Floor Plate Cells Research Articles

Restricted expression of protocadherin 2A in the developing mouse brain

Protocadherins are cell–cell adhesion molecules that are thought to be involved in neural development. Here, we report the expression pattern of protocadherin 2A (Pc2A) in the developing mouse brain as determined by the in situ hybridization technique. In the postnatal day 2 brain, various regions expressed Pc2A including the cerebellar cortex, ventral posterior thalamic nucleus, dorsal lateral geniculate nucleus, hippocampus and cerebellum (Purkinje cells). In particular, some ependymal cells that form the lining of the lateral ventricle and the third ventricle and floor plate cells lining the fourth ventricle showed prominent expression. In the adult brain, strong expression was restricted to the Purkinje cells. Expression in other areas of the adult brain was down-regulated to a faint level, and only a weak signal was detected in regions such as the retina, olfactory bulb, dentate gyrus of the hippocampus, and in some parts of the medial eminence. These observations suggest that Pc2A is expressed in various regions of the brain in a developmentally regulated manner.

Zebrafish tiggy-winkle hedgehog promoter directs notochord and floor plate green fluorescence protein expression in transgenic zebrafish embryos.

Zebrafish tiggy-winkle hedgehog (twhh) is a member of the hedgehog gene family that plays an important role in patterning brain, neural tube, somites, and eyes. To better understand the regulation of its tissue-specific expression, the activity of the twhh promoter was determined in zebrafish embryos by transient and transgenic expression analysis. Transient expression studies revealed that the 5.2-kb twhh promoter drove green fluorescence protein (GFP) expression in the notochord, floor plate, and branchial arches. Deletion analysis showed that distinct regions of the twhh promoter regulated the respective notochord or floor plate specific expression. To confirm the tissue specificity of the twhh promoter, transgenic zebrafish containing the twhh-GFP transgene were generated. GFP expression was analyzed in the F1, F2, and F3 generations of the transgenic embryos. The results confirmed the tissue-specific expression of the transgene in the notochord, floor plate, and branchial arches. In addition, GFP expression was also found in the pectoral fin buds, retina, and epithelial lining cells of the Kupffer's vesicle in the transgenic fish embryos. The expression pattern of the twhh-GFP transgene mimicked the expression of the endogenous twhh mRNAs in the floor plate, fin buds, branchial arches, retina, and epithelial lining cells of the Kupffer's vesicle. The expression in the notochord, however, did not mimic the pattern of the endogenous twhh expression. To determine whether no tail (ntl) or floating head (flh) mutants that have developmental defect in the notochord or the Kupffer's vesicle may affect the GFP expression in these regions, GFP expression was analyzed in ntl or flh transgenic embryos. No GFP expression could be detected in the midline region of the ntl transgenic embryos. However, in flh transgenic embryos, although GFP expression was affected in the midline region, its expression in the Kupffer's vesicle appeared normal. Together, these data indicated that the 5.2-kb twhh promoter contains regulatory elements for tissue-specific expression of twhh in the floor plate, pectoral fin bud, branchial arches, retina, and Kupffer's vesicle.

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.

Zebrafishsmoothenedfunctions in ventral neural tube specification and axon tract formation

Sonic hedgehog (Shh) signaling patterns many vertebrate tissues. shh mutations dramatically affect mouse ventral forebrain and floor plate but produce minor defects in zebrafish. Zebrafish have two mammalian Shh orthologs, sonic hedgehog and tiggy-winkle hedgehog, and another gene, echidna hedgehog, that could have overlapping functions. To examine the role of Hedgehog signaling in zebrafish, we have characterized slow muscle omitted (smu) mutants. We show that smu encodes a zebrafish ortholog of Smoothened that transduces Hedgehog signals. Zebrafish smoothened is expressed maternally and zygotically and supports specification of motoneurons, pituitary cells and ventral forebrain. We propose that smoothened is required for induction of lateral floor plate and a subpopulation of hypothalamic cells and for maintenance of medial floor plate and hypothalamic cells.

The floor plate cells from bovine express the mRNA encoding for SCO-spondin and its translation products

The floor plate (FP) is a transient structure of the embryonic central nervous system (CNS) which plays a key role in development driving cell differentiation and patterning in the ventral neural tube. The fact that antisera raised against subcommissural organ (SCO) secretion immunostain FP cells and react with high-molecular-mass proteins in FP extracts, prompted us to investigate the expression of a SCO-related polypeptide in FP cells. RNA from bovine FP was analyzed by means of reverse transcriptase polymerase chain reaction (RT-PCR), using primers derived from the 3′ end of SCO-spondin which revealed products of 233, 237, 519 and 783 bp. Sequence analysis of the 233 bp PCR fragment confirmed the identity between this FP product and SCO-spondin. FP-translation of the SCO-spondin encoded polypeptide(s) was demonstrated by Western blot analysis and immunocytochemistry, using antisera raised against (i) the glycoproteins secreted by the bovine SCO, and (ii) a peptide derived from the open reading frame of the major SCO secretory protein, SCO-spondin, respectively. Additional evidence pointing to active transcription and translation of a SCO-spondin related gene was obtained in long term FP organ cultures. On the basis of partial sequence homologies of SCO-spondin with protein domains implicated in cell–cell contacts, cell–matrix interactions and neurite outgrowth it is possible to suggest that the SCO-spondin secreted by the FP is involved in CNS development.

Differential Responsiveness to the Chemorepellent Semaphorin 3A Distinguishes Ipsi- and Contralaterally Projecting Axons in the Chick Midbrain

In the chick dorsal mesencephalon, the optic tectum, the developing axons must choose between remaining on the same side of the midline or growing across it. The ipsilaterally projecting axons, forming the tectobulbar tract, course circumferentially toward the ventrally situated floor plate but before reaching the basal mesencephalon, the tegmentum, gradually turn caudally. Here, they follow the course of the medial longitudinal fasciculus (MLF), located parallel to the floor plate. By in vivo labeling of tectal axons, we could demonstrate that these axons arise primarily in the dorsal tectum. To test the idea that chemorepellent molecules are involved in guidance of the nondecussating axons, we performed coculture experiments employing tectal explants from various positions along the dorso-ventral axis. Axons emanating from dorsal tectal explants were strongly repelled by diencephalic tissue containing the neurons that give rise to the MLF whereas ventral tectal axons showed only a moderate response. This inhibitory effect was substantially neutralized by the addition of anti-neuropilin-1 antibodies. A similar differential response of axons was observed when tectal explants were cocultured with cell aggregates secreting the chemorepellent Semaphorin 3A (Sema3A). Sema3B and Sema3C, respectively, did not inhibit growth of tectal axons. In addition, neither the floor plate nor Slit2-secreting cell aggregates influenced outgrowth of dorsal fibers. In Sema3A-deficient mice, DiI-labeling revealed that dorsal mesencephalic axons cross the MLF instead of turning posteriorly upon reaching the fiber tract, thus behaving like the ventrally originating contralaterally projecting axons. A differential responsiveness of tectal axons to Sema3A most likely released by the MLF thus contributes to pathfinding in the ventral mesencephalon.

Crossing the Floor Plate Triggers Sharp Turning of Commissural Axons

During development of the vertebrate CNS, commissural axons initially grow circumferentially toward the ventral midline floor plate. After crossing the floor plate, they abruptly change their trajectory from the circumferential to the longitudinal axis. Although recent studies have unraveled the mechanisms that control navigation of these axons along the circumferential axis, those that result in the transition from circumferential to longitudinal trajectory remain unknown. Here, we examined whether an interaction with the floor plate is a prerequisite for the initiation of trajectory transition of commissural axons, using in vitro preparations of the rat metencephalon. We found that commissural axons in the metencephalon, once having crossed the floor plate, turned sharply to grow longitudinally. In contrast, axons extending in floor plate-deleted preparations, continued to grow circumferentially, ignoring the hypothetical turning point. These results suggest that a prior interaction of commissural axons with floor plate cells is a key step for these axons to activate a navigation program required for their change in axonal trajectory from the circumferential to the longitudinal axis.

A morpholino phenocopy of the cyclops mutation.

Mutations of the cyclops locus produce a characteristic pattern of abnormalities, including prechordal plate and notochord defects, loss of ventral forebrain tissue with cyclopia, deletion of the medial floor plate cells of the spinal cord, and malformation of the tail (“curly tail down morphology”) (Hatta et al., 1991; Thisse et al., 1994; Yan et al., 1995; Odenthal et al., 2000). The various cyclops alleles can be classified according to the severity of the central nervous system (CNS) and eye defects (Brand et al., 1996). However, cyclops mutations have little or no effect on cardiac left–right asymmetry, which stands in sharp contrast to the strong laterality phenotype of many other midline or curly tail down mutations (Chen et al., 1997; Bisgrove et al., 2000; Chin et al., 2000). The cyclops locus itself encodes a member of the nodal-related subclass of the TGF-b superfamily (Rebagliati et al., 1998; Sampath et al., 1998). Molecular and/or genetic data suggest that the weak and strong cyclops mutations are hypomorphic or null alleles, respectively (Hatta et al., 1991; Halpern et al., 1997; Rebagliati et al., 1998; Sampath et al., 1998; Talbot et al., 1998; Gritsman et al., 1999; Miller and Rebagliati, unpublished data). Consistent with this, we report here that injection of an inhibitory cyclops morpholino (CycMO: 59-GCGACTCCGAGCGTGTGCATGATG-39) reproduces the range of phenotypes seen in existing weak and strong cyclops alleles in a dose-dependent fashion (Table 1). Cyc-MO-injected embryos could be placed into two phenotypic categories: a weak class [cyclopia and loss of ventral forebrain similar to that caused by the weak cylops allele, cyc (Brand et al., 1996); Fig. 1B] and a strong class [more severe cyclopia and more extensive ventral forebrain loss, resembling that of the strong cyclops alleles, cyc (Hatta et al., 1991), cyc (Brand et al., 1996) and cyc (Schier et al., 1996), Fig. 1C–E]. These classes correlated well with the dose of injected morpholino (Table 1). A high dose (8 ng) of a negative control morpholino (GeneTool negative control MO: 59-CCTCTTACCTCAGTTACAATTTATA-39) had no effect (n 5 69, data not shown). Variable CNS necrosis was seen, and was most frequent at the two highest Cyc-MO doses (4 and 8 ng). All cyclopic embryos from the weak and strong classes also showed the curly tail down morphology (Fig. 1G). To determine the occurrence of floor plate defects, Cyc-MO-injected embryos were analyzed by in situ hybridization for netrin expression, which marks the medial floor plate and parts of the brain (Strahle et al., 1997). The cyclops morpholino produced the expected floor plate deficit, as evidenced by the partial or complete loss of netrin staining within the embryonic spinal cord [compare Fig. 1H (uninjected) versus Fig. 1I (Cyc-MO-injected)]. Mutations in squint, another zebrafish nodal-related gene, also cause cyclopia and midline defects. This raises the question as to whether the Cyc-MO could also be

Sonic hedgehog and tiggy-winkle hedgehog cooperatively induce zebrafish branchiomotor neurons.

genesisVolume 30, Issue 3 p. 170-174 ArticleFree Access Sonic hedgehog and tiggy-winkle hedgehog cooperatively induce zebrafish branchiomotor neurons Stephanie Bingham, Stephanie Bingham Division of Biological Sciences, University of Missouri, Columbia, MissouriSearch for more papers by this authorAidas Nasevicius, Aidas Nasevicius Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MinnesotaSearch for more papers by this authorStephen C. Ekker, Stephen C. Ekker Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MinnesotaSearch for more papers by this authorAnand Chandrasekhar, Corresponding Author Anand Chandrasekhar [email protected] Division of Biological Sciences, University of Missouri, Columbia, MissouriDivision of Biological Sciences, University of Missouri, Room 205 Lefevre Hall, Columbia, Missouri 65211Search for more papers by this author Stephanie Bingham, Stephanie Bingham Division of Biological Sciences, University of Missouri, Columbia, MissouriSearch for more papers by this authorAidas Nasevicius, Aidas Nasevicius Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MinnesotaSearch for more papers by this authorStephen C. Ekker, Stephen C. Ekker Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MinnesotaSearch for more papers by this authorAnand Chandrasekhar, Corresponding Author Anand Chandrasekhar [email protected] Division of Biological Sciences, University of Missouri, Columbia, MissouriDivision of Biological Sciences, University of Missouri, Room 205 Lefevre Hall, Columbia, Missouri 65211Search for more papers by this author First published: 23 July 2001 https://doi.org/10.1002/gene.1057Citations: 11AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat LITERATURE CITED Beattie CE, Hatta K, Halpern ME, Liu H, Eisen JS, Kimmel CB. 1997. Temporal separation in the specification of primary and secondary motoneurons in zebrafish. Dev. Biol. 187: 171– 182. Chandrasekhar A, Moens CB, Warren Jr. JT, Kimmel CB, Kuwada JY. 1997. Development of branchiomotor neurons in zebrafish. Development 124: 2633– 2644. Chandrasekhar A, Warren Jr. JT, Takahashi K, Schauerte HE, van Eeden FJM, Haffter P, Kuwada JY. 1998. Role of sonic hedgehog in branchiomotor neuron induction in zebrafish. Mech Dev 76: 101– 115. Currie PD, Ingham PW. 1996. Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish. Nature 382: 452– 455. Ekker SC, Ungar AR, Greenstein P, von Kessler DP, Porter JA, Moon RT, Beachy PA 1995. Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr Biol 5: 944– 955. Etheridge LA, Wu T, Liang JO, Ekker SC, Halpern ME. 2001. Floor plate develops upon depletion of Tiggy-winkle and Sonic hedgehog. Genesis (this issue). Higashijima S, Hotta Y, Okamoto H. 2000. Visualization of cranial motor neurons in live transgenic zebrafish expressing green fluorescent protein under the control of the islet-1 promoter/enhancer. J Neurosci 20: 206– 218. Ingham PW. 1998. Transducing Hedgehog: the story so far. The EMBO Jour. 17: 3505– 3511. Krauss S, Concordet JP, Ingham PW. 1993. A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75: 1431– 1444. Lauderdale JD, Pasquali SK, Fazel R, van Eeden FJM, Schauerte HE, Haffter P, Kuwada JY. 1998. Regulation of netrin-1a Expression by Hedgehog Proteins. Mol Cell Neurosci 11: 194- 205. Nasevicius A, Ekker SC. 2000. Effective targeted gene “knockdown” in zebrafish. Nat Genet 26: 216– 220. Schauerte HE, van Eeden FJM, Fricke C, Odenthal J, Strahle U, Haffter P. 1998. Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish. Development 125: 2983– 2993. van Eeden FJ, Granato M, Schach U, Brand M, Furutani-Seiki M, Haffter P, Hammerschmidt M, Heisenberg CP, Jiang YJ, Kane DA, Kelsh RN, Mullins MC, Odenthal J, Warga RM, Allende ML, Weinberg ES, Nusslein-Volhard C. 1996. Mutations affecting somite formation and patterning in the zebrafish, Danio rerio. Development 123: 153– 164. Wicking C, Smyth I, Bale A. 1999. The hedgehog signalling pathway in tumorigenesis and development. Oncogene 18: 7844– 7851. Citing Literature Volume30, Issue3July 2001Pages 170-174 ReferencesRelatedInformation

Expression of Pcdh15 in the inner ear, nervous system and various epithelia of the developing embryo.

We previously determined that Protocadherin 15 (Pcdh15) is associated with the Ames waltzer mutation in the mouse. Here we describe where the Pcdh15 gene is expressed at specific times during mouse development using RNA in situ hybridization. The expression of Pcdh15 is found in the sensory epithelium in the developing inner ear, in Rathke's pouch, and broadly throughout the brain with the highest level of expression being detected at embryonic day 16 (E16). Pcdh15 transcripts are also found in the developing eye, dorsal root ganglion, and the dorsal aspect of the neural tube, floor plate and ependymal cells adjacent to the neural canal. Additionally, expression is also detected in the developing glomeruli of the kidney, surface of the tongue, vibrissae, bronchi of the lung, and in the epithelium of the olfactory apparatus, gut and lung.

A Hedgehog-Insensitive Form of Patched Provides Evidence for Direct Long-Range Morphogen Activity of Sonic Hedgehog in the Neural Tube

Cell pattern in the ventral neural tube is organized by Sonic hedgehog (Shh) secreted by floor plate cells. To assay the range of direct Shh action, we developed a general method for blocking transduction of Hedgehog (Hh) signals through ectopic expression of a deleted form of the Hh receptor Patched (Ptc), termed Ptc Δloop2. We validated this method in Drosophila and used mouse Ptc1 Δloop2 (mPtc1 Δloop2) to block Shh transduction in the chick neural tube. mPtc1 Δloop2 expression caused cell-autonomous ventral-to-dorsal switches in progenitor identity and neuronal fate throughout the ventral neural tube, supporting a gradient mechanism whereby Shh acts directly and at long range. mPtc1 Δloop2 expression also caused the abnormal spread of Shh to more dorsal cells, indicating that Shh in the neural tube, like Hh in Drosophila, induces a feedback mechanism that limits its range of action.

Overexpression of a Slit Homologue Impairs Convergent Extension of the Mesoderm and Causes Cyclopia in Embryonic Zebrafish

Slit is expressed in the midline of the central nervous system both in vertebrates and invertebrates. In Drosophila, it is the midline repellent acting as a ligand for the Roundabout (Robo) protein, the repulsive receptor which is expressed on the growth cones of the commissural neurons. We have isolated cDNA fragments of the zebrafish slit2 and slit3 homologues and found that both genes start to be expressed by the midgastrula stage well before the axonogenesis begins in the nervous system, both in the axial mesoderm, and slit2 in the anterior margin of the neural plate and slit3 in the polster at the anterior end of the prechordal mesoderm. Later, expression of slit2 mRNA is detected mainly in midline structures such as the floor plate cells and the hypochord, and in the anterior margins of the neural plates in the zebrafish embryo, while slit3 expression is observed in the anterior margin of the prechordal plate, the floorplate cells in the hindbrain, and the motor neurons both in the hindbrain and the spinal cord. To study the role of Slit in early embryos, we overexpressed Slit2 in the whole embryos either by injection of its mRNA into one-cell stage embryos or by heat-shock treatment of the transgenic embryos which carries the slit2 gene under control of the heat-shock promoter. Overexpression of Slit2 in such ways impaired the convergent extension movement of the mesoderm and the rostral migration of the cells in the dorsal diencephalon and resulted in cyclopia. Our results shed light on a novel aspect of Slit function as a regulatory factor of mesodermal cell movement during gastrulation.

Hindbrain floor plate of the rat: ultrastructural changes occurring during development.

Most of the molecular and experimental studies on the floor plate (FP) have been performed on the FP region extending along the spinal cord. However, little is known about the hindbrain FP. The FP undergoes regional and temporal changes throughout development, but information with respect to the ultrastructural correlate of such changes is missing. The present investigation was focused on the ultrastructural developmental changes occurring in the FP of the rat hindbrain. The FP cells of the hindbrain secrete a material reacting with antibodies against the secretory glycoproteins of the subcommissural organ (AFRU). This antibody was used to perform an ultrastructural immunocytochemical analysis of the rat FP. From E-12 on, there is a progressive increase in the development of the rough endoplasmic reticulum (RER), so that by E-18, it has reached a high degree of hypertrophy. A unique feature of the hindbrain FP cells is the presence of tubular formations and 140-nm vesicles that appear to originate from RER cisternae. The labelling of these two structures with AFRU and Concanavalin A strongly suggests that they are pre-Golgi compartments containing secretory material. Since these structures are present in the basal process and in the apical cell pole of the FP cells, the possibility that they release their content at these sites, is discussed. It is proposed that a secretory mechanism bypassing the Golgi apparatus (constitutive secretion?) operates in the FP cells. The presence of apoptotic cells within the FP of E-20 embryos and newborns suggests that death, and not re-differentiation, is the fate of the FP cells.

Vertebrate floor plate transiently expresses a compound recognized by antisera raised against subcommissural organ secretion.

Located along the ventral midline of the neural tube, the floor plate (FP) performs an essential role in central nervous system development, especially in the patterning of the ventral region of the neural tube and axonal guidance. Several studies have been directed to the identification of molecules mediating some of the functions of the FP. Most of the models proposed for floor plate actions involve contact-mediated- and/or gradients of diffusible-signals acting throughout the nervous tissue. This report presents and discusses findings showing that the FP cells secrete a novel compound, which is recognized by antisera raised against secretory products of the subcommissural organ (SCO). This immunoreactive compound appears to be very similar to one of the glycoproteins secreted by the SCO. This immunoreactivity is expressed transiently during central nervous system development, and its rostro-caudal extension along the anterior-posterior axis of the FP displays some species variations. However, a constant feature in all species investigated is that this immunoreactive compound is highly expressed in the FP located in the mesencephalic-metencephalic boundary. The distribution of this compound is compatible with basal and apical pathways of release from FP cells. The former might participate in the formation of some brain commissures. The latter might involve the use of the cerebrospinal fluid as a route for performing actions on distant targets, a pathway somehow disregarded by most models accounting for morphogen actions.

Neural patterning: the role of Nkx genes in the ventral spinal cord.

The mammalian nervous system is extremely complex, consisting of many thousands of distinct neuronal cell types, any one of which may require multiple cell interactions to generate a functional circuit. The elaboration of this circuitry is dependent in no small part on the logic of the developmental program that ensures that appropriate cell types are generated at specific positions in the brain or spinal cord, in the correct numbers, and at the correct stage of development. Given the relative simplicity of the spinal cord circuitry, it has become the model of choice for addressing these problems. Only 7 years ago our understanding of neural development in the spinal cord was rudimentary. A number of studies had highlighted the importance of regional organizing centers, both outside and within the developing neural plate and neural tube, in generating the early pattern (for review, see Edlund and Jessell 1999). However, none of these putative inductive signals had been identified. Consequently, there was no molecular framework within which to construct a plausible model for generating cell type diversity in the developing spinal cord. As we fast-forward to the present, the situation is dramatically different. The first major inroad came with the discovery of Sonic hedgehog (Shh; Echelard et al. 1993; Krauss et al. 1993; Riddle et al. 1993; Roelink et al. 1994). Shh encodes a ventralizing signal produced by two ventral midline structures, the notochord, which underlies the ventral neural plate, and the floor plate, a specialized population of glial cells at the ventral midline of the developing hindbrain and spinal cord. It is now clear that Shh is both necessary and sufficient for the induction of the floor plate (notwithstanding evidence that, in some organisms, cell lineage and not simply induction may account for some floor plate cell fates; Le Douarin and Halpern 2000; Placzek et al. 2000), motor neurons, and a number of distinct ventral interneurons (for review, see Jessell 2000). Moreover, there is strong evidence that the concentration of Shh dictates the position where distinct ventral cell identities arise; developmentally naive intermediate neural plate explants respond to small incremental changes in the external concentration of Shh protein to give rise to distinct cell fates (Ericson et al. 1997). At least five distinct cell identities are thought to arise as a primary response to Shh signaling (Fig. 1). These are (from ventral to dorsal) floor plate, V3 interneurons, motor neurons, V2 interneurons, and V1 interneurons. This general model leads to three central questions. How is the Shh concentration gradient generated? How is this gradient translated into distinct transcriptional responses? And finally, what are the transcriptional regulators that lock in a particular neuronal or glial fate? Little progress has been made in addressing the first question. Shh undergoes a unique processing event that appends a cholesterol moiety to the carboxy-terminal amino acid of the amino-terminal signaling moiety (Porter et al. 1996). As a result, the modified protein appears to be retained at the cell surface. However, antibody blocking experiments demonstrate that at least some protein does travel beyond Shh-expressing cells and into the ventral field of Shh-responsive cells (Ericson et al. 1996). This conclusion is also supported indirectly by the observed transcriptional up-regulation of the gene encoding a multipass membrane protein, Patched-1 (Goodrich et al. 1996; Marigo and Tabin 1996), which serves as a general Hedgehog (Hh) receptor and is a general transcriptional target of the pathway. Many lines of evidence indicate that transduction of an Hh signal modulates the activity and/or expression of the Gli/Ci family of zinc-finger DNA binding proteins (for review, see Ruiz i Altaba 1999). In the mouse, which has three Gli family members, Gli2 loss-of-function mutants have a complete absence of floor plate development, consistent with a specific role for Gli2 in mediating the role of Shh in floor plate induction (Ding et al. 1998; Matise et al. 1998). The simplest model would then indicate that other Gli members may direct the development of other ventral cell identities, either uniquely or in combination. However, this does not appear to be the case, at least as far as current publications on the analysis of combinatorial mutants in Gli genes indicate. If this result holds, this leaves open two opposing interpretations. In the first, Shh plays a less direct role in inducing all ventral cell fates, that is, there may be secondary signals. Evidence has been presented for 1E-MAIL amcmahon@mcb.harvard.edu; FAX (617) 496-3763. Article and publication are at www.genesdev.org/cgi/doi/10.1101/ gad.840800.

The metencephalic floor plate of chick embryos expresses two secretory glycoproteins homologous with the two glycoproteins secreted by the subcommissural organ.

The nature and the function of the compounds secreted by the floor plate (FP) of the metencephalon are little known. The FP cells of the hindbrain react with antibodies (AFRU) against the glycoproteins secreted by the subcommissural organ (SCO). One of the these proteins, RF-Gly I, is a 540-kDa core glycosylated protein. The aims of the present investigation were to identify by immunoblot the AFRU-immunoreactive compound secreted by the FP of chick embryos, to establish temporal and regional patterns of this secretory activity, and to obtain information about the fate of these compounds. It was established that the SCO and FP of chick embryos secrete two AFRU-immunoreactive compounds of 540 and 230 kDa. The two compounds secreted by the FP have been designated as FP-Gly I and FP-Gly II. The expression of these proteins was circumscribed to the metencephalic FP, and occurred from HH 29 to HH 36. Within the FP cells, FP-Gly I and FP-Gly II were confined to the supranuclear and apical regions, which under the electron microscope displayed numerous cisternae of the rough endoplasmic reticulum and granules. Aggregates of AFRU-immunoreactive material appeared on the free surface of the FP. The possibility that FP-Gly I and FP-Gly II are released into the ventricle to reach distant targets is discussed.

Complementary expression of transmembrane ephrins and their receptors in the mouse spinal cord: a possible role in constraining the orientation of longitudinally projecting axons.

In the developing spinal cord, axons project in both the transverse plane, perpendicular to the floor plate, and in the longitudinal plane, parallel to the floor plate. For many axons, the floor plate is a source of long- and short-range guidance cues that govern growth along both dimensions. We show here that B-class transmembrane ephrins and their receptors are reciprocally expressed on floor plate cells and longitudinally projecting axons in the mouse spinal cord. During the period of commissural axon pathfinding, B-class ephrin protein is expressed at the lateral floor plate boundaries, at the interface between the floor plate and the ventral funiculus. In contrast, B-class Eph receptors are expressed on decussated commissural axon segments projecting within the ventral funiculus, and on ipsilaterally projecting axons constituting the lateral funiculus. Soluble forms of all three B-class ephrins bind to, and induce the collapse of, commissural growth cones in vitro. The collapse-inducing activity associated with B-class ephrins is likely to be mediated by EphB1. Taken together, these data support a possible role for repulsive B-class Eph receptor/ligand interactions in constraining the orientation of longitudinal axon projections at the ventral midline.

Two Distinct Cell Populations in the Floor Plate of the Zebrafish Are Induced by Different Pathways

The floor plate is a morphologically distinct structure of epithelial cells situated along the midline of the ventral spinal cord in vertebrates. It is a source of guidance molecules directing the growth of axons along and across the midline of the neural tube. In the zebrafish, the floor plate is about three cells wide and composed of cuboidal cells. Two cell populations can be distinguished by the expression patterns of several marker genes, including sonic hedgehog (shh) and the fork head-domain gene fkd4: a single row of medial floor plate (MFP) cells, expressing both shh and fkd4, is flanked by rows of lateral floor plate (LFP) cells that express fkd4 but not shh. Systematic mutant searches in zebrafish embryos have identified a number of genes, mutations in which visibly reduce the floor plate. In these mutants either the MFP or the LFP cells are absent, as revealed by the analysis of the shh and fkd4 expression patterns. MFP cells are absent, but LFP cells are present, in mutants of cyclops, one-eyed pinhead, and schmalspur, whose development of midline structures is affected. LFP cells are absent, but MFP cells are present, in mutants of four genes, sonic you, you, you-too, and chameleon, collectively called the you-type genes. This group of mutants also shows defects in patterning of the paraxial mesoderm, causing U- instead of V-shaped somites. One of the you-type genes, sonic you, was recently shown to encode the zebrafish Shh protein, suggesting that the you-type genes encode components of the Shh signaling pathway. It has been shown previously that in the zebrafish shh is required for the induction of LFP cells, but not for the development of MFP cells. This conclusion is supported by the finding that injection of shh RNA causes an increase in the number of LFP, but not MFP cells. Embryos mutant for iguana, detour, and umleitung share the lack of LFP cells with you-type mutants while somite patterning is not severely affected. In mutants that fail to develop a notochord, MFP cells may be present, but are always surrounded by LFP cells. These data indicate that shh, expressed in the notochord and/or the MFP cells, induces the formation of LFP cells. In embryos doubly mutant for cyclops (cyc) and sonic you (syu) both LFP and MFP cells are deleted. The number of primary motor neurons is strongly reduced in cyc;syu double mutants, while almost normal in single mutants, suggesting that the two different pathways have overlapping functions in the induction of primary motor neurons.

Axon guidance

Axon guidance

Ventral midline cells are required for the local control of commissural axon guidance in the mouse spinal cord.

Specialized cells at the midline of the central nervous system have been implicated in controlling axon projections in both invertebrates and vertebrates. To address the requirement for ventral midline cells in providing cues to commissural axons in mice, we have analyzed Gli2 mouse mutants, which lack specifically the floor plate and immediately adjacent interneurons. We show that a Dbx1 enhancer drives tau-lacZ expression in a subpopulation of commissural axons and, using a reporter line generated from this construct, as well as DiI tracing, we find that commissural axons projected to the ventral midline in Gli2(-/-) embryos. Netrin1 mRNA expression was detected in Gli2(-/-) embryos and, although much weaker than in wild-type embryos, was found in a dorsally decreasing gradient. This result demonstrates that while the floor plate can serve as a source of long-range cues for C-axons in vitro, it is not required in vivo for the guidance of commissural axons to the ventral midline in the mouse spinal cord. After reaching the ventral midline, most commissural axons remained clustered in Gli2(-/-) embryos, although some were able to extend longitudinally. Interestingly, some of the longitudinally projecting axons in Gli2(-/-) embryos extended caudally and others rostrally at the ventral midline, in contrast to normal embryos in which virtually all commissural axons turn rostrally after crossing the midline. This finding indicates a critical role for ventral midline cells in regulating the rostral polarity choice made by commissural axons after they cross the midline. In addition, we provide evidence that interactions between commissural axons and floor plate cells are required to modulate the localization of Nr-CAM and TAG-1 proteins on axons at the midline. Finally, we show that the floor plate is not required for the early trajectory of motoneurons or axons of the posterior commissure, whose projections are directed away from the ventral midline in both WT and Gli2(-/-) embryos, although they are less well organized in Gli2(-/-)mutants.