To determine whether the mouse Msx genes could influence roof-plate differentiation in chick neural tube, we examined three roof-plate markers: Bmp4 (Liem et al., 1995), Wnt1 (Parr et al., 1993) and Lmx1 (Millonig et al., 2000; Yuan and Schoenwolf, 1999) in electroporated embryos. Activation of Bmp signaling by ca-Bmpr1 induced the expression of Bmp4, Wnt1 and Lmx1 in the dorsal neural tube after 24 hours (Fig. 5A,F,K,P). The Lmx1 family members (Lmx1a and Lmx1b) are expressed in the roof plate, floor plate and dI5 population of dorsal interneurons (Gross et al., 2002; Millonig et al., 2000; Muller et al., 2002; Pierani et al., 2001). At 24 hours post electroporation, the induction of Lmx1 expression most probably reflects an induction of roof-plate cell fate because differentiation of dorsal interneurons has not occurred at this stage (Fig. 5K, see control side). Overexpression of Msx1, but not Msx3, also induced all three roof-plate markers after 24 hours, resulting in a ventral expansion of the roof-plate marker expression (Fig. 5, compare B,G,L with C,H,M). However, Bmp signaling is more potent in its ability to induce roof- plate development than Msx1, with more cells ectopically expressing the roof-plate markers and the ectopic expression extended more ventrally, even though the electroporation efficiency in all experiments is comparable, as indicated by the control GFP expression (Fig. 5A-E, insets).
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Understanding when and how multipotent progenitors segregate into diverse fates is a key question during embryonic development. The neural crest (NC) is an exemplary model system with which to investigate the dynamics of progenitor cell specification, as it generates a multitude of derivatives. Based on ‘ in ovo ’ lineage analysis, we previously suggested an early fate restriction of premigratory trunk NC to generate neural versus melanogenic fates, yet the timing of fate segregation and the underlying mechanisms remained unknown. Analysis of progenitors expressing a Foxd3 reporter reveals that prospective melanoblasts downregulate Foxd3 and have already segregated from neural lineages before emigration. When this downregulation is prevented, late-emigrating avian precursors fail to upregulate the melanogenic markers Mitf and MC/1 and the guidance receptor Ednrb2, generating instead glial cells that express P0 and Fabp. In this context, Foxd3 lies downstream of Snail2 and Sox9 , constituting a minimal network upstream of Mitf and Ednrb2 to link melanogenic specification with migration. Consistent with the gain-of-function data in avians, loss of Foxd3 function in mouse NC results in ectopic melanogenesis in the dorsal tube and sensory ganglia. Altogether, Foxd3 is part of a dynamically expressed gene network that is necessary and sufficient to regulate fate decisions in premigratory NC. Their timely downregulation in the dorsal neural tube is thus necessary for the switch between neural and melanocytic phases of NC development.
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as the brain ventricles, the roof plate is expanded: paired lateral ventricles form in the telencephalon, the third ventricle in the diencephalon and the fourth ventricle in the hindbrain. Within these regions, the roof plate comprises a pseudostratified roof plate boundary (at the interface with the neuroepithelium) that borders a broadened single cell layer roof plate epithelium (Landsberg et al., 2005). As embryonic development progresses, this expanded single cell layer roof plate differentiates into a specialised epithelium, which establishes a close relationship with an ingrowing, dense and fenestrated vasculature (Hunter and Dymecki, 2007). The resulting choroid plexuses are a series of ventricular, secretory interfaces that form the blood-cerebrospinal fluid (CSF) barrier (Johansson et al., 2008). Thus, at ventricle regions of the brain, the roof plate is, as in other regions of the CNS, an early embryonic organiser of neuroepithelial development, but later develops into the epithelial component of the choroid plexus. As in the adult brain, the embryonic ventricle- CSF system serves several functions, including the distribution of nutrients, carriage of metabolites and the production of a fluid cushion for its physical protection (Redzic et al., 2005). Additionally, a growing body of evidence implicates the choroid plexus in signalling to the developing neural tube to stimulate proliferation or differentiation of neural progenitors. For example, the fourth ventricle choroid plexus has been shown to induce neurite outgrowth in cerebellar explants via its production of retinoic acid (Yamamoto et al., 1996). More recently it has been shown that CSF-borne Sonic Hedgehog (Shh) regulates the proliferation of cerebellar radial glial cells and production of progenitors of inhibitory neurons (Huang et al., 2010), whereas CSF-borne insulin-like growth factor 2 stimulates the proliferation of cortical neuronal progenitors (Lehtinen et al., 2011).
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progressive recovery of the population (Fig. 4B). By contrast, the numbers of dI2/dP2 cells were only fully recovered after HH stage 18 EP, whereas dI1/dP1 cells were always strongly reduced after Smad6 transfection (Fig. 4B; Fig. 5A,B). These data are consistent with a model in which later blockades of signalling allow higher levels of BMP activity to be reached and therefore more dorsal populations to be specified. Importantly, unlike the removal of Bmp4 in our ex vivo experiments, which resulted in sustained levels of signalling (Fig. 2B), transfection of Smad6 led to a marked Fig. 4. Dorsal progenitors and interneurons require different durations of BMP signalling. (A) Upper panel: expression of Olig3 (dP1- 3) at E4, after electroporation (EP) of Smad6 at the indicated stages (transfected side is on the right of each image). Brackets indicate the extent of Olig3 expression. Lower panel: the graph shows the relative numbers of Olig3 cells after Smad6 EP at the indicated stages. The ratios were calculated by counting the number of Olig3 cells expressing Smad6 and normalizing this to the number in control GFP-transfected embryos. All sections were taken at brachial levels, except for HH stage 12 for which both lumbar (L) and brachial (B) levels were analysed. (B) Relative numbers of each dorsal neuronal subtype after Smad6 EP at the indicated stages (the ratios were calculated as for Olig3 in A). Dorsal interneuron populations were identified as follows: dI1, Lhx2/9 + ; dI2, Lim1/2 + Lbx1 - ;
By initially characterizing a set of positive and negative ac- tivities involving regulated BMP signaling and Hes/Foxd3 interactions, our results provide novel insights into the dy- namic events leading to the transition from the NC to the RP phase of NT development. Four main processes are noteworthy: first, our finding that RP progenitors initially respond to BMP yet lose competence upon relocation to their definitive dorsal midline position in the NT, where they finally consolidate their identity; second, that BMP signaling induces Hes transcription, which in turn down- regulates BMP responsiveness, likely through modulation of Alk3 receptor transcription; third, that downstream of BMP, a cross-repressive interaction between Foxd3 (an NC marker) and Hairy1 (an RP marker) accounts for the temporal and spatial segregation of both lineages; and fourth, that in spite of being refractory, the definitive RP continues producing BMP, which is likely to act upon dor- sal interneurons. The precise time-dependent activities of BMP emanating from the early (NC stage) versus late (RP stage) dorsal NT remain to be defined. These multiple roles of BMP signaling indicate that its function is context dependent and dictated by the regulatory state and com- petence of the target cells. We also notice that RP on- togeny bears significant resemblance to the development of the FP, initiated by Shh signaling in the ventral NT, both in terms of signal duration/intensity followed by re- fractoriness. Future research should focus on unveiling additional genes and interactions that comprise the differ- ential molecular networks underlying the sequential func- tions of BMP on NC, RP, and interneuron development.
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4C,D), which is consistent with a requirement for Wnt/-catenin signaling for the proper expression of this gene in the neural tube (Bonner et al., 2008). Conversely, induction of ectopic notum1a in transgenic embryos eliminates msxc expression in the dorsal neural tube (n25/28), while such a loss of msxc expression was never observed in heat-shocked non-transgenic embryos (Fig. 4E,F). Depletion of Notum 1a had effects limited to the most dorsal domains of zebrafish neural tube. No alterations in the expression of markers of ventral or intermediate neural progenitors such as pax3a, pax6a or foxa were detected in Notum 1a-depleted embryos (supplementary material Fig. S6E-H,K,L) (Dirksen and Jamrich, 1995; Seo et al., 1998; Krauss et al., 1991). Additionally, the domains of markers of differentiated oligodendrocytes and motoneurons in the ventral neural tube, such as lhx3, were not affected in notum1a MO-injected embryos (supplementary material Fig. S6I,J) (Appel et al., 1995). We also examined the consequence of loss of notum1a on cell proliferation and found no difference in the mitotic indices, as assessed by phospho-Histone H3 staining at 24 hpf (control1.9±0.6%, n8 embryos; MO1.9±1.0%, n8 embryos; P0.99) or in the total cell counts (control61.1±1.2 cells/section/embryo, n8 embryos, 2565 cells; MO61.6±1.2, n8 embryos, 5853 cells, P0.73) between the neural tubes of control and notum1a-MO-injected embryos. Furthermore, we did not see alterations in the activity or expression of components of other signaling pathways that may contribute to neural tube patterning in morphant embryos, such as bmp4 (Hammerschmidt et al., 1996). These data indicate that although the depletion of Notum 1a results in an expansion in the domains of dorsally expressed, Wnt- dependent markers located near the source of Wnt signals, such as msxc and notum1a itself, more ventrally expressed markers or overall cell proliferation within the neural tube are not affected. The enhancement of the domains of expression of Wnt-reporter genes, including notum1a itself, following Notum 1a loss of function reveals that it contributes to an autoregulatory loop restricting Wnt signaling close to its source within the dorsal neural tube. Manipulation of Notum 1a activity provides a novel means with which to misregulate Wnts locally and to investigate their role in neural tube development.
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The distinct temporal and spatial characteristics of Neurog2 expression throughout the developing neural tube is regulated by multiple, separable regulatory sequences located 5 ⬘ and 3 ⬘ of the Neurog2 coding region (Scardigli et al., 2001; Simmons et al., 2001). To achieve the complexity of Neurog2 expression, these regulatory sequences are likely targets of field- and lineage-specific transcription factors. In the dorsal neural tube, Neurog2 is expressed in cells that give rise to multiple dorsal interneuron populations, including dI2 and dI4. However, the only known enhancer with activity in the dorsal neural tube (TgN2-4) is active only in progenitors that give rise to the dI4 population. This is evident from the complete loss of β -gal signal from the TgN2-4 reporter in the Ptf1a mutants in which dI4 neurons are lost but the other dorsal interneurons are still present (Glasgow et al., 2005). Not all Neurog2 cells in the dP4/dI4 domain have detectable levels of β -gal from the TgN2-4 transgene (Fig. 2), possibly indicating that regulatory elements even for this restricted domain are missing from this enhancer. These regulatory elements, and the regulatory sequences directing Neurog2 expression to the other dorsal interneuron lineages, have yet to be located and must be in gene regions not yet tested.
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Ascl1 and Ptf1a bind largely distinct sites within neural tube chromatin and have distinct E-box sequence preferences In the dorsal spinal cord, progenitor cells transiently express Ascl1 in the ventricular zone (Fig. 1A,B). As these cells begin to differentiate, a subset of these now postmitotic cells express Ptf1a as they migrate laterally toward the mantle zone (Fig. 1A-B 00 ). Thus, overlap of Ptf1a and Ascl1 expression can be found in a subpopulation of the developing dorsal spinal cord near the ventricular and mantle zone border. The progenitor cells, expressing Ascl1 alone, or with the subsequent expression of Ptf1a, result in the activation of different TFs and neuronal fates in the spinal cord (Fig. 1C) (Glasgow et al., 2005; Nakada et al., 2004). In order to uncover mechanisms by which two neural class II bHLH factors regulate different sets of gene programs that give rise to distinct subtypes of neurons in the dorsal neural tube, we compared and contrasted the genome-wide binding sites of Ascl1 and Ptf1a by ChIP-Seq in E12.5 mouse neural tubes. ChIP-Seq for Ascl1 and Ptf1a have been recently published (Meredith et al., 2013; Sun et al., 2013), but were re-evaluated here and compared using the peak- calling software Homer (Heinz et al., 2010). Using the parameters of a false discovery rate (FDR) cutoff of 0.001, a 4-fold enrichment of sequence tags in the target experiment over control and a cumulative Poisson P-value threshold of 0.0001, Ascl1 was found to bind 4082 sites and Ptf1a was found at 7749 sites, with 1588 of those sites bound by both factors (Fig. 2A; supplementary material Table S1). Heat maps show the binding profiles of Ascl1 and Ptf1a (Fig. 2A). The stringent criteria for peak calling discard many low-affinity Ptf1a- and Ascl1-binding events, and visual inspection of the heat maps (Fig. 2A) suggests that the 1588 overlapping sites might be an
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We have followed the expression of the zic1-4 genes dur- ing early chick development, resulting in a comprehen- sive side-by-side study of zic1-4 gene expression throughout neurulation and somitogenesis. We find that the zic1-3 genes are expressed in partially overlapping domains in the dorsal neural tube and in dorsal portions of somites. In addition, the zic2 gene is uniquely expressed along the entire early neural plate and zic3 is uniquely expressed in the surrounding presomitic meso- derm, suggesting that Zic2 and Zic3 specifically regulate developmental genes during initial formation of the neu- ral tube and somites, respectively. Further, zic2 is expressed in the periotic mesoderm and in limb buds and both zic2 and zic3 are expressed in developing eyes, sug- gesting involvement of these genes in regulating the for- mation of these tissues. We also show that the zic4 gene is expressed in dorsal regions of the future head, but does not appear to be expressed in the chick hindbrain or trunk. Overall, zic gene expression in chick and other organisms shows significant similarities, indicating that the particular strengths of the chick developmental sys- tem will complement current studies of zic genes in other organisms. At the same time, the species-specific differ- ences in zic gene expression that we observe may point to important evolutionary differences, which are of interest in their own right.
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in later stages of somite development. Following somite formation, the ventral somite halves de-epithelialize under the influence of signals from the notochord and ventral neural tube (reviewed by Christ et al., 2004). In the dorsal compartment, the somitic epithelium remains intact under the influence of the neural tube and the surface ectoderm, thus forming the dermomyotome (Kenny- Mobbs and Thorogood, 1987; Christ et al., 1992; Spence et al., 1996). Molecular studies have revealed that the formation of the medial dermomyotome depends on Wnt1 and Wnt3a signaling from the dorsal neural tube (Dietrich et al., 1997; Fan et al., 1997; Marcelle et al., 1997; Wagner et al., 2000). Accordingly, in mouse, Wnt1/3a knockout mice lose the medial aspect of the dermomyotome (Ikeya and Takada, 1998). The lateral dermomyotome is known to depend on signals from the surface ectoderm (Dietrich et al., 1997; Fan and Tessier-Lavigne, 1994; Fan et al., 1997). In recent studies, Wnt6 has been identified as an epithelialization factor from the surface ectoderm which is required for dermomyotome formation and maintenance (Schmidt et al., 2004; Linker et al., 2005). Wnt6 maintains the epithelial morphology of dermomyotomal cells by promoting Paraxis expression via Frizzled7 and ␤ -catenin intracellular signaling (Linker et al., 2005). Until embryonic day 3 the dermomyotome is a continuous epithelial sheet of approximately rectangular shape. At either margin, the epithelial cells form lip-like structures in which cells de-epithelialize and emigrate to form muscle (Gros et al., 2004) and endothelium (Wilting et al., 1995). Later on, the central region of the dermomyotome (CD) deepithelializes completely to give rise to dorsally emigrating dermal and subcutaneous precursor cells, and ventrally emigrating proliferative muscle progenitor cells and satellite cells (Gros et al., 2005; Relaix et al., 2005; Ben-Yair and Kalcheim, 2005). By contrast, the DML and VLL persist as two separate epithelial proliferation zones which are required for ongoing mediolateral growth of the dermomyotome and its derivatives. At embryonic day 7, when the entire dermomyotome has developed into definite tissues, the DML and VLL disintegrate (Ordahl et al., 2001; Venters and Ordahl, 2002). The molecular basis for the differential timing of dermomyotomal de-epithelialization in the margins and the CD has remained elusive.
The NCCs has been a fascinating group of cells because of its multipotency, long range migration through embryo and its capacity to generate a prodigious number of differentiated cell types . However, the biological studies of human NCCs are extremely challenging due to the ethical issues. NCCs derived from hESCs offer an effective approach to study the human neural crest development and the pathology of related-diseases . RBPs play vital functions in a range of biological processes and are involved in many diseases , including multifactorial developmental anomalies. To investigate the roles of RBPs in FAD induced NCC dysfunction, we built three types of FA deficient H9-NCCs models in vitro and screened out the differentially expressed RBPs. Additionally, a FAD mouse model was used to verify the expression of RBPs in the dorsal neural tube parts of embryos enriching neural crest-derived cells and further narrowed down the differentially expressed RBPs into hnRNPC, LAPR6 and RCAN2. Furthermore, knocking down hnRNPC in FAD culture medium promoted H9-NCC viability but was negatively correlated with H9-NCC migration; while LARP6-knockdown H9-NCCs exhibited significantly increased cell viability without significant effects on H9-NCC migration. Knocking down RCAN2 promoted H9-NCC viability as well as migration under FAD conditions. In addition, overexpression of LARP6 could partially mimic the effects of FAD by decreasing the viabilities of H9-NCCs cultured in normal condition. Our results provide insights into the RBPs function in FAD-induced dysfunction of NCCs.
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Interestingly, GPC4 is also expressed in the DML. This observation gave us the opportunity to test whether GPC4 also plays a role in the receiving, responding cells of the DML. We electroporated the medial somites of E2.5 embryos with the GPC4 siRNA. As observed in the dorsal neural tube (see above), this led to a significant decrease in the level of GPC4 transcripts in the DML (Fig. 4G-I; n=5/5). However, this did not affect WNT11 mRNA expression in this structure (Fig. 4J-L; n=6/7), as compared with a Luciferase siRNA that did not alter GPC4 expression (Fig. 4A-C) nor WNT11 expression in the DML (Fig. 4D-F) (n=4/5). Altogether, this indicates that GPC4 does not play any significant role in the DML to regulate WNT11 expression. A similar function of Dlp restricted to the donor, and not in the receiving cells, has been described in the Drosophila embryo (Franch-Marro et al., 2005). In this organism, it was suggested that Dally and Dlp have complementary, yet distinct, functions in Wingless signaling: Dally plays the role of co-receptor in the donor cell, whereas Dlp plays a role in transmitting the signal to the neighboring cell (Franch-Marro et al., 2005).
There are at least three hypothetical scenarios that address the questions posed above (Figure 1B-D). First, repulsive signals from the roof plate or dorsal neural tube may act to repel and/or polarize neural crest cells towards the medioventral direction (Figure 1B). Second, tracking of endothelial cells through their intersomitic journey has revealed the exciting possibility that trunk neural crest cells use endothelial cells as a scaffold and are perhaps directed by endothelial cell signaling to move along the medioventral pathway (Figure 1C). Third, a local secreted or membrane bound factor within the tissue near the dorsal neural tube or somitic mesoderm may attract neural crest cells to move along a medioventral pathway (Figure 1D). Here, we discuss data that support and identify limitations of these three scenarios and develop and simulate a computational model based on a chemotactic model of trunk neural crest cell migration.
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An epithelial-mesenchymal transition (EMT) is the process whereby epithelial cells become mesenchymal cells, and is typified by the generation of neural crest cells from the neuroepithelium of the dorsal neural tube. To investigate the neural crest EMT, we performed live cell confocal time-lapse imaging to determine the sequence of cellular events and the role of cell division in the EMT. It was observed that in most EMTs, the apical cell tail is retracted cleanly from the lumen of the neuroepithelium, followed by movement of the cell body out of the neural tube. However, exceptions to this sequence include the rupture of the neural crest cell tail during retraction (junctional complexes not completely downregulated), or translocation of the cell body away from the apical surface while morphologically rounded up in M phase (no cell tail retraction event). We also noted that cell tail retraction can occur either before or after the redistribution of apical-basolateral epithelial polarity markers. Surprisingly, we discovered that when an EMT was preceded by a mitotic event, the plane of cytokinesis does not predict neural crest cell fate. Moreover, when daughter cells are separated from the adherens junctions by a parallel mitotic cleavage furrow, most re-establish contact with the apical surface. The diversity of cellular mechanisms by which neural crest cells can separate from the neural tube suggests that the EMT program is a complex network of non-linear mechanisms that can occur in multiple orders and combinations to allow neural crest cells to escape from the neuroepithelium.
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A model of patterned vessel ingression into the neural tube that only considers VEGF-A, however, is obviously not sufficient to explain the stereotypical pattern we observed. VEGF-A RNA expression is not localized to areas of ingression, but is broadly expressed, with no observable differences along the dorsal-ventral axis at stages when blood vessels ingress at specific medial locations. Moreover, staining for the heparan sulfate proteoglycans that bind VEGF165 and VEGF189 showed uniform expression along the lateral edge of the neural tube, suggesting that VEGF protein is not preferentially localized to ingression points via matrix binding (J.M.J. and V.L.B., unpublished). We thus conclude that VEGF-A is necessary but not sufficient to pattern the angiogenic blood vessels that enter the developing neural tube. Although it is formally possible that the endothelial cells at the ingression points are uniquely able to respond to the VEGF-A signal due to cell- autonomous differences between them and neighboring endothelial cells, our data do not support such a model, as all PNVP endothelial cells seem capable of responding to ectopic expression of heparin- binding VEGF-A. Likewise, a model whereby egression of motor neurons and/or ingression of DRG neurons physically blocks blood vessel ingression does not account for the extensive areas of the floor plate, ventral neural tube, and dorsal neural tube that do not support ingression of the adjacent PNVP vessels. Our data best support a model in which the positive signals emanating from the neural tube are balanced by negative spatial cues that are also produced by the neural tube and prevent ingression both dorsally and ventrally (Fig. 7B-E). Several signaling pathways are candidates to coordinate with VEGF signaling to pattern vessel ingression into the neural tube, based on the expression of the ligands and their ability to negatively influence vessel migration (see review by Eichmann et al., 2005). Among these are the semaphorins that signal through plexins, the slits that signal through robo receptors and netrins that signal through UNC and DCC receptors. Thus, VEGF-A signaling is predicted to provide a positive spatial cue that, when balanced by a negative spatial cue, is neutralized. However, this balance can be tipped in favor of VEGF-A and vessel ingression by ectopic expression of VEGF-A. In our model endothelial cells are capable of a sophisticated reading of incoming cues, and of integrating these cues to produce a behavior that leads to proper neurovascular communication. Moreover, pathologies such as the CCMs (cerebral cavernous malformations) disrupt a unique communication between the neural and vascular compartment (for reviews, see McCarty, 2005; Lok et al., 2007) that begins at the earliest stages of development.
Studies of Stbm genes and the proteins that they encode in mice, flies, frogs and fish have shown that they have a crucial role in regulating planar cell polarity and convergent extension movements . In fly mutated embryos, the polarity of the ommatidia of the compound eye and the hairs of the wing and thorax are disrupted, such that rather than pointing in the same direction, they point in multiple directions . In zeb- rafish, trilobite mutant embryos (loss of Stbm) have defects in gastrulation movements and posterior migra- tion of hindbrain neurons , resulting in ectopic neural progenitor accumulations and NTDs . In Xenopus, the homolog of Stbm is called xstbm. The xstbm can regulate convergent extension in both dorsal mesoderm and neural tissue by either increasing or decreasing the Vangl2 function due to its optimal retard of convergent extension movements . Reduction of xstbm function using a morpholino antisense oligo also causes the trunk shortening .
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Neurulation varies across species, both with regard to the number of closure points, their timing and the order in which these points close. In mice, the initial neural tube (NT) closure point (closure 1; see Glossary, Box 1) is at the hindbrain/cervical boundary, with a second independent initiation point (closure 2) at the forebrain/midbrain boundary. Closure ( ‘ zippering ’ ) proceeds both rostrally and caudally from these sites (Fig. 2A). A third initiation point (closure 3) is located at the most rostral end of the forebrain and closure proceeds backwards from this site towards closure 2 (Fig. 2A) (Copp and Greene, 2010). These multiple closure initiation sites create three neuropores (open regions of the NT): the anterior and hindbrain neuropores in the cranial region and the posterior neuropore (PNP) in the low spinal region. Neurulation in humans appears to be slightly different: human embryos between Carnegie stages 8 and 13 display two closure initiation sites, corresponding to mouse closures 1 and 3, whereas there is no apparent equivalent to closure 2 in humans (O ’ Rahilly and Muller, 2002). In non-mammalian vertebrates, there is progressively more divergence in the neurulation process with increasing evolutionary separation. The chick, for example, has two points of closure initiation: at the level of the future midbrain and at the hindbrain-cervical boundary, with bi-directional zippering between the sites (Fig. 2B) (Van Straaten et al., 1996). By contrast, Xenopus embryos exhibit closure almost simultaneously along the entire body axis and in teleost fish there is no formation of neural folds at all; rather, the NP cells coalesce to form a neural keel and the NT lumen opens subsequently within this structure.
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Fig. 6. Inhibition of intracellular trafficking rescues the Ovo1 morphant phenotype. (A-C) In situ hybridization for sox10 at 24 hpf. Dorsal views, anterior to the left. Percentages of embryos with NC cell aggregates at the dorsal midline (arrows) with or without BFA treatment are indicated at the bottom of each panel. (A) A severe example with >5 cells per aggregate over both the midbrain and hindbrain. (C) A less severe example with <5 cells per aggregate exclusively located over the hindbrain. (D-I) Ncad:Gfp injections. Ncad:Gfp localizes to the membranes (arrows) of untreated (D) and BFA-treated control cells (G). In Ovo1 morphant cells, Ncad:Gfp also accumulates in the cytoplasm (E,F; arrowheads), but is largely restored to the membrane in BFA-treated Ovo1 morphant cells (H,I). n, nucleus; ot, otic vesicle.
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Our observation of MtPtch1;Ptch1 –/– ;Hhip1 –/– embryos provides strong evidence for this model; in the ventral half of the neural tube, many cells adopted more ventral fates than expected for their relative DV position along the neuraxis, indicating that Hh pathway activity was abnormally heightened in these cells [Fig. 7D, part a). In addition, the total range of Shh signaling in these mutants, shown by Ptch1 LacZ reporter or Nkx6.1 induction, was extended to encompass the entire DV extent of the neural tube. These striking phenotypes of MtPtch1;Ptch1 –/– ;Hhip1 –/– spinal cord underscore the importance of feedback LDA in controlling both the magnitude and range of Shh signaling (Fig. 7D). Two arguments support the assumption that the Hh signaling in MtPtch1;Ptch1 –/– ;Hhip1 –/– neural tube is indeed reflective of the presence of the ligand. First, Ptch1 LacZ reporter expression showed that LIA is intact in MtPtch1;Ptch1 –/– embryos, whereas the ubiquitous and strong activation of the Hh pathway in the Ptch1 –/– neural tube indicates that Ptch1 is the key regulator of LIA, and Hhip1 plays no role in this activity. Given this fact, LIA in MtPtch1;Ptch1 –/– ;Hhip1 –/– embryos should be equivalent to that of MtPtch1;Ptch1 –/– embryos, and the observed changes in ventral specification in the former reflect attenuated LDA. Second, the progenitor domains in MtPtch1;Ptch1 –/– ;Hhip1 –/– neural tube maintain their normal relative positions, and Ptch1 LacZ reporter activity also retains a ventral to dorsal gradient. This is consistent with the idea that they are induced by Shh emanating from the ventral midline sources and thus ventral pattern has a vector that reflects a ventral to dorsal movement of Shh ligand.
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Sequential signalling cascades operate to pattern the developing neural plate and neural tube. For simplicity, only the neural plate is depicted. High levels of SHH (pink arrows) provided by notochord (N) induce floor plate cells (F) in the overlying neuroepithelium. These, in turn, express high levels of SHH and can recruit adjacent cells to adopt a floor plate fate (F ′ ). However, further patterning does not appear to be mediated solely by the lateral propagation of a cascade of inductive signals, as cells depicted in region X cannot induce motor neurons . Instead, a long-range ventralizing signal (green arrows), which may be SHH itself, appears to spread laterally over time to ventralize the neural tube and induce motor neuron differentiation. The spread of this signal at the time of interneuron differentiation is unclear (fading green arrow). However, motor neurons can seemingly induce the differentiation of Engrailed1- expressing interneurons (I) in neural tissue that has not been exposed to a ventralizing signal. It is unclear whether the induction of Engrailed1- expressing interneurons is a direct effect (blue arrows) or whether it is mediated by secondary cell types that are also induced by motor neurons.