of the intervertebral disk (IVD). It is likely that hedgehog genes and the BMP inhibitors are key factors involved in spinal joint formation. Radioactive in situ hybridization with mRNA probes for pax-1, SHH, IHH and Noggin gene was performed on mouse embryo and adult tissue. Immunohistochemistry was performed to localize hedgehog receptor, “patched” (ptc). From 14.5 dpc until birth pax-1 mRNA was expressed in the developing anulus fibrosus (AF). During the same developmental period Noggin mRNA is highly expressed throughout the spine, in the developing AF, while ptc protein and SHH mRNA were expressed in the developing nucleus pulposus (NP). IHH mRNA was expressed by condensing chondrocytes of the vertebral bodies and later becomes confined to the vertebral endplate. We show for the first time that pax-1 is expressed in the adult intervertebral disk. Ptc expression in the NP is an indicator of hedgehog protein signaling in the developing IVD. The expression pattern of the BMP inhibitor Noggin appears to be important for the normal formation of the IVD and may prove to play a role in its segmental pattern formation.

Analysis of spatial and temporal expression patterns of bone morphogenetic protein family members in the rat uterus over the estrous cycle.

Recent studies have demonstrated that bone morphogenetic proteins (BMPs) play fundamental roles in female fertility. This is particularly evident in terms of the ovary. One major question that is just beginning to be addressed is the role of BMPs in the non-pregnant uterus. To help fill this gap, we used in situ hybridization to investigate the expression of BMP family members in the rat uterus over the estrous cycle. We found that the endometrial/uterine cycle is accompanied by the expression of several components of the BMP pathway - including ligands, receptors and antagonists. The mRNAs encoding BMP receptors are expressed in the epithelial (BMP-RIA, -RIB and -RII), periluminal stroma (BMP-RIA and -RII) and smooth muscle cells (BMP-RIA and -RII). The expression of all three receptors showed clear cyclic variations. The mRNAs encoding BMP ligands were highly expressed in the periluminal stroma (BMP-2 and -7) and glandular epithelium (BMP-7). The expression of BMP-2, but not BMP-7, was cyclical. Notably, the periluminal stroma expressed noggin mRNA. In the blood vascular system, BMP-4, -6 and -RII mRNAs were expressed in myometrial endothelial cells. Interestingly, follistatin, noggin, and BMP-4, -6 and -7 mRNAs were expressed in eosinophilic leukocytes, suggesting unexpected roles for eosinophil-derived BMPs in uterine function. We conclude that BMP ligands, receptors and antagonists are expressed in spatially and temporally restricted patterns that are consistent with a physiological role for these regulatory molecules in promoting uterine cellular processes including cell proliferation, differentiation and apoptosis during the cycle.

A combined in situ hybridization and RT-PCR method to detect spatial and temporal patterns of Noggin gene expression in embryonic and postnatal rat hippocampus

Recent studies indicate that Noggin not only plays an important role in the early development of the nervous system, but may also plays a role in postnatal central nervous system (CNS) development. In this study, we examined the relative levels and localization of Noggin mRNA in the hippocampus of rats of different developmental stages with reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. RT-PCR showed that the peak level of expression of Noggin mRNA was observed at embryonic day 13 (E13), subsequently gradually declined at 1–3 months (P1-3M) postnatal, and was detected only at a low level at P18M. In situ hybridization revealed that at embryonic stages, Noggin mRNA was localized throughout all hippocampal regions, whereas at early postnatal ages, Noggin mRNA was primarily localized in the anterior subiculum. At later postnatal ages, Noggin mRNA expression was obviously observed in the dentate gyrus and in the CA1-CA3 pyramidal cell layers. Taken together, our results demonstrate that Noggin is expressed in embryonic and postnatal hippocampus, and that the temporal and spatial patterns of its expression is developmentally regulated.

Spatial and temporal patterns of expression of Noggin and BMP4 in embryonic and postnatal rat hippocampus

Recent studies indicate that bone morphogenetic protein-4 (BMP4) and Noggin not only play an important role in the early development of the nervous system, but may also play a role in postnatal central nervous system (CNS) development. In this study, we examined the relative levels and localization of Noggin and BMP4 mRNA in the hippocampus of rats of different developmental stages with reverse transcriptase–polymerase chain reaction (RT–PCR) and in situ hybridization (ISH). RT–PCR showed that the temporal changes in the levels of expression of Noggin and BMP4 were different. The peak level of expression of Noggin mRNA was observed at embryonic day 13 (E13), subsequently gradually declined at 1–3 months (P1–3M) postnatal, and was detected only at a low level at P18M. In contrast, the expression of BMP4 mRNA increased gradually during embryonic development, reached a maximal level at 3 weeks (W) postnatal, and declined only slightly through P18M. In situ hybridization revealed that at embryonic stages, Noggin mRNA was localized throughout all hippocampal regions, whereas at early postnatal ages, Noggin mRNA was primarily localized in the anterior subiculum. At embryonic and early postnatal stages, no significant BMP4 mRNA expression was detectable in the hippocampus. At later postnatal ages, however, Noggin and BMP4 mRNA expression was observed in similar patterns. At 1–3 months postnatal, expression of BMP4 was observed mainly in the dentate gyrus and in the CA1–CA3 pyramidal cell layers. Lower hybridization signals were observed in the hilus and subiculum. Taken together, our results demonstrate that Noggin and BMP4 are expressed in embryonic and postnatal hippocampus, and that the temporal and spatial patterns of their expression are developmentally regulated. These data suggest that Noggin and BMP4 play important roles in hippocampal development.

Temporal and spatial expression profiles of BMP receptors and noggin during BMP-2-induced ectopic bone formation.

The mechanism of ectopic bone formation has not been clear. After BMP-2 implantation into the back muscles of 198 mice, expression of BMPR-1A, -2, and Noggin was increased during the early phase of the reaction. The results suggest that positive and negative feedback mechanisms modulate ectopic osteogenesis induced by this growth factor. The expression of bone morphogenetic protein receptors (BMPRs) and Noggin during ectopic bone formation after implantation of BMP-2 into the back muscles of adult mice was investigated in this study. One hundred ninety-eight male ddy mice were divided into groups and received either collagen disks containing BMP-2, collagen disks alone, or sham surgery with no disk implantation. Changes in the temporal and spatial expression profiles of BMPRs and Noggin were examined by Northern blotting, in situ hybridization, Western blotting, and immunohistochemistry. In the BMP group, expression of BMPR-1A, -2, and Noggin mRNA and protein was enhanced 2-4 days after implantation in undifferentiated mesenchymal cells and regenerating muscle fibers located close to the BMP-retaining implants. On day 7, the expression was also observed in cartilage cells, and after day 14, in the osteoblastic cells around bone tissue. The level of expression peaked at day 4 after implantation and persisted at a much lower level during the bone forming process. No significant expression of BMPR-1B was detected at the mRNA and protein levels during the bone-forming reaction. In the BMP free control groups, a mild enhancement of BMPR-2 expression was also noted around the implant, but this was not observed for BMPR-1A, -1B, or Noggin. Upregulated expression of BMPR-1A, -2, and Noggin in undifferentiated mesenchymal cells and regenerating muscle fibers occurs during the early phase of BMP-2-induced bone formation. The coordinate expression of these positive and negative regulators of BMP signaling points to a potential regulatory mechanism for bone induction.

BMP Responsiveness in Human Mesenchymal Stem Cells

Bone morphogenetic proteins (BMPs) are well known to induce bone formation in animal models and can promote osteogenesis in cultures of multipotential mesenchymal stem cells (MSC) isolated from rat and mouse bone marrow. However, clinical trials of BMPs suggest that BMPs are relatively ineffective inducers of osteogenesis in humans. Recent studies from our lab indicate that when human bone marrow MSC are placed in primary culture, osteogenesis can be induced by dexamethasone (Dex), but not by BMP-2, -4, or -7. We have therefore investigated components of BMP signaling pathways in human MSC. First passage cells, derived from the bone marrow of patients undergoing hip replacement surgery, were cultured with ascorbate phosphate and treated with 100 nM dexamethasone (Dex), 100 ng/ml BMP, or both. After 6 days, alkaline phosphatase activity of cell extracts was measured, and RNA was extracted for RT-PCR analysis of mRNA levels. Among human MSC samples from more than a dozen patients, only one patient sample showed significantly elevated alkaline phosphatase after exposure to BMP; the rest responded to Dex but not BMP. Analysis of mRNA from cultured human MSC indicated that, while Dex treatment caused increased levels of mRNA for alkaline phosphatase, BMP did not. Noggin is a BMP-binding protein that is upregulated by BMPs. BMP-treated human MSC cultures that did not show increased alkaline phosphatase did express elevated levels of noggin mRNA, indicating that the cells are capable of some BMP response. Our results suggest that BMP signaling in mesenchymal stem cells utilizes more than one system for transcriptional activation. The inability of most human MSC to activate transcription of the alkaline phosphatase gene implies that a defect exists in the system required for induction of the osteoblast phenotype.

The roles of three signaling pathways in the formation and function of the Spemann Organizer

Since the three main pathways (the Wnt, VegT and BMP pathways) involved in organizer and axis formation in the Xenopus embryo are now characterized, the challenge is to understand their interactions. Here three comparisons were made. Firstly, we made a systematic comparison of the expression of zygotic genes in sibling wild-type, VegT-depleted (VegT(-)), beta-catenin-depleted (beta-catenin(-)) and double depleted (VegT(-)/beta-catenin(-)) embryos and placed early zygotic genes into specific groups. In the first group some organizer genes, including chordin, noggin and cerberus, required the activity of both the Wnt pathway and the VegT pathway to be expressed. A second group including Xnr1, 2, 4 and Xlim1 were initiated by the VegT pathway but their dorsoventral pattern and amount of their expression was regulated by the Wnt pathway. Secondly, we compared the roles of the Wnt and VegT pathways in producing dorsal signals. Explant co-culture experiments showed that the Wnt pathway did not cause the release of a dorsal signal from the vegetal mass independent from the VegT pathway. Finally we compared the extent to which inhibiting Smad 1 phosphorylation in one area of VegT(-), or beta-catenin(-) embryos would rescue organizer and axis formation. We found that BMP inhibition with cm-BMP7 mRNA had no rescuing effects on VegT(-) embryos, while cm-BMP7 and noggin mRNA caused a complete rescue of the trunk, but not of the anterior pattern in beta-catenin(-) embryos.

Localized BMP4–Noggin Interactions Generate the Dynamic Patterning of Noggin Expression in Somites

Interactions between BMP4 and its inhibitor, noggin, regulate patterning of somites and neural crest. During mesoderm development, noggin mRNA is expressed in the intermediate mesoderm. Upon segmentation, it is detected in the lateral portion of epithelial somites becoming progressively medialized as they mature. In dissociated segments, noggin becomes transiently confined to the dorsomedial lip of the dermomyotome. Here, we investigated the factor(s) that control this lateral-to-medial shift in transcription of somitic noggin. Inhibition of BMP activity in the caudal lateral plate/intermediate mesoderm prevented noggin transcription in the lateral somite. Further rostrally (or later in development), inhibition of tube-derived BMP, but not of Wnt activity, prevented initial noggin expression in the dorsomedial lip of the dermomyotome. Moreover, BMP4 was sufficient to trigger initial expression of noggin even in the absence of ectoderm and/or neural tube, suggesting a direct action on the dorsomedial somite. Thus, the patterns of noggin transcription in somites are directly regulated by BMP4 activities emanating first from the mesoderm and later from the neural tube. Expression patterns of BMP4 and of type IA BMP receptors are spatiotemporally compatible with this lateral-to-medial shift. These results highlight the existence in the neural tube–mesoderm complex of a regulatory loop by which BMP positively regulates transcription of noggin, which in turn represses further ligand activity.

Synthesis and release of activin and noggin by cultured human amniotic epithelial cells.

Recent studies suggest that extra-embryonic tissues may be essential sources of early organizing signals for the mouse embryo. In vitro studies of human amniotic epithelial cells (HAEC) have shown that the amnion can produce various biologically active substances. In this study, the synthesis and release of activin A and noggin, and the activin signaling pathway, was investigated in HAEC. Conditioned medium from cultured HAEC contained activin A which was functionally active in Xenopus laevis animal cap assays. Immunohistochemistry, western blotting and reverse transcription-polymerase chain reaction confirmed that HAEC also synthesize and release noggin. Noggin transcripts were induced by the addition of recombinant activin A, and activin A was inhibited by activin antibody except in the presence of cycloheximide (CHX). These data demonstrate that noggin mRNA expression is induced directly by activin A without new protein synthesis, indicating that noggin is a primary response gene. The results suggest that there is an activin signaling pathway in HAEC, and that the human amnion might therefore be involved in neural formation during early development.

Cloning and characterization of the T-box gene Tbx6 in Xenopus laevis.

Tbx6 is a member of the T-box gene family. Studies of knockout mice indicate that Tbx6 is involved in somite differentiation. In the present study, we cloned Tbx6 from another vertebrate species, namely Xenopus laevis, and studied its roles in development. The expression of Tbx6 in Xenopus started from the early gastrula stage, reached a peak during the late gastrula to neurula stages and then declined. Initial expression of Tbx6 was observed in the paraxial mesoderm during the gastrula stage. The Tbx6-expressing region spread anteriorly and ventrally in the neurula stage. In the tailbud stage, the area of expression shrank caudally and was finally restricted to the tip of the tailbud. Overexpression of Tbx6 mRNA in dorsal blastomeres caused atrophy of the neural tube and inhibited differentiation of the notochord. Animal cap explants overexpressing Tbx6 or Tbx6VP16 mRNA, but not Tbx6EnR mRNA, differentiated mainly into ventral mesodermal tissues. This suggests that Tbx6 is a transcriptional activator. Higher doses of Tbx6 or Tbx6VP16 mRNA caused hardly any muscular differentiation. However, coinjection of Tbx6 mRNA with noggin mRNA elicited marked muscle differentiation. These results suggest that Tbx6 is implicated in ventral mesoderm specification but is involved in muscle differentiation when acting together with the dorsalizing factor noggin.

Noggin is required for induction of the hair follicle growth phase in postnatal skin.

During postnatal development, the hair follicle (HF) shows cyclic activity with periods of relative resting, active growth (anagen), and regression. We demonstrate that similar to the HF induction in embryonic skin, initiation of a new hair growth phase in postnatal skin requires neutralization of the inhibitory activity of bone morphogenetic protein 4 (BMP4) by the BMP antagonist noggin. In the resting HF, BMP4 mRNA predominates over noggin in the epithelium and mesenchyme, and the BMP receptor IA is prominently expressed in the follicular germ. Anagen development is accompanied by down-regulation of the BMP4 and increased noggin mRNA in the HF. Furthermore, administration of noggin protein induces new hair growth phase in postnatal telogen skin in vivo. In contrast, BMP4 induces selective arrest of anagen development in the non-tylotrich (secondary) HF. As a hair growth inducer, noggin increases Shh mRNA in the HF whereas BMP4 down-regulates Shh. This suggests that modulation of BMP4 signaling by noggin is essential for hair growth phase induction in postnatal skin and that the hair growth-inducing effect of noggin is mediated, at least in part, by Shh.

Colocalization of noggin and bone morphogenetic protein-4 during fracture healing.

The regulation of callus formation during fracture repair involves the coordinate expression of growth factors and their receptors. This article describes the temporal and spatial expression of noggin gene, an antagonist to bone morphogenetic protein (BMP), during the fracture repair process. Noggin expression was examined by means of Northern blotting and in situ hybridization and compared with the expression pattern of BMP-4 in a model of fracture repair in adult mice. Expression levels of noggin messenger RNA (mRNA) were enhanced in the early phase of fracture callus formation. The localization of the noggin mRNA was similar to that of BMP-4 mRNA. Distinct noggin mRNA signals were located predominantly in cells lining the periosteum and the cortical endosteum near the fracture site at 2 days after fracture. At 5, 10, and 21 days after fracture, noggin mRNA was detected in the chondrocytes and osteoblasts in the newly formed callus. The pattern of localization was indistinguishable from that of BMP-4. These results suggest that the noggin/BMP-4 balance could be an important factor in the regulation of callus formation during fracture healing.

Characterization of the Functionally Related Sites in the Neural Inducing Gene Noggin

Previously we have shown that blocking bone morphogenetic protein (BMP) receptor signaling by a dominant negative BMP receptor causes neurogenesis in Xenopus animal caps (ACs), whereas the physiological neural inducer noggin acts as a homodimer physically binding to BMP-4 and disrupting its signaling at the ligand level. The present study attempted to elucidate the relationship between the structure and function of noggin. By replacing some cysteine residues with serine residues through a site-directed mutagenesis strategy, we generated three noggin mutants, C145S, C205S, and C(218, 220, 222)S (3CS). Although mRNAs encoded by these mutants were translated as efficiently as wild-type (WT) noggin mRNA, they behaved differently when expressed in vivo. Expression of WT noggin or C205S in Xenopus ACs converted the explants (prospective ectoderm) into neural tissue, indicated by the neural-like morphology and expression of the pan neural marker NCAM in the ACs. In contrast, ACs expressing C145S or 3CS sustained an epidermal fate like the control caps. Similar results were observed in the mesoderm where C205S (but not C145S and 3CS) displayed dorsalizing activity as well as WT noggin. Altogether, our results suggest that Cys145 alone or Cys(218, 220, 222) as a whole in noggin protein is required for the biological activities of noggin, probably participating in the dimerization of noggin with BMP-4 or itself.

Coordinated Expression of Noggin and Bone Morphogenetic Proteins (BMPs) During Early Skeletogenesis and Induction of Noggin Expression by BMP-7

Coordinated regulation of the activities of bone morphogenetic protein (BMP) and its inhibitors is essential for skeletal development since loss-of-function experiments show that both BMPs and BMP inhibitory signals, such as noggin, are required to establish proper formation of skeletal tissues. In this paper, we asked how and when noggin would be functional to interact with BMPs during skeletogenesis in mammals. For this purpose, we first analyzed the spatial and temporal patterns of noggin, BMP-2, BMP-4, and BMP-7 expression during early skeletogenesis in mouse embryos. In situ hybridization study revealed that noggin expression was detected at a low level in limb mesenchyme, whereas BMP-7 was expressed at a high level throughout limb mesenchyme 10.5 days postcoitum (dpc) in mouse embryos. One day later, noggin mRNA was expressed at a high level in the prechondrogenic condensations in appendicular and axial skeletal primordia, where sox9 transcripts were also expressed. At this stage, noggin-expressing cells were surrounded by those expressing BMP-7. The chondrogenic cell condensation continued to express noggin transcripts in 12.5 dpc and 13.5 dpc embryos, and again the noggin-expressing cells within the cartilaginous tissue were surrounded by those expressing BMP-7. We further examined interaction of noggin and BMPs by using organ cultures of 11.5 dpc mouse forelimbs and found that implantation of carriers containing BMP-7 protein into the forelimb explants induced noggin expression in the limb mesenchyme. BMP-7 also induced type II collagen and sox9 mRNAs in the same cell population, indicating that noggin induction occurred in the chondrogenic precursor cells. BMP-7 effects on noggin expression were observed in a dose-dependent manner within a dose range of 10-100 ng/microliter. These results suggest that BMP-7 induced expression of noggin transcripts within skeletal cell condensation and that this noggin expression in turn could act antagonistically to attenuate BMP action in the early skeletogenesis.

Noggin and Bone Morphogenetic Protein-4 Coordinately Regulate the Progression of Chondrogenic Differentiation in Mouse Clonal EC Cells, ATDC5

Here we report the gene expression and regulation and the function of noggin in clonal mouse chondrogenic EC cells, ATDC5. In ATDC5 cells, the expression of Noggin mRNA increased in parallel with the progression of chondrogenic differentiation. The treatment with conditioned medium of noggin-transfected COS-7 cells decreased the levels of type II and type X collagen gene transcripts of differentiated ATDC5 cells in a dose-dependent manner, and this inhibitory action was reversed by exogenously administered BMP-4 in a dose-dependent manner. The steady-state level of noggin gene transcripts was markedly upregulated by exogenously administered BMP-4 in time- and dose-dependent manners. Furthermore, this stimulatory effect of BMP-4 was attenuated by treatment with actinomycin D, but not with cycloheximide. These results indicate that noggin and BMP-4 coordinately regulate the progression of chondrogenic differentiation in ATDC5 cells.

Noggin expression in a mesodermal pluripotent cell line C1 and its regulation by BMP

Osteoblasts and chondrocytes are derived from mesodermal stem cells and their differentiation is under the control of coordinated interaction among signaling molecules. Noggin is one of the signaling molecules which bind to and inactivate BMPs to induce neural tissues and dorsal mesoderm in Xenopus. However, its expression and regulation in mammalian cells has not been known. In this study, we investigated expression of noggin in murine pluripotent mesodermal cell line, C1. Noggin expression was very low in these C1 cells before they were induced to differentiate. When C1 cells were induced to differentiate into chondrocytes in aggregate cultures in the presence of dexamethasone(dex), noggin expression was significantly increased. In a sharp contrast, when the C1 cells were induced to differentiate into osteoblastic cells by the treatment with beta glycerophosphate (betaGP) and ascorbic acid (AA), noggin mRNA expression remained to be barely detectable. Noggin expression was also observed in the developing cartilage of vertebrae in 15.5 dpc mouse embryos. The noggin mRNA level in C1 cells in monolayer cultures was enhanced significantly by the treatment with BMP4/7 in a dose-dependent manner with a maximal effect at 100 ng/ml. The BMP4/7 effect on noggin expression was time dependent starting within 12 h and peaked at 24 h. These results indicate that noggin is expressed in the pluripotent mesodermal cell line C1 and that its expression is regulated by BMP.

Bone morphogenetic proteins induce the expression of noggin, which limits their activity in cultured rat osteoblasts.

Bone morphogenetic proteins (BMPs) induce the differentiation of cells of the osteoblastic lineage and enhance the function of the osteoblast. Growth factors are regulated by binding proteins, but there is no information about binding proteins for BMPs in skeletal cells. Noggin specifically binds BMPs, but its expression by cells of the osteoblastic lineage has not been reported. We tested for the expression of noggin and its induction by BMP-2 in cultures of osteoblast-enriched cells from 22-d-old fetal rat calvariae (Ob cells). BMP-2 caused a time- and dose-dependent increase in noggin mRNA and polypeptide levels, as determined by Northern and Western blot analyses. The effects of BMP-2 on noggin transcripts were dependent on protein, but independent of DNA synthesis. BMP-2 increased the rates of noggin transcription as determined by nuclear run-on assays. BMP-4, BMP-6, and TGF-beta1 increased noggin mRNA in Ob cells, but basic fibroblast growth factor, platelet- derived growth factor BB, and IGF-I did not. Noggin decreased the stimulatory effects of BMPs on DNA and collagen synthesis and alkaline phosphatase activity in Ob cells. In conclusion, BMPs induce noggin transcription in Ob cells, a probable mechanism to limit BMP action in osteoblasts.

Neural induction and patterning by fibroblast growth factor, notochord and somite tissue in Xenopus.

Two natural neural inducing sources have been used, the notochord and the somites together with the growth factor bFGF, to investigate the anterior/posterior patterning of neural tissue in an animal cap explant model in Xenopus laevis. Notochord and somite tissue from stages 12.5/13 and 16, respectively, were manually isolated, and combined heterochronically with responding animal cap ectoderm aged to gastrula stages. Somite recombinants were also constructed with animal caps injected with noggin mRNA. The responses of the ectoderm were analyzed by reverse transcription polymerase chain reaction (RT-PCR) detection of marker gene expression, and in some cases by in situ hybridization. The requirement for FGF receptor function was analyzed using the dominant negative FGF receptor (XFD). The experiments showed that bFGF is capable of direct neural induction in caps aged to stage 10.5. It was also shown that notochords are capable of inducing anterior neural tissue in gastrula stage animal cap ectoderm, and this induction is sensitive to XFD in the responding tissue. Injection of noggin mRNA results in the induction of anterior neural differentiation, and it was demonstrated that this induction was insensitive to the expression of XFD in the responding tissue. It was also shown that somite tissue recombined with gastrula stage animal cap ectoderm, can induce both anterior and posterior nervous tissue and can also posteriorize noggin-induced anterior neural tissue when combined with noggin-injected animal cap ectoderm. This response is partially sensitive to XFD expression. The results shed light on the role of competence of animal cap ectoderm and the signals from postgastrulation axial and paraxial mesoderm in the patterning of the amphibian nervous system.

Pax-6 gene expression in newt eye development.

pax-6 is thought to be a master control gene of eye development in species ranging from insects to mammals. We have isolated a pax-6 cDNA homolog of the newt, Cynops pyrrhogaster. RT-PCR and sequence analyses predicted four alternatively spliced forms derived from inclusion or exclusion of the region corresponding to exons 5a and 12 in the human pax-6 ortholog. This gene shared extensive sequence identitiy and similar expression patterns with those of mouse and zebrafish. pax-6 signal was first detected at the anterior ridge of the neural plate, and later at the eye and nasal primordium and in the central nervous system - except for the midbrain. The injection of sonic hedgehog (shh) RNA inhibited the expression of pax-6 within the optic vesicle and disturbed eye cup formation. A similar suppressive effect of shh was also observed in the conjugation of the animal caps preloaded with exogenous shh and noggin mRNA, which was used as an inducer of pax-6. In contrast, shh injection had no effect on the expression of pax-6 in the surface ectoderm overlying the optic cup, suggesting that the expression of pax-6 in the surface ectoderm is not regulated by shh in vivo. Moreover, we found transient activation of pax-6 in animal cap explants at the sibling stage of mid-late gastrula. This observation raises the possibility that the ectoderm is competent to the lens-inducing signal at a stage as early as mid gastrula.

Induction of the prospective neural crest of Xenopus

The earliest sign of the prospective neural crest of Xenopus is the expression of the ectodermal component of Xsna (the Xenopus homologue of snail) in a low arc on the dorsal aspect of stage 11 embryos, which subsequently assumes the horseshoe shape characteristic of the neural folds as the convergence-extension movements shape the neural plate. A related zinc-finger gene called Slug (Xslu) is expressed specifically in this tissue (i.e. the prospective crest) when the convergence extension movements are completed. Subsequently, Xslu is found in pre- and post-migratory cranial and trunk neural crest and also in lateral plate mesoderm after stage 17. Both Xslu and Xsna are induced by mesoderm from the dorsal or lateral marginal zone but not from the ventral marginal zone. From stage 10.5, explants of the prospective neural crest, which is underlain with tissue, are able to express Xslu. However expression of Xsna is not apparently specified until stage 12 and further contact with the inducer is required to raise the level of expression to that seen later in development. Xslu is specified at a later time. Embryos injected with noggin mRNA at the 1-cell stage or with plasmids driving noggin expression after the start of zygotic transcription express Xslu in a ring surrounding the embryo on the ventroposterior side. We suggest this indicates (a) that noggin interacts with another signal that is present throughout the ventral side of the embryo and (b) that Xslu is unable to express in the neural plate either because of the absence of a co-inducer or by a positive prohibition of expression. The ventral co-inducer, in the presence of overexpressed noggin, seems to generate an anterior/posterior pattern in the ventral part of the embryo comparable to that seen in neural crest of normal embryos. We suggest that the prospective neural crest is induced in normal embryos in the ectoderm that overlies the junction of the domains that express noggin and Xwnt-8. In support of this, we show animal cap explants from blastulae and gastrulae, treated with bFGF and noggin express Xslu but not NCAM although the mesoderm marker Xbra is also expressed. Explants treated with noggin alone express NCAM only. An indication that induction of the neural plate border is regulated independently of the neural plate is obtained from experiments using ultraviolet irradiation in the precleavage period. At certain doses, the cranial crest domains are not separated into lateral masses and there is a reduction in the size of the neural plate.