SHH-N upregulates Sfrp2 to mediate its competitive interaction with WNT1 and WNT4 in the somitic mesoderm

Dorsoventral polarity of the somitic mesoderm is established by competitive signals originating from adjacent tissues. The ventrally located notochord provides the ventralizing signals to specify the sclerotome, while the dorsally located surface ectoderm and dorsal neural tube provide the dorsalizing signals to specify the dermomyotome. Noggin and SHH-N have been implicated as the ventralizing signals produced by the notochord. Members of the WNT family of proteins, on the other hand, have been implicated as the dorsalizing signals derived from the ectoderm and dorsal neural tube. When presomitic explants are confronted with cells secreting SHH-N and WNT1 simultaneously, competition to specify the sclerotome and dermomyotome domains within the naive mesoderm can be observed. Here, using these explant cultures, we provide evidence that SHH-N competes with WNT1, not only by upregulating its own receptor Ptc1, but also by upregulating Sfrp2 (Secreted frizzled-related protein 2), which encodes a potential WNT antagonist. Among the four known Sfrps, Sfrp2 is the only member expressed in the sclerotome and upregulated by SHH-N recombinant protein. We further show that SFRP2-expressing cells can reduce the dermomyotome-inducing activity of WNT1 and WNT4, but not that of WNT3a. Together, our results support the model that SHH-N at least in part employs SFRP2 to reduce WNT1/4 activity in the somitic mesoderm.     

Anti-apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis

In vertebrates the neural tube, like most of the embryonic organs, shows discreet areas of programmed cell death at several stages during development. In the chick embryo, cell death is dramatically increased in the developing nervous system and other tissues when the midline cells, notochord and floor plate, are prevented from forming by excision of the axial-paraxial hinge (APH), i.e. caudal Hensen's node and rostral primitive streak, at the 6-somite stage ( Charrier, J. B., Teillet, M.-A., Lapointe, F. and Le Douarin, N. M. (1999). Development 126, 4771-4783). In this paper we demonstrate that one day after APH excision, when dramatic apoptosis is already present in the neural tube, the latter can be rescued from death by grafting a notochord or a floor plate fragment in its vicinity. The neural tube can also be recovered by transplanting it into a stage-matched chick embryo having one of these structures. In addition, cells engineered to produce Sonic hedgehog protein (SHH) can mimic the effect of the notochord and floor plate cells in in situ grafts and transplantation experiments. SHH can thus counteract a built-in cell death program and thereby contribute to organ morphogenesis, in particular in the central nervous system.     

Dorsoventral differential distribution of collagen type XIV around the spinal cord is regulated by the ectoderm

Regional specification in the nervous system is a critical issue in nervous system morphogenesis. Along the dorsoventral axis of the spinal cord, ventral inductive signals of the notochord and floor plate, and dorsal ones of the epidermal ectoderm are essential. Collagen type XIV is uniquely distributed around the spinal cord with a gradient of dorsal high and ventral low at the early developmental stages of the chick embryo. In the present study it was found that collagen type XIV expression around the spinal cord was entirely regulated by the ectoderm and that even the ventralizing tissues, the notochord and floor plate, themselves could be influenced to express this molecule by the ectoderm.     

Effects of retinoic acid on the distribution of glycoconjugates during mouse tail bud development

Retinoic acid (RA), a potent teratogen of caudal axial development in rodents, has been shown to alter glycoconjugates in a variety of embryonic tissues and teratocarcinomas. In this study, we examined its effects on the expression of cell surface and extracellular matrix glycoconjugates during tail bud development in mouse embryos by using lectin histochemistry. The lectins WGA, sWGA, and PNA showed striking differences in binding between RA-exposed and control embryos. Computer-assisted densitometry revealed a significant increase in binding of all three lectins to the extracellular material of the luminal and abluminal borders of the secondary neural tube and surrounding the notochord in RA-exposed embryos. RA-treated embryos also showed an increased binding affinity for the lectins sWGA and PNA to the cells of the notochord, while WGA showed increased binding to the neuroepithelial cells of the secondary neural tube. The results suggest that RA affects the expression of lectin binding sites during the early development of RA-induced caudal axial defects.     

Imaging of nitric oxide in a living vertebrate using a diamino-fluorescein probe

Numerous approaches have been described to identify nitric oxide (NO), a free radical involved in various physiological and pathophysiological processes. One of these approaches is based on the use of chemical probes whose transformation by NO generates highly fluorescent derivatives, permitting detection of NO down to nanomolar concentrations. Here, we show that the cell-permeant diamino-fluorophore 4-amino-5-methylamino-2'-7'-difluoro-fluorescein diacetate (DAF-FM-DA) can be used to detect NO production sites in a living vertebrate, the zebrafish Danio rerio. The staining pattern obtained in larvae includes the bulbus arteriosus, forming bones, the notochord, and the caudal fin. The specificity of the signal was confirmed by its decrease in animals exposed to a NO scavenger or a NO synthase inhibitor and its increase in the presence of a NO donor. Using this method, NO production was observed to change along development in the notochord and the caudal fin whereas it remained stable in the bulbus arteriosus. Local changes in NO production in response to stressful conditions were also detected by this method. Altogether, labeling with DAF-FM DA is an efficient method to monitor changes in NO production in live zebrafish under physiological as well as pathophysiological conditions, suggesting applications to drug screening and molecular pharmacology.     

Defining subregions of Hensen's node essential for caudalward movement, midline development and cell survival.

Hensen's node, also called the chordoneural hinge in the tail bud, is a group of cells that constitutes the organizer of the avian embryo and that expresses the gene HNF-3(&bgr;). During gastrulation and neurulation, it undergoes a rostral-to-caudal movement as the embryo elongates. Labeling of Hensen's node by the quail-chick chimera system has shown that, while moving caudally, Hensen's node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endodermal cells. In this work, we demonstrate that the node can be divided into functionally distinct subregions. Caudalward migration of the node depends on the presence of the most posterior region, which is closely apposed to the anterior portion of the primitive streak as defined by expression of the T-box gene Ch-Tbx6L. We call this region the axial-paraxial hinge because it corresponds to the junction of the presumptive midline axial structures (notochord and floor plate) and the paraxial mesoderm. We propose that the axial-paraxial hinge is the equivalent of the neuroenteric canal of other vertebrates such as Xenopus. Blocking the caudal movement of Hensen's node at the 5- to 6-somite stage by removing the axial-paraxial hinge deprives the embryo of midline structures caudal to the brachial level, but does not prevent formation of the neural tube and mesoderm located posteriorly. However, the whole embryonic region generated posterior to the level of Hensen's node arrest undergoes widespread apoptosis within the next 24 hours. Hensen's node-derived structures (notochord and floor plate) thus appear to produce maintenance factor(s) that ensures the survival and further development of adjacent tissues.     

Effects of rat Axin domains on axis formation in Xenopus embryos

Wnt signaling plays an important role in axis formation in early vertebrate development. Axin is one Wnt signaling regulator that inhibits this pathway. The effects of the injection of mRNA of several rat Axin (rAxin) mutants on axis formation in Xenopus embryos were examined. It was found that rAxin mutants containing only a regulation of G-protein signaling (RGS) domain fragment or with deletion of the RGS domain induced axis formation. Because the RGS domain is a major adenomatous polyposis coli gene product (APC)-binding domain, APC association with glycogen synthase kinase 3beta (GSK3beta) on the Axin molecule may be important in inhibition of axis formation. The ventralizing activities of wild-type rAxin and a mutant in which the Dishevelled and Axin (DIX) domain was deleted (deltaDIX mutant) were examined. Histological examination and gene expression revealed that the ventralizing activity of the deltaDIX mutant was weaker than that of wild-type rAxin. This finding suggests that the C-terminus of rAxin contributes to the inhibition of Wnt signaling in Xenopus embryos. Furthermore, an rAxin mutant that contained both the RGS and GSK3beta-binding domains affected both the dorsal and ventral sides of blastomeres, mediated ectodermal fate and induced expansion of notochord and/or endoderm, but did not induce axis formation.     

Anatomical and Histological Investigations on the Caudal Skeleton of Apteronotus leptorhynchus (Apteronotidae,Gymnotoidei)

The caudal skeleton of Apteronotus leptorhynchus was studied at various stages from hatching to the adult stage using anatomical and histological techniques. The caudal skeleton that supports the lepidotrichia is reduced to a rhomboid caudal plate (caudal cartilage) that extends the vertebral axis. This cartilage appears for the first time in 8 day old fish, postero-ventral to the notochord. During its growth, perichondral and endochondral ossification occurs, beginning at the anterior end of the cartilage. Comparison with the anatomy and ontogeny of the typical caudal skeleton of teleosts allows us to interpret the caudal cartilage of A. leptorhynchus as an hypuro-opisthural component that is homologous to the cartilage that occurs at the tip of the axial skeleton in Eigenmannia virescens.     

Involvement of EphA2 in the formation of the tail notochord via interaction with ephrinA1

Eph receptors have been implicated in cell-to-cell interaction during embryogenesis. We generated EphA2 mutant mice using a gene trap method. Homozygous mutant mice developed short and kinky tails. In situ hybridization using a Brachyury probe found the notochord to be abnormally bifurcated at the caudal end between 11.5 and 12.5 days post coitum. EphA2 was expressed at the tip of the tail notochord, while one of its ligands, ephrinA1, was at the tail bud in normal mice. In contrast, EphA2-deficient notochordal cells were spread broadly into the tail bud. These observations suggest that EphA2 and its ligands are involved in the positioning of the tail notochord through repulsive signals between cells expressing these molecules on the surface.     

Embryonic development and newly hatched larvae of the little dragon sculpinBlepsias cirrhosus

This paper describes both embryonic development and newly hatched larval morphology of the little dragon sculpinBlepsias cirrhosus. The eggs ofB. cirrhosus are almost spherical, 3.0–3.2 mm in diameter, and have a yolk color of burnt orange. Development is very slow, being especially sluggish once the embryo appears. The embryo begins forming from the 10th day. In size, the early embryo is less than 1/6 of the yolk’s circumference. Incubation at 10°C takes about 200 days, 50 days shorter than the incubation period in a natural environment, with a mean water temperature of 11°C. The notochord length of newly-hatched larvae averages 11.1 mm. The larvae are developed so fully that the notochord is already flexing and the caudal and pectoral rays are forming.     

Mutations affecting tail and notochord development in the ascidian Ciona savignyi.

Ascidians are among the most distant chordate relatives of the vertebrates. However, ascidians share many features with vertebrates including a notochord and hollow dorsal nerve cord. A screen for N-ethyl-N-nitrosourea (ENU)-induced mutations affecting early development in the ascidian Ciona savignyi resulted in the isolation of a number of mutants including the complementing notochord mutants chongmague and chobi. In chongmague embryos the notochord fails to develop, and the notochord cells instead adopt a mesenchyme-like fate. The failure of notochord development in chongmague embryos results in a severe truncation of tail, although development of the tail muscles and caudal nerve tracts appears largely normal. Chobi embryos also have a truncation of the tail stemming from a disruption of the notochord. However, in chobi embryos the early development of the notochord appears normal and defects occur later as the notochord attempts to extend and direct elongation of the tail. We find in chobi tailbud embryos that the notochord is often bent, with cells clumped together, rather than extended as a column. These results provide new information on the function and development of the ascidian notochord. In addition, the results demonstrate how the unique features of ascidians can be used in genetic analysis of morphogenesis.     

Molecular cloning of a human Vent-like homeobox gene

We have isolated a previously unknown human homeobox-containing cDNA, VENT-like homeobox-2 (VENTX2), using PCR with a bone marrow cDNA library and primers designed from the VENTX1 (alias HPX42) homeobox sequence. Here we describe the molecular cloning, chromosomal localization to 10q26.3, and functional analysis of this gene. The 2.4-kb human VENTX2 cDNA encoded a protein with a predicted molecular weight of 28 kDa containing a homeodomain with 65% identity to the Xenopus laevis ventralizing gene Xvent2B. VENTX2 antisera detected a 28-kDa protein in cells transfected with a VENTX2 expression construct, in a human erythroleukemic cell line and in bone marrow samples obtained from patients in recovery phase after chemotherapy. The similarity of the homeodomains from VENTX2 and the X. laevis Vent gene family places them in the same homeodomain class. Consistent with this structural classification, overexpression of VENTX2 in zebrafish embryos led to anterior truncations and failure to form a notochord, which are characteristics of ventralization.     

Morphology,mechanics, and locomotion: the relation between the notochord and swimming motions in sturgeon

Synopsis To examine the relation between morphology and performance, notochordal morphology was correlated with notochordal mechanics and with steady swimming motions in white sturgeon, Acipenser transmontanus. In a still-water tank, motions of four sturgeon varied with changes in swimming speed and axial position along the body. For a 1..34 m sturgeon, slow and fast swimming modes were distinguished, with speeds at the fast mode more than two times those at the slow mode without changes in tailbeat frequency. This increase in speed is correlated with an increase in the body's maximal midline curvature (m^–1), suggesting a role for curvature-related mechanical properties of the notochord. Maximal midline curvature also varied with axial position, and surprisingly was uncorrelated with axial changes in the notochord's cross-sectional shape - as measured by height, width, inner diameter, and lateral thickness of the sheaths. On the other hand, maximal midline curvature was negatively correlated with the axial changes in the notochord's angular stiffness (N m rad^–1) and change in internal pressure (% change from baseline of 58.6 kPa), both of which were measured during in vitro bending tests. In vivo curvature and in vitro angular stiffness were then used to estimate the bending moments (Nm) in the notochord during swimming. In the precaudal notochord, the axial pattern of maximal stiffness moments was congruent with the pattern of maximal notochordal curvature in the precaudal region, but in the caudal notochord maximal angular stiffness was located craniad to maximal curvature. One interpretation of this pattern is that the precaudal notochord resists bending moments generated by the muscles and that the caudal notochord resists bending moments generated by hydrodynamic forces acting on the tail.     

BMP and non-canonical Wnt signaling are required for inhibition of secondary tail formation in zebrafish

The role of bone morphogenetic protein (BMP) signaling in specifying cell fate in the zebrafish tailbud has been well established. In addition to a loss of ventral tissues, such as ventral tailfin and cloaca, some embryos with compromised BMP signaling produce an additional phenotype: a ventrally located secondary tail containing both somitic muscle and notochord. This phenotype has been proposed to reflect a fate-patterning defect due to a change in a hypothesized BMP activity gradient. Here, we show that a defect in morphogenetic movements, not fate patterning, underlies the formation of secondary tails in BMP-inhibited embryos. Our data indicate that BMP signaling is activated in the ventroposterior tailbud to promote cell migration during tailbud protrusion, and that defective migration of these cells in BMP mutants ultimately leads to bifurcation of the caudal notochord. Additionally, we show that non-canonical Wnt signaling is also required for proper tail morphogenesis, possibly by maintaining cohesion of notochord progenitors by regulation of cadherin localization. We propose a model in which BMP and the non-canonical Wnt pathway regulate tail morphogenesis by controlling cell migration and cell adhesion within the tailbud.     

Discontinuity of primary and secondary neural tube in spina bifida induced by retinoic acid in mice

This report shows by light microscopy the appearance of secondary neurulation separated from primary neurulation and its developmental fate in the spinal cord of mice exposed to retinoic acid in utero. The embryos and fetuses were derived from pregnant mice (ICR strain) given 60, 40, or 0 mg/kg of retinoic acid in olive oil on day 8 of gestation orally and killed 1, 2, or 10 days later. Separation of the primary neural fold from the secondary neural tube was seen in 9- and 10-day-old embryos: the caudal part of the neuroepithelium of the primary neural fold was disarranged with non-closed posterior neuropore, and underneath it the secondary neural tissue extended caudally with abnormal notochord. At term, fetuses showed spina bifida, including myeloschisis, myelocele, and diplomyelia (diastematomyelia) with abnormal distribution of ganglionic cells. These cord lesions were located between the third lumbar and second coccygeal levels. The former two cord anomalies were associated with diplomyelia and split the dorsal and ventral portions of the spinal cord with an overlapping zone between the third lumbar and third sacral levels. These findings suggest that the separation from primary neurulation is due to the lesions in both primary neural folds and notochord induced by retinoic acid and that the spinal cord caudal to the third lumbar level originates from both neuroectoderm and mesenchyme-like cells while that caudal to the third sacral level originates from mesenchyme-like cells only.     

Continued growth and cell proliferation into adulthood in the notochord of the appendicularian Oikopleura dioica

The appendicularian urochordate Oikopleura dioica retains a free-swimming chordate body plan throughout life, in contrast to ascidian urochordates, whose metamorphosis to a sessile adult form involves the loss of chordate structures such as the notochord and dorsal nerve cord. Development to adult stages in Oikopleura involves a lengthening of the tail and notochord and an elaboration of the repertoire of tail movements. To investigate the cellular basis for this lengthening, we have used confocal microscopy and BrdU labeling to examine the development of the Oikopleura notochord from hatching through adult stages. We show that as the notochord undergoes the typical urochordate transition from a stacked row of cells to a tubular structure, cell number begins to increase. Addition of new notochord cells continues into adulthood, multiplying the larval complement of 20 cells by about 8-fold by the third day of life. In parallel, the notochord lengthens by about 4-fold. BrdU incorporation and a cell-cycle marker confirm that notochord cells continue to proliferate well into adulthood. The extensive postlarval proliferation of notochord cells, together with their arrangement in four circumferentially distributed longitudinal rows, presumably provides the Oikopleura tail with the necessary mechanical support for the complex movements exhibited at adult stages.     

Effects of retinoic acid on chick tail bud development

The present study describes the teratogenic effects of retinoic acid (RA) on the development of the chick tail bud. Chick embryos were recovered 48 hours after treatment at HH stages 11 to 16 with various dosages of RA by subblastodermal injection. At the gross level, RA treatment resulted in varying degrees of caudal regression, scoliosis, limb malformations, and open posterior neuropores among the survivors. Histological examination of tail buds from treated embryos revealed defects which included total dysplasia of caudal structures, the presence of accessory neural tube and notochord tissue, and abnormal fusions of the notochord to the neural tube and tailgut. The incidence, severity, and location of the defects were dependent on the dose of the teratogen, and the stage of development at the time of treatment. The defects resembled those induced in previous studies by treatment with sialic acid binding lectins such as wheat germ agglutinin and limulus polyphemus lectin (Griffith and Wiley, '90b).