Table of contents

Consequences of Hedgehog expression in notochord and precordal mesoderm on neural development

By using the quail-chicken chimera system, it has been shown that during development of the spinal cord, floor plate cells are inserted between neural progenitors giving rise to the alar plates. These cells are derived from the regressing Hensen's node or chordoneural hinge (HN-CNH). This common population of HN-CNH cells gives rise to three types of midline descendants: notochord, floor plate, and dorsal endoderm. HNF3beta, an important gene in the development of the midline structures, is continuously expressed in the HN-CNH cells and their derivatives: floor plate, notochord, and dorsal endoderm. Experiments in which the notochord was removed in the posterior region of either normal chicken or of quail-chicken chimeras in which a quail HN had been grafted demonstrate that the floor plate develops in a cell-autonomous manner in the absence of notochord. Sonic hedgehog released from the notochord has been thought to be an inducer of floor plate. Absence of floor plate observed at the posterior level of the excision results from removal of HN-CNH material, including the future floor plate, and not from the lack of an inductive signal of notochord origin. This cell lineage analysis of gastrulation and neurulation thus leads to revision of the model previously proposed, according to which the neural plate forms first as a continuous sheet of epithelial cells in which the notochord induces, via Sonic hedgehog the overlying ectodermal cells to become the floor plate. In fact, the neural plate forms according to a more complex process, since its midline component, the floor plate, has an embryonic origin different from its lateral ones that yield the basal and alar plates of the neural tube and the neural crest (Teillet, 1998).

Pax6 is a paired-type homeobox gene expressed in discrete regions of the central nervous system. In the spinal cord of 7- to 10-somite-stage chicken embryos, Pax6 is not detected within the caudal neural plate, but is progressively upregulated in the neuroepithelium neighbouring each newly formed somite. This initial activation of Pax6 is controlled via the paraxial mesoderm in correlation with somitogenesis. High levels of Pax6 expression occur independent of the presence of SHH-expressing cells when neural plates are maintained in culture in the presence of paraxial mesoderm. Grafting a somite caudally under a neural plate that has not yet expressed the gene induces a premature activation of Pax6. Furthermore, after the graft of a somite, a period of incubation corresponding to the individualization of a new somite in the host embryo produces an appreciable activation of Pax6. Conversely, Pax6 expression is delayed under conditions where somitogenesis is retarded, i.e., when the rostral part of the presomitic mesoderm is replaced by the same tissue isolated more caudally. Finally, Pax6 transcripts disappear from the neural tube when a somite is replaced by presomitic mesoderm, suggesting that the somite is also involved in the maintenance of Pax6 expression in the developing spinal cord. All together these observations lead to the proposal that Pax6 activation is triggered by the paraxial mesoderm in phase with somitogenesis in the cervical spinal cord. With respect to the role of SHH in Pax6 induction, prospective neural plates isolated caudal to Hensen’s node and maintained in vitro display Pax6 expression in the absence of SHH producing notochordal and floor plate cells. Also, the presence of the notochord expressing SHH is not sufficient to upregulate and maintain Pax6 expression in the cervical spinal cord after removal of a somite. Consequently, it is proposed that mechanisms others than SHH signaling may be involved in regulating Pax6 expression in the neural tube. Molecular evidence suggests that a developmental clock may be linked to somitogenesis of the paraxial mesoderm. The developing spinal cord has no obvious anteroposterior landmarks, but genes such as Pax6 are activated at precise times and locations along the rostrocaudal axis and such an activation correlates with somitogenesis. It is therefore tempting to speculate that, at least in some regions of the developing spinal cord, somitogenesis may be used as a clock to activate specific genes in a temporally and spatially appropriate manner. Together, these data argue in favour of a model in which Pax6 is activated in the cervical spinal cord via a positive signal from the somite, this signal being maintained at least for the next few hours to stabilize the gene expression. The nature of the signaling molecule mediating Pax6 upregulation remains unknown. The fact that a preincubation of the somite in blocking anti-SHH antibodies does not abolish the activity of the somite suggests that the factor is not SHH, even if this molecule is well known for its ability to upregulate Pax6 expression (Pituello, 1999)

Ventral midline cells at different rostrocaudal levels of the central nervous system exhibit distinct properties but share the ability to pattern the dorsoventral axis of the neural tube. Ventral midline cells acquire distinct identities in response to the different signaling activities of underlying mesoderm. Signals from prechordal mesoderm control the differentiation of rostral diencephalic ventral midline cells, whereas notochord induces floor plate cells caudally. Sonic hedgehog (SHH) is expressed throughout axial mesoderm and is required for the induction of both rostral diencephalic ventral midline cells and floor plate. However, prechordal mesoderm also expresses BMP7, whose function is required coordinately with SHH to induce rostral diencephalic ventral midline cells. BMP7 acts directly on neural cells, modifying their response to SHH so that they differentiate into rostral diencephalic ventral midline cells rather than floor plate cells. These results suggest a model whereby axial mesoderm both induces the differentiation of overlying neural cells and controls the rostrocaudal character of the ventral midline of the neural tube (Dale, 1997).

The neural plate is subdivided into distinct anterior-posterior domains that have different responses to inductive signals from the prechordal plate, Sonic Hedgehog, the anterior neural ridge and FGF8. For example, Engrailed 2 is induced by beads placed more posteriorly than those that induce BF1. The induced BF1-expression domain is delineated posteriorly by a sharp boundary, which may be orthogonal to the long axis of the explants. The posterior boundary of BF1 and the anterior boundary of En2 are nearly adjacent. In sum, these results suggest that regionalization of the forebrain primordia is established by several distinct patterning mechanisms: (1) anterior-posterior patterning creates transverse zones with differential competence within the neural plate; (2) patterning along the medial-lateral axis generates longitudinally aligned domains and (3) local inductive interactions, such as a signal(s) from the anterior neural ridge, further define the regional organization (Shimamura, 1997).

Zebrafish neurogenin1 (Drosophila homolog: Atonal) encodes a basic helix-loop-helix protein that shares structural and functional characteristics with proneural genes in Drosophila melanogaster. neurogenin1 is expressed in the early neural plate in domains comprising more cells than the primary neurons known to develop from these regions; its expression is modulated by Delta/Notch signaling, suggesting that it is a target of lateral inhibition. Misexpression of neurogenin1 in the embryo results in development of ectopic neurons. Markers for different neuronal subtypes are not ectopically expressed in the same patterns in neurogenin1-injected embryos suggesting that the final identity of the ectopically induced neurons is modulated by local cues. Induction of ectopic motor neurons by neurogenin1 requires coexpression of a dominant negative regulatory subunit of protein kinase A, an intracellular transducer of Hedgehog signals. Inhibition of ngn1 expression in the lateral plate in embryos injected with constitutively active PKA suggests that PKA may act as a dominant repressor of ngn1 expression. The pattern of endogenous neurogenin1 expression in the neural plate is expanded in response to elevated levels of Hedgehog (Hh) signaling or abolished as a result of inhibition of Hh signaling. Other factors induced by Hedgehogs must be required in addition to Neurogenin1 for development of motor neurons. It is possible that ngn1 expression in the lateral neural plate is controlled by BMP4/7 and that the interplay of the two signaling centers causes the striped pattern of Ngn1 in the posterior neural plate. Together these data suggest that Hh signals regulate neurogenin1 expression and subsequently modulate the type of neurons produced by Neurogenin1 activity (Blader, 1997).

Sonic Hedgehog signaling is required to specify motor neuron identity. SHH activity is required for induction of floor plate differentiation by the notochord and independently, later, for the induction of motor neurons by both the notochord and midline neural cells. Motor neuron generation depends on two critical periods of SHH signaling: an early period during which naive neural plate cells are converted into ventralized progenitors and a late period that extends well into S phase of the final progenitor cell division, during which SHH drives the differentiation of ventralized progenitors into motor neurons. During the early period, high SHH exposure results in the extinction of pax7 (See gooseberry-distal for a Drosophila paired homeodomain protein involved in neurogenesis) in neural plate cells close to the notochord resulting in an interneuron fate. The expression of other homeobox genes, notably pax3, is also repressed by the notochord and by SHH. The ambient SHH concentration during the late period, generated by both notochord and floor plate, determines whether ventral progenitors differentiate into motor neurons (intermediate SHH) or interneurons (low SHH), thus defining the pattern of neural cell types generated in the neural tube. Interneurons are characterized by subsequent expression of Lim1/2, while motor neurons express Lim domain protein Isl1/2 (See Apterous for a Drosophila Lim homeodomain protein involved in neurogenesis) (Ericson, 1996).

At the time the motor neuron is generated, the extracellular matrix protein vitronectin and its mRNA are both present in the embryonic chick notochord, floor plate and in the ventral neural tube. Chick vitronectin has 453 amino acids : overall amino acid identity between chick and mammalian vitronectins ranges from 53% to 56%. However, homology increases significantly around the known functional domains, such as the RGD domain involved in the binding to integrin receptors, and the basic region that binds to heparin. When added to cultures of neural tube explants of developmental stage 9, vitronectin promotes the generation of motor neurons in the absence of either notochord or exogenously added Sonic hedgehog. Conversely, the neutralization of endogenous vitronectin with antibodies inhibits over 90% motor neuron differentiation in co-cultured neural tube/notochord explants, neural tube explants cultured in the presence of Sonic hedgehog, and in committed (stage 13) neural tube explants. Treatment of embryos with anti-vitronectin antibodies results in a substantial and specific reduction in the number of motor neurons generated in vivo. These results demonstrate that vitronectin stimulates the differentiation of motor neurons in vitro and in vivo. The treatment of stage 9 neural tube explants with Sonic hedgehog results in induction of vitronectin mRNA expression before the expression of floor plate marker HNF-3beta, a gene regulating the differentiation of the floor plate, and also before that of Islet-1, necessary for motor neuron differentiation. It is concluded that vitronectin may act either as a downstream effector in the signaling cascade induced by Sonic hedgehog, or as a synergistic factor that increases Shh-induced motor neuron differentiation (Martinez-Morales, 1997).

Sonic hedgehog (Shh) is thought to control the generation of motoneurons (MNs) and interneurons in the ventral CNS. Shh mutant mice lack floor plate cells and MNs thus establishing an essential role for Shh in the generation of certain ventral neurons. In addition to MNs, however, ventral progenitors give rise to four major classes of interneurons that can be defined by homeodomain protein expression. V0 and V1 neurons derive from the dorsal region of the ventral neural tube and express Evx1/2 (V0) and En1 (V1), respectively. A more ventral class of V2 neurons expresses Chx10 (a paired-type homeodomain protein related to Aristaless). Finally, the region between floor plate cells and MNs generates V3 neurons, defined by expression of Nkx2.2 (a homolog of Drosophila Vnd). Elimination of the Eyeless homolog Pax6 perturbs the differentiation of both V1 interneurons and MNs, but the broad domain of Pax6 expression is suggestive of a more general patterning function. A pair of homeobox genes, Dbx1 and Dbx2, are expressed by progenitor cells in a domain of the ventral spinal cord that appears to overlap the position of V0 and V1 neuron generation, raising the possibility that they contribute to the generation of V0 and/or V1 neurons (Pierani, 1999 and references).

Dbx homeodomain proteins are related to a novel homeobox gene from Drosophila, designated H2.0 [see Barad, M., Erlebacher, A., McGinnis, W. (1991). Dev. Genet. 12: 206-211]. H2.0 has the most diverged homeobox so far characterized in metazoa, and, in contrast to all previously isolated homeobox genes, H2.0 exhibits a tissue-specific pattern of expression. The cells that accumulate transcripts for this novel gene correspond to the visceral musculature and its anlagen. It has been shown that a small deletion, which eliminates H2.0, has no detectable effect on normal gut morphogenesis, visceral muscle actin organization, or larval peristalsis (Pierani, 1999).

To define progenitor populations within the intermediate region of the developing spinal cord, antibodies were generated that react selectively with Dbx1 and Dbx2. In chick, Dbx1 and Dbx2 are first expressed at approximately stage 13 at caudal hindbrain and cervical levels, whereas more caudally, only Dbx1 expression is detected. By stage 15, however, Dbx1 and Dbx2 were expressed along the rostrocaudal axis of the spinal cord. From the outset, the expression of Dbx1 and Dbx2 is restricted to the intermediate region of the neural tube, but the domain of expression of Dbx2 extends more ventrally than that of Dbx1. Within the central domain where both Dbx1 and Dbx2 are expressed, cells that express high levels of Dbx1 expressed low levels of Dbx2 and vice versa. The relationship between ventral Dbx progenitors and the position of generation of V0 and V1 neurons was examined. At early developmental stages, neural progenitors migrate and differentiate into neurons in a strict mediolateral progression. Ventral (v) Evx1/2 (V0) neurons are generated from stages 17-18 and are found initially within the ventral domain of expression of Dbx1 and Dbx2, although they later migrated ventrally. From stage 21, a more dorsal (d) set of Evx1/2 neurons is detected. These neurons lack Lim1/2 expression but coexpress LH2A/B and thus are D1 neurons. En1, Lim1/2 (V1) neurons are generated from stage 17, and most appear ventral to the domain of Dbx1 expression. The expression of Dbx2 is extinguished as progenitors leave the cell cycle, whereas Dbx1 expression persists in neurons for a brief period. Many neurons that coexpressed Dbx1 and Lim1/2 and occasional Dbx1, Evx1/2 neurons were detected, supporting the idea that Dbx1 progenitors give rise to V0 neurons. Dbx expression is not detected in En1 neurons, possibly because of the relatively late onset of expression of En1. Together, these findings suggest that V0 neurons derive from the ventral domain of Dbx1 and Dbx2 expression, whereas V1 neurons derive primarily from more ventral Dbx2on, Dbx1off progenitors (Pierani, 1999).

A Shh-independent pathway of interneuron generation operates in the ventral spinal cord. Evidence for this parallel pathway emerged from an analysis of the induction of ventral progenitors that express the Dbx homeodomain proteins and of Evx1/2 (V0) and En1 (V1) neurons. Shh signaling is sufficient to induce Dbx cells and V0 and V1 neurons but is not required for their generation in vitro or in vivo. These neurons are generated in culture even when Shh is eliminated with anti Shh antiserum. These neurons are also generated in Shh mutant mice. These results confirm the existence of a Shh-independent signaling pathway and suggest that this parallel pathway is involved selectively in the induction of Dbx progenitors and V0 and V1 neurons. Retinoids are expressed at high levels by caudal paraxial mesoderm and also by notochord precursors. Retinoids appear to mediate the parallel pathway for the generation of V0 and V1 neurons, acting in this inductive capacity from the paraxial mesoderm. Shh-mediated induction of Dbx1 and Dbx2 cells is not inhibited by addition of retinoid receptor antagonists. Conversely, explants exposed to retinoids do not express Shh (Pierani, 1999). The spatial restriction in the pattern of Dbx expression appears to be controlled by two repressive pathways, mediated by high-level Shh signaling and BMPs. Importantly, the domain of the ventral neural tube within which parallel Shh and retinoid signaling can operate is likely to be defined by the graded nature of Shh signaling and its consequent effects on Dbx expression. In this view, the key step in establishing a domain of neurogenesis that depends exclusively on Shh signaling is the high level of Shh signaling activity that operates in more ventral regions of the neural tube and is sufficient to repress Dbx expression, despite ongoing retinoid signaling. In a similar manner, the dominant inhibitory action of BMPs may underlie the exclusion of Dbx expression from dorsal extremes of the neural tube, despite exposure of these cells to retinoid signals. The enhanced sensitivity of Dbx1 to repression by Shh and BMPs may underlie the establishment of the distinct boundaries of expression of Dbx1 and Dbx2 within the neural tube. Together, these results support the idea that Shh and retinoids induce Dbx progenitors through independent pathways. These findings reveal an unanticipated Shh-independent signaling pathway that controls progenitor cell identity and interneuron diversity in the ventral spinal cord (Pierani, 1999).

Neural tube closure is a fundamental embryonic event whose molecular regulation is poorly understood. As mouse neurulation progresses along the spinal axis, there is a shift from midline neural plate bending to dorsolateral bending. Midline bending is not essential for spinal closure: in its absence, the neural tube can close by a 'default' mechanism involving dorsolateral bending, even at upper spinal levels. Midline and dorsolateral bending are regulated by mutually antagonistic signals from the notochord and surface ectoderm. Notochordal signaling induces midline bending and simultaneously inhibits dorsolateral bending. Sonic hedgehog is both necessary and sufficient to inhibit dorsolateral bending, but is neither necessary nor sufficient to induce midline bending, which seems likely to be regulated by another notochordal factor. Attachment of surface ectoderm cells to the neural plate is required for dorsolateral bending, which ensures neural tube closure in the absence of sonic hedgehog signaling (Ybot-Gonzalez, 2002).

Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks

Human embryonic stem cells (hESCs) offer a platform to bridge what has been learned from animal studies to human biology. Using oligodendrocyte differentiation as a model system, this study shows that sonic hedgehog (SHH)-dependent sequential activation of the transcription factors OLIG2, NKX2.2 and SOX10 is required for sequential specification of ventral spinal OLIG2-expressing progenitors, pre-oligodendrocyte precursor cells (pre-OPCs) and OPCs from hESC-derived neuroepithelia, indicating that a conserved transcriptional network underlies OPC specification in human as in other vertebrates. However, the transition from pre-OPCs to OPCs is protracted. FGF2, which promotes mouse OPC generation, inhibits the transition of pre-OPCs to OPCs by repressing SHH-dependent co-expression of OLIG2 and NKX2.2. Thus, despite the conservation of a similar transcriptional network across vertebrates, human stem/progenitor cells may respond differently to those of other vertebrates to certain extrinsic factors (Hu, 2009).

Inaccessibility to human embryo experimentation calls for an alternative, in vitro model to study human cells or tissues directly. In recent years, directed neural differentiation from human embryonic stem cells (hESCs) has allowed a re-examination of the fundamental principles of early neural development learned from vertebrate studies. The present study revealed that human oligodendrocyte development involves a transcriptional network of nearly identical sequence to that observed in vertebrate models. Following expression of OLIG2 and genesis of motoneurons, the human OLIG2 progenitors become pre-OPCs by co-expressing NKX2.2, and finally differentiate into OPCs by activation of SOX10 and PDGFRα. Blocking OLIG2 expression inhibits OPC production, confirming the requirement of OLIG2 for human OPC specification. The vast majority of human OLIG2 progenitors are generated in a SHH-dependent manner, because cultures without exogenous SHH, or those in which endogenous SHH signaling has been blocked with cyclopamine, have few OLIG2 progenitors and OPCs. It was also found that SHH is not only crucial for efficiently inducing pre-OPCs, but it is also required for the transition from pre-OPCs to OPCs, for which endogenous SHH signaling is sufficient. Thus, the SHH-dependent signaling network underlying vertebrate OPC development is conserved in humans (Hu, 2009).

This study also reveals unique aspects of human OPC generation. In human OPC differentiation cultures, NKX2.2 is the first OPC-related transcription factor that is co-expressed with OLIG2 at the fifth week, preceding the expression of PDGFRα. This expression pattern resembles that in the chick and in the mouse hindbrain, but differs from that in the mouse spinal cord, where NKX2.2 expression is induced after PDGFRα+ OPCs are formed. This might be partly related to the hindbrain/cervical spinal identity of the human progenitors patterned by RA, although species differences cannot be excluded. A protracted transition period was found from pre-OPCs at the fifth week to human OPCs at the fourteenth week. In chick and mouse brainstem, the OLIG2+ NKX2.2+ progenitors quickly express PDGFRα and become migrating OPCs. OLIG2 progenitors differentiated from mESCs also co-express PDGFRα and NG2 shortly following motoneuron differentiation. It is possible that the serum-free culture conditions are not optimal for acquisition of the OPC identity at an earlier time. The addition of FGF2, EGF, SHH or noggin, however, did not accelerate the transition of pre-OPCs to OPCs. Similarly, removal of PDGF, NT3 and/or IGF1 did not alter the time course of OPC generation either. Alternatively, this protracted transition might be intrinsically controlled. The long OPC-specification process (3 months) appears to match the earliest appearance of OPCs in human embryos at the end of the first trimester. One potential explanation for the late appearance of OPCs is a need for expanding neurogenic progenitors, as large numbers of neurons are needed for the evolutionarily enlarged human brain and spinal cord (Hu, 2009).

FGF2 is a mitogen for rodent and human neural stem/progenitor cells and enhances the generation of OPCs in rodents. In the present study, it is interesting that following the induction of OLIG2, FGF2 nearly completely blocked motoneuron differentiation and preferentially increased the proportion of OLIG2 progenitors. The inhibition of motoneuron differentiation is not attributable to the mitogenic effect of FGF2 preventing the OLIG2 progenitors from exiting the cell cycle. Nor does FGF2 preferentially promote the survival of the OLIG2 progenitors. It is likely that FGF2 enhances the transition from the neurogenic OLIG2 progenitors to pre-OPCs. Although fine dissection of the mechanism is not possible with the available system, this finding provides, as it stands, a simple way of switching neurogenesis to gliogenesis from a pool of progenitors (Hu, 2009).

Continued use of FGF2 inhibited the generation of OPCs from hESCs. This is reminiscent of observations in the past decade that human neural stem/progenitor cells, following expansion with FGF2, or FGF2 and EGF, rarely produce oligodendrocytes in vitro. Even when the human neural progenitor cultures are enriched for OPCs, the OPCs quickly disappear after culturing in the presence of FGF2. The present finding that co-expression of OLIG2 and NKX2.2 is suppressed by FGF2 in the pre-OPCs explains the phenomenon. The inhibitory effect appears specific to FGF2, as EGF did not affect the co-expression of OLIG2 and NKX2.2 and subsequent generation of OPCs. FGF2 induces OPC generation from mouse neural stem/progenitor cells by activating endogenous SHH signaling or by as yet unknown SHH-independent pathways. In the human cell differentiation system, FGF2 inhibits endogenous SHH expression and significantly increases the level of GLI2 and GLI3, which are downstream repressors of SHH signaling, thus disrupting co-expression of OLIG2 and NKX2.2 in pre-OPCs. This results in the loss of pre-OPCs and the conversion to progenitors expressing OLIG2 (at a low level) or NKX2.2, which generates astrocytes or neurons. Nevertheless, the possibility was not excluded that FGF does this through other oligodendroglial transcription factors, such as SOX10 and ASCL1, or by selectively promoting the survival/expansion of neuronal progenitors in the long-term cultures (Hu, 2009).

The present study has developed an effective strategy for reproducibly directing hESCs to an enriched population of OPCs with an unequivocal oligodendrocyte identity and myelination potential. In the previous reports of OPC differentiation from hESCs, SHH was not applied and the expression of OLIG2 in neural progenitors was not examined by single-cell-resolution immunocytochemistry, but instead by PCR on bulk cultures. Based on these findings, it is believed that the OPCs described in those reports are differentiated from OLIG2 progenitors spontaneously induced by endogenous or alternatively activated SHH signaling. The near pure population of 'OPCs' generated from hESCs without application of SHH cannot be explained by the current model, nor has the result been replicated by in other reports. The identity of the OPCs in that report has not been unequivocally verified either (Hu, 2009).

The SHH-dependent transcriptional network underlying human OPC specification and the time course of OPC generation, consistent with that in human embryo development, revealed in the present study suggest that the hESC differentiation system is a useful tool for understanding the biology of human cells. The divergent responses to common growth factors such as FGF2 between human and other vertebrate cells might be related to the in vitro system, but may also reflect the nature of normal human development. Similar to the present finding, the maintenance of human and mouse ESCs depends on the same transcriptional network. However, the common extrinsic factor, BMP, maintains the self-renewal of mESCs but induces cell differentiation of hESCs. This seemingly 'trivial' deviation has slowed down the translation of findings from mESCs to the establishment of hESCs. Similarly, over the past decade, many laboratories have stumbled in trying to replicate the finding in rodents, so as to differentiate human neural stem/progenitors to OPCs. Thus, the confirmation of conserved principles and the revelation of 'nuances' using the hESC differentiation system might bear significant consequences (Hu, 2009).

Sonic hedgehog and floor plate structure

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 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 that 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, wherein 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-shaped instead of V-shaped somites. One of the you-type genes, sonic you, encodes the zebrafish Shh protein, suggesting that the you-type genes encode components of the Shh signaling pathway. 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, 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 (Odenthal, 2000).

Reported here is the expression of the zebrafish zic1 gene, also known as opl, a homolog to other vertebrate Zic genes and the Drosophila odd-paired gene. zic1 expression starts during epiboly stages in lateral parts of the neural plate and eventually comes to lie in dorsal regions of the developing brain following the morphogenetic movements of neural tube formation. To determine whether BMP2 signaling affects the extent of zic1 expression, swirl and chordino mutant embryos were examined. Expanded Zic1 expression in swirl and reduced expression in chordino as well as in bmp2 injected embryos suggest that BMP2 and its antagonists define the extent of zic1 expression in the neural plate. By searching for factors responsible for the dorsal restriction of Zic1 expression, it was found that zic1 expression is eliminated in sonic hedgehog (shh) injected embryos. However, the most rostral expression is not affected by Shh, suggesting that Shh plays a different role in dorso-ventral patterning of the future telencephalon. During somitogenesis zic1 is expressed in the dorsal most part of the developing somites. Here zic1 marks cells that are distinct from the main adaxial somite portion, the future myomere. zic1 expression in the somites is expanded in swirl but reduced in shh injected embryos, suggesting these factors have opposing activity in dorsoventral patterning of the somites. Later, a growing mass of zic1 expressing cells occurs in a dorsal mesenchyme that eventually invades the dorsal fin fold, suggesting a somitic contribution to the dorsal fin mesenchyme (Rohr, 1999).

To begin to reconcile models of floor plate formation in the vertebrate neural tube, experiments aimed at understanding the development of the early floor plate were performed in the chick embryo. Using real-time analyses of cell behavior, evidence is provided that the principal contributor to the early neural midline, the future anterior floor plate, exists as a separate population of floor plate precursor cells in the epiblast of the gastrula stage embryo, and does not share a lineage with axial mesoderm. Analysis of the tissue interactions associated with differentiation of these cells to a floor plate fate reveals a role for the nascent prechordal mesoderm, indicating that more than one inductive event is associated with floor plate formation along the length of the neuraxis. Nr1, a chick nodal homolog, is expressed in the nascent prechordal mesoderm, and evidence is provided that Nodal signalling can cooperate with Shh to induce the epiblast precursors to a floor-plate fate. These results indicate that a shared lineage with axial mesoderm cells is not a pre-requisite for floor plate differentiation and suggest parallels between the development of the floor plate in amniote and anamniote embryos (Patten, 2003).

The secreted ligand Sonic Hedgehog (Shh) organizes the pattern of cellular differentiation in the ventral neural tube. For the five neuronal subtypes, increasing levels and durations of Shh signaling direct progenitors to progressively more ventral identities. This study demonstrates that this mode of action is not applicable to the generation of the most ventral cell type, the nonneuronal floor plate (FP). In chick and mouse embryos, FP specification involves a biphasic response to Shh signaling that controls the dynamic expression of key transcription factors. During gastrulation and early somitogenesis, FP induction depends on high levels of Shh signaling. Subsequently, however, prospective FP cells become refractory to Shh signaling, and this is a prerequisite for the elaboration of their identity. This prompts a revision to the model of graded Shh signaling in the neural tube, and provides insight into how the dynamics of morphogen signaling are deployed to extend the patterning capacity of a single ligand. In addition, evidence is provided supporting a common scheme for FP specification by Shh signaling that reconciles mechanisms of FP development in teleosts and amniotes (Ribes, 2010).

Following initial induction of FP specification, Shh signaling is attenuated in presumptive FP cells. Maintaining Shh signaling at this time converts FP cells to ventral neural progenitors, demonstrating that the down-regulation of signaling is a prerequisite for the elaboration of FP identity. Thus, the specification of the FP and other ventral neuronal progenitors depends on distinct timing and duration of Shh signaling. Consistent with this, it was demonstrated that presumptive FP cells display a dynamic transcriptional code that distinguishes FP precursors from ventral neural progenitors. Evidence is provided that FoxA2 is required for FP specification and the inhibition of p3 fate. Taken together, the data indicate that, in all vertebrates, FP induction takes place in a brief time window during the course of gastrulation, and the extrinsic signals involved in this process regulate FoxA2 expression. The difference between species resides mainly in the relative contribution of each signal. It is therefore tempting to hypothesize that both Shh and Nodal signals were involved in FP specification in the common ancestor of vertebrates. Subsequently, the relative importance of each signal changed during the evolution of individual species. Detailed analysis of the regulatory elements directing expression of FoxA2 in different species should shed further light on this hypothesis (Ribes, 2010).

Table of contents

hedgehog continued: Biological Overview | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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