The cytokine leukemia inhibitory factor (LIF) drives self-renewal of mouse embryonic stem (ES) cells by activating the transcription factor STAT3. In serum-free cultures, however, LIF is insufficient to block neural differentiation and maintain pluripotency. Bone morphogenetic proteins act in combination with LIF to sustain self-renewal and preserve multilineage differentiation, chimera colonization, and germline transmission properties. ES cells can be propagated from single cells and derived de novo without serum or feeders using LIF plus BMP. The critical contribution of BMP is to induce expression of Id genes via the Smad pathway. Forced expression of Id liberates ES cells from BMP or serum dependence and allows self-renewal in LIF alone. Upon LIF withdrawal, Id-expressing ES cells differentiate but do not give rise to neural lineages. It is concluded that blockade of lineage-specific transcription factors by Id proteins enables the self-renewal response to LIF/STAT3 (Ying, 2003).

Temporal changes in progenitor cell responses to extrinsic signals play an important role in development, but little is known about the mechanisms that determine how these changes occur. In the rodent CNS, expression of epidermal growth factor receptors (EGFRs) increases during embryonic development, conferring mitotic responsiveness to EGF among multipotent stem cells. Cell-cell signaling controls this change. Whereas EGF-responsive stem cells develop on schedule in explant and aggregate cultures of embryonic cortex, co-culture with younger cortical cells delays their development. Exogenous BMP4 mimics the effect of younger cells, reversibly inhibiting changes in EGFR expression and responsiveness. Moreover, blocking endogenous BMP receptors in progenitors with a virus transducing dnBMPR1B accelerates changes in EGFR signaling. This involves a non-cell-autonomous mechanism, suggesting that BMP negatively regulates signal(s) that promotes the development of EGF-responsive stem cells. FGF2 is a good candidate for such a signal: it antagonizes the inhibitory effects of younger cortical cells and exogenous BMP4. These findings suggest that a balance between antagonistic extrinsic signals regulates temporal changes in an intrinsic property of neural progenitor cells (Lillien, 2000).

What then triggers the onset of the appearance of EGF-responsive stem cells? It is appealing to consider a feedback mechanism, whereby cells that produce BMPs or FGF2 achieve appropriate numbers or states of maturation at mid-embryonic development, resulting in the generation of a net positive signal. BMPs are produced by radial glial cells and by the choroid plexus. FGF2 is made by progenitor cells and choroid plexus. The numbers of these cells do not change in an appropriate manner at mid-embryonic development to provide a trigger, suggesting that the cellular event(s) that initiates the change in EGFR expression may be more complex and involve a change in the level of expression of FGF, BMP and/or their receptors. It has been reported that the level of FGF2 increases during mid-late stages of embryonic development. Thus, an increase in FGF2 expression could be the trigger. The level of expression of BMPRs in the brain appears to decline during embryonic development, suggesting that BMP signaling might decline. Given the observation that BMPs in the limb negatively regulate the expression of FGFs, together with the finding that dnBMPRs have a non-cell-autonomous positive effect, it is possible that a reduction in BMP signaling triggers the increase in FGF2 expression in the CNS. In explant cultures, it has been observed that a lower concentration of FGF2 (1 ng/ml) has a greater effect on E15 explants than on E12 explants. This observation is consistent with an increase in the level of an endogenous positive signal, such as FGF2 with age, or with a decline in the level or effectiveness of endogenous BMPs (Lillien, 2000).

In addition to the change in EGFR expression and responsiveness, cortical progenitor cells and stem cells change in several other ways during mid-embryonic development. Early progenitor cells and stem cells tend to generate more neuronal progeny, while later progenitor cells and stem cells tend to generate more glial progeny. Early and late progenitor cells also differ in their competence to generate deep layer neurons. The developmental change in the latter property also appears to be controlled by cell-cell signaling. Studies using viral transduction of EGFRs suggest that mitotic stimulation of progenitor cells with EGF does not control developmental changes in the ratio of neuronal and glial progeny. It will be interesting to determine whether the signals that have been identified as regulators of EGFR expression and responsiveness also control other properties of progenitor cells that change during embryonic development, or whether these properties are controlled by distinct mechanisms (Lillien, 2000).

A large number of neurons are born everyday in the adult subventricular zone (SVZ) and they migrate to the olfactory bulb. Unlike the embryonic brain, where neurogenesis is a transient phenomenon, the adult SVZ must retain the neurogenic environment for an extended period of time, probably for the entire life of the animal. During development, Noggin and BMPs are thought to act in temporally restricted inductive events. BMP and Noggin are retained in the SVZ region in adult life, and these molecules are important regulators of adult neurogenesis. The molecular niche of SVZ stem cells is poorly understood. BMP antagonist Noggin is expressed by ependymal cells adjacent to the SVZ. SVZ cells were found to express BMPs as well as their cognate receptors. BMPs potently inhibit neurogenesis both in vitro and in vivo. BMP signaling cell-autonomously blocks the production of neurons by SVZ precursors by directing glial differentiation. Purified mouse Noggin protein promotes neurogenesis in vitro and inhibits glial cell differentiation. Ectopic Noggin promotes neuronal differentiation of SVZ cells grafted to the striatum. It is thus proposed that ependymal Noggin production creates a neurogenic environment in the adjacent SVZ by blocking endogenous BMP signaling (Lim, 2000).

Little is known about how neural stem cells are initially formed during development. Could a default mechanism of neural specification regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells? ES cells cultured in defined, low-density conditions readily acquire a neural identity. A novel primitive neural stem cell was characterized as a component of neural lineage specification that is negatively regulated by TGFß-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment (Tropepe, 2001).

Given that very few of the cultured ES cells generated sphere colonies (0.2%), attempts were made to determine if the release of endogenous BMP from the ES cells inhibits neural sphere colony formation, as would be predicted from the neural default model. Given that BMP4 and BMP-receptor-1 are expressed by undifferentiated ES cells, tests were made to see whether BMP could inhibit ES sphere colony formation by adding BMP4 (5 ng/ml) to ES cell cultures containing LIF and FGF2. A >50% decrease in the number of colonies generated was observed, and this effect appeared to be maximal since a 5-fold increase in BMP4 concentration did not further significantly attenuate the number of sphere colonies generated. Addition of the BMP4 protein antagonist Noggin (100 µg/ml) to the primary ES cell cultures caused a 50% increase in the number of sphere colonies generated. This increase appeared to be maximal since an increase in Noggin concentration from 10 µg/ml to 100 µg/ml resulted in no additional increase in the numbers of colonies generated (Tropepe, 2001).

It is evident that although Noggin can enhance the numbers of ES cells that differentiate into neural colony-forming stem cells, the effect is moderate. Noggin is known to be less effective than Chordin in neural induction assays in Xenopus, and targeted null mutations in both Noggin and Chordin in mouse are required to reveal anterior neural deficits in vivo. Thus, the effects of Noggin alone may underestimate the role for BMP-mediated inhibition of neural stem cell colony formation. To determine more directly the effect of blocking BMP signaling, an ES cell line with a targeted null mutation in the Smad4 gene, a critical intracellular transducer of multiple TGFß-related signaling pathways, was used. Smad4-/- ES cells cultured in the presence of LIF generate a 3- to 4-fold increase in the numbers of colonies, compared to the wild-type E14K cell line used to generate the targeted mutation. Interestingly, the rate of proliferation between wild-type and Smad4-/- cells in high or low serum concentration is similar, indicating that the increase in the number of colonies from mutant ES cells is likely not a result of a general increase in proliferation. Taken together, these results indicate that BMP4 signaling has a specific effect in limiting the numbers of single ES cells that differentiate into colony-forming neural stem cells and that inhibition of this pathway is sufficient to enhance neural stem cell colony formation (Tropepe, 2001).

The differentiation, survival, and proliferation of developing sympathetic neuroblasts are all coordinately promoted by neurotrophins. Bone morphogenetic protein 4 (BMP4), a factor known to be necessary for the differentiation of sympathetic neurons, conversely reduces both survival and proliferation of cultured E14 sympathetic neuroblasts. The anti-proliferative effects of BMP4 occur more rapidly than the pro-apoptotic actions and appear to involve different intracellular mechanisms. BMP4 treatment induces expression of the transcription factor Msx-2 and the cyclin-dependent kinase inhibitor p21CIP1/WAF1 (p21). Treatment of cells with oligonucleotides antisense to either of these genes prevents cell death after BMP4 treatment but does not significantly alter the anti-proliferative effects. Thus Msx-2 and p21 are necessary for BMP4-mediated cell death but not for promotion of exit from cell cycle. Although treatment of cultured E14 sympathetic neuroblasts with neurotrophins alone does not alter cell numbers, BMP4-induced cell death was prevented by co-treatment with either neurotrophin-3 (NT-3) or nerve growth factor (NGF). This suggests that BMP4 may also induce dependence of the cells on neurotrophins for survival. Thus, sympathetic neuron numbers may be determined in part by factors that inhibit the proliferation and survival of neuroblasts and make them dependent upon exogenous factors for survival (Gomes, 2001).

Bone morphogenetic proteins (BMPs) promote astrocytic differentiation of cultured subventricular zone stem cells. To determine whether BMPs regulate the astrocytic lineage in vivo, transgenic mice were constructed that overexpress BMP4 under control of the neuron-specific enolase (NSE) promoter. Overexpression of BMP4 was first detectable by Western analysis on embryonic day 16 and persists into the adult. The overexpression of BMP4 results in a remarkable 40% increase in the density of astrocytes in multiple brain regions accompanied by a decrease in the density of oligodendrocytes ranging between 11% and 26%, depending on the brain region and the developmental stage. No changes in neuron numbers or the pattern of myelination were detected, and there were no gross structural abnormalities. Similar phenotypes were observed in three independently derived transgenic lines. Coculture of transgenic neurons with neural progenitor cells significantly enhances astrocytic lineage commitment by the progenitors; this effect is blocked by the BMP inhibitor Noggin, indicating that the stimulation of astrogliogenesis is due to BMP4 release by the transgenic neurons. These observations suggest that BMP4 directs progenitor cells in vivo to commit to the astrocytic rather than the oligodendroglial lineage. Further, differentiation of radial glial cells into astrocytes is accelerated, suggesting that radial glia are a source of at least some of the supernumerary astrocytes. Therefore, BMPs are likely important mediators of astrocyte development in vivo (Gomes, 2003).

Bone morphogenetic protein (BMP) signaling inhibits the generation of oligodendroglia and enhances generation of astrocytes by neural progenitor cells both in vitro and in vivo. This study examined the mechanisms underlying the effects of BMP signaling on glial lineage commitment. Treatment of cultured neural progenitor cells with BMP4 induces expression of all four members of the inhibitor of differentiation (ID) family of helix-loop-helix transcriptional inhibitors and blocks oligodendrocyte (OL) lineage commitment. Overexpression of Id4 or Id2 but not Id1 or Id3 in cultured progenitor cells reproduces both the inhibitory effects of BMP4 treatment on OL lineage commitment and the stimulatory effects on astrogliogenesis. Conversely, decreasing the levels of Id4 mRNA by RNA interference enhances OL differentiation and inhibits the effects of BMP4 on glial lineage commitment. This suggests that induction of Id4 expression mediates effects of BMP signaling. Bacterial two-hybrid and co-immunoprecipitation studies demonstrate that ID4, and to a lesser extent ID2, complexes with the basic-helix-loop-helix transcription (bHLH) factors OLIG1 and OLIG2, which are required for the generation of OLs. By contrast, ID1 and ID3 do not complex with the OLIG proteins. In addition, the OLIG and ID proteins both interacted with the E2A proteins E12 and E47. Further, exposure of cultured progenitor cells to BMP4 changes the intracellular localization of OLIG1 and OLIG2 from a predominantly nuclear to a predominantly cytoplasmic localization. These observations suggest that the induction of ID4 and ID2, and their sequestration of both OLIG proteins and E2A proteins mediate the inhibitory effects of BMP signaling on OL lineage commitment and contribute to the generation of astrocytes (Samanta, 2004).

Bone morphogenetic protein (BMP) and leukemia inhibitory factor (LIF) signaling both promote the differentiation of neural stem/progenitor cells into glial fibrillary acidic protein (GFAP) immunoreactive cells. This study compares the cellular and molecular characteristics, and the potentiality, of GFAP(+) cells generated by these different signaling pathways. Treatment of cultured embryonic subventricular zone (SVZ) progenitor cells with LIF generates GFAP(+) cells that have a bipolar/tripolar morphology, remain in cell cycle, contain progenitor cell markers and demonstrate self-renewal with enhanced neurogenesis - characteristics that are typical of adult SVZ and subgranular zone (SGZ) stem cells/astrocytes. By contrast, BMP-induced GFAP(+) cells are stellate, exit the cell cycle, and lack progenitor traits and self-renewal - characteristics that are typical of astrocytes in the non-neurogenic adult cortex. In vivo, transgenic overexpression of BMP4 increases the number of GFAP(+) astrocytes but depletes the GFAP(+) progenitor cell pool, whereas transgenic inhibition of BMP signaling increases the size of the GFAP(+) progenitor cell pool but reduces the overall numbers of astrocytes. It is concluded that LIF and BMP signaling generate different astrocytic cell types, and it is proposed that these cells are, respectively, adult progenitor cells and mature astrocytes (Bonaguidi, 2005).

DPP homologs and neural patterning and development

The role of the bone morphogenetic protein (BMP) pathway during neural tissue formation was examined in the ascidian (phylum Urochordata) embryo. The ortholog of the BMP antagonist, chordin, was isolated from the ascidian Halocynthia roretzi. While both the expression pattern and the phenotype observed by overexpressing chordin or BMPb (the dpp-subclass BMP) do not suggest a role for these factors in neural induction, BMP/CHORDIN antagonism is found to affect neural patterning. Overexpression of BMPb induces ectopic sensory pigment cells in the brain lineages that do not normally form pigment cells and suppresses pressure organ formation within the brain. Reciprocally, overexpressing chordin suppresses pigment cell formation and induces ectopic pressure organ. Pigment cell formation occurs in three steps. (1) During cleavage stages ectodermal cells are neuralized by a vegetal signal that can be substituted by bFGF. (2) At the early gastrula stage, BMPb secreted from the lateral nerve cord blastomeres induces those neuralized blastomeres in close proximity to adopt a pigment cell fate. (3) At the tailbud stage, among these pigment cell precursors, BMPb induces the differentiation of specifically the anterior type of pigment cell, the otolith; while posteriorly, CHORDIN suppresses BMP activity and allows ocellus differentiation (Darras, 2001).

Inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the activation of extracellular signal-regulated kinase (Erk) following engagement of integrin/fibronectin receptors and Raf is the critical target of PI 3-kinase regulation. To investigate how PI 3-kinase regulates Raf, sites on Raf1 required for regulation by PI 3-kinase were examined and the mechanisms involved in this regulation were explored. Serine 338 (Ser338), which 1s critical for fibronectin stimulation of Raf1, is phosphorylated in a PI 3-kinase-dependent manner following engagement of fibronectin receptors. In addition, fibronectin activation of a Raf1 mutant containing a phospho-mimic mutation (S338D) is independent of PI 3-kinase. Furthermore, integrin-induced activation of the serine/threonine kinase Pak-1, which has been shown to phosphorylate Raf1 Ser338, is also dependent on PI 3-kinase activity, and expression of a kinase-inactive Pak-1 mutant blocks phosphorylation of Raf1 Ser338. These results indicate that PI 3-kinase regulates phosphorylation of Raf1 Ser338 through the serine/threonine kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Ras leads to strong activation of Raf1 kinase activity by integrins (Chaudhary, 2000).

In Xenopus embryos, fibroblast growth factors (FGFs) and secreted inhibitors of bone morphogenetic protein (BMP)-mediated signalling have been implicated in neural induction. The precise roles, if any, that these factors play in neural induction in amniotes remains to be established. To monitor the initial steps of neural induction in the chick embryo, an in vitro assay of neural differentiation in epiblast cells was developed. Using this assay, evidence was found that neural cell fate is specified in utero, before the generation of the primitive streak or Hensen's node. Early epiblast cells express both Bmp4 and Bmp7, but the expression of both genes is downregulated as cells acquired neural fate. During prestreak and gastrula stages, exposure of epiblast cells to BMP4 activity in vitro is sufficient to block the acquisition of neural fate and to promote the generation of epidermal cells. Fgf3 is expressed in the early epiblast, and ongoing FGF signalling in epiblast cells is required for acquisition of neural fate and for the suppression of Bmp4 and Bmp7 expression. It is concluded that the onset of neural differentiation in the chick embryo occurs in utero, before the generation of Hensen's node. Fgf3, Bmp4 and Bmp7 are each expressed in prospective neural cells, and FGF signalling appears to be required for the repression of Bmp expression and for the acquisition of neural fate. Subsequent exposure of epiblast cells to BMPs, however, can prevent the generation of neural tissue and induce cells of epidermal character (Wilson, 2000).

The secretion of Sonic hedgehog (Shh) from the notochord and floor plate appears to generate a ventral-to-dorsal gradient of Shh activity that directs progenitor cell identity and neuronal fate in the ventral neural tube. In principle, the establishment of this Shh activity gradient could be achieved through the graded distribution of the Shh protein itself, or could depend on additional cell surface or secreted proteins that modify the response of neural cells to Shh. Cells of the neural plate differentiate from a region of the ectoderm that has recently expressed high levels of BMPs, raising the possibility that prospective ventral neural cells are exposed to residual levels of BMP activity. Whether modulation of the level of BMP signaling regulates neural cell responses to Shh, and thus might contribute to the patterning of cell types in the ventral neural tube, has been examined. Based on results from an in vitro assay of neural cell differentiation, BMP signaling has been shown to markedly alter neural cell responses to Shh signals, eliciting a ventral-to-dorsal switch in progenitor cell identity and neuronal fate (Reim, 2000).

BMP signaling is regulated by secreted inhibitory factors, including noggin and follistatin, both of which are expressed in or adjacent to the neural plate. Conversely, follistatin but not noggin produces a dorsal-to-ventral switch in progenitor cell identity and neuronal fate in response to Shh both in vitro and in vivo. Since patched is likely to be a direct target of Shh signaling, the ability of BMPs to block the Shh-induced elevation in ptc expression provides one line of evidence that the modulatory action of BMPs on neural cell responses to Shh is exerted at a proximal step in the Shh signal transduction pathway. Exposure of neural cells to BMPs also blocks the Shh-mediated induction of HNF3 beta, thought to be induced as a direct response to hedgehog signaling. These results suggest that the specification of ventral neural cell types depends on the integration of Shh and BMP signaling activities. The net level of BMP signaling within neural tissue may be regulated by follistatin and perhaps other BMP inhibitors secreted by mesodermal cell types that flank the ventral neural tube (Riem, 2000).

The role of bone morphogenetic protein 4 (BMP-4) and a BMP antagonist, chordin (Drosophila homolog: Short gastrulation), has been investigated in primitive streak formation and neural induction in amniote embryos. Both BMP-4 and chordin are expressed before primitive streak formation, and BMP-4 expression is downregulated as the streak starts to form. When BMP-4 is misexpressed in the posterior area pellucida, primitive streak formation is inhibited. Misexpression of BMP-4 also arrests further development of Hensen's node and axial structures. Chick chordin is 60% homologous to Xenopus chordin and is 27 % homologous to Drosophila Sog. At pre-primitive streak stages, chordin mRNA is found in the epiblast just anterior to Koller's sickle and in underlying middle layer cells. Both of these cell populations contribute to the organizer. As soon as the primitive streak forms (and concomitant with downregulation of BMP-4 and BMP-7 in the area pellucida) chordin is strongly expressed at the anterior tip of the primitive streak and subsequently appears in Hensen's node, where it persists at least until stage 23. The head process and notochord express chordin at high levels, as soon as the cells emerge from the node. Misexpression of chordin in the anterior area pellucida generates an ectopic primitive streak that expresses mesoderm and organizer markers. No evidence is found for a neuralization of non-neural epiblast by chordin or for a chordin directed inhibition of neural induction, which is carried out by BMP-4 and BMP-7. Rather, the chordin/BMP system appears to act either downstream of, or in conjunction with other factors produced by the organizer. Similar conclusions are reached in Drosophila, where the sog mutant phenotype in the nervous system is detectable only during mid-gastrulation. Thus sog mutants have neural progenitors despite some reduction in the size of the domains of rhomboid, lethal of scute and thick veins, which has lead to the proposal that the major role of sog in the nervous system is to stabilize or maintain a subdivision of the primary ectoderm into neural and non-neural territories, established previously by other signals (Streit, 1998).

Thus chordin is not sufficient to induce neural tissue in the chick. Misexpression of chordin in regions outside the future neural plate does not induce the early neural markers L5, Sox-3 or Sox-2. Furthermore, neither BMP-4 nor BMP-7 interfere with neural induction when misexpressed in the presumptive neural plate before or after primitive streak formation. However, chordin can stabilise the expression of early neural markers in cells that have already received neural inducing signals. These results suggest that the regulation of BMP signaling by chordin plays a role in primitive streak formation and that chordin is not sufficient to induce neural tissue (Streit, 1998).

Although Sonic Hedgehog (Shh) plays a critical role in brain development, its actions on neural progenitor cell proliferation and differentiation have not been clearly defined. Transcripts for the putative Shh-receptor genes patched (Ptc) and smoothened (Smo) are expressed by embryonic, postnatal, and adult progenitor cells, suggesting that Shh can act directly on these cells. The recombinant human amino-terminal fragment of Shh protein (Shh-N) alone does not support the survival of cultured progenitor cells, but treatment with Shh-N in the presence of bFGF increases progenitor cell proliferation. Furthermore, treatment of embryonic rat progenitor cells propagated either in primary culture or after mitogen expansion significantly increases the proportions of both beta-tubulin- (neuronal marker) and O4- (oligodendroglial marker) immunoreactive cells and reduces the proportion of nestin- (uncommitted neural progenitor cell marker) immunoreactive cells. By contrast Shh-N has no effect on the elaboration of GFAP- (astroglial marker) immunoreactive cells. Cotreatment with Shh-N and bone morphogenetic protein-2 (BMP2) inhibits the anti-proliferative, astroglial-inductive, and oligodendroglial-suppressive effects of BMP2. These observations suggest that Shh-N selectively promotes the elaboration of both neuronal and oligodendroglial lineage species and inhibits the effects of BMP2 on progenitor cell proliferation and astroglial differentiation (Zhu, 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).

Ventral midline cells in the chick neural tube have distinct properties at different rostrocaudal levels, apparently in response to differential signaling by axial mesoderm. Floor plate cells are induced by sonic hedgehog (SHH) secreted from the notochord, whereas ventral midline cells of the rostral diencephalon (RDVM cells) appear to be induced by the dual actions of SHH and bone morphogenetic protein 7 (BMP7) from prechordal mesoderm. Examined have been the cellular and molecular events governing the program of differentiation of RDVM cells, as carried out under the influence of the axial mesoderm. Fate mapping has shown that prospective RDVM cells migrate rostrally within the neural plate, passing over the rostral notochord before establishing register with prechordal mesoderm at stage 7. Despite the co-expression of SHH and BMP7 by the rostral notochord, prospective RDVM cells appear to be specified initially as caudal ventral midline neurectodermal cells and to acquire RDVM properties only at stage 7. Evidence is provided that over this period, the signaling properties of axial mesoderm are regulated by the BMP antagonist, chordin. As the axial mesoderm extends, Chordin is expressed throughout, but the gene is downregulated in prechordal mesoderm coincident with the onset of RDVM cell differentiation. Addition of chordin to conjugate explant cultures of prechordal mesoderm and neural tissue prevents the rostralization of ventral midline cells by prechordal mesoderm. Chordin may thus act to refine the patterning of the ventral midline along the rostrocaudal axis (Dale, 1999).

Bone morphogenetic proteins (Bmps) are key regulators of dorsoventral (DV) patterning. Within the ectoderm, Bmp activity has been shown to inhibit neural development, promote epidermal differentiation and influence the specification of dorsal neurons and neural crest. The patterning of neural tissue was examined in mutant zebrafish embryos with compromised Bmp signaling activity. Although Bmp activity does not influence anteroposterior (AP) patterning, it does affect DV patterning at all AP levels of the neural plate. Thus Bmp activity is required for specification of cell fates around the margin of the entire neural plate, including forebrain regions that do not form neural crest. Surprisingly, Bmp activity is also required for patterning neurons at all DV levels of the CNS. In swirl/bmp2b minus embryos, laterally positioned sensory neurons are absent, whereas more medial interneuron populations are hugely expanded. somitabun minus embryos carry a mutation in Smad5, an intracellular transducer of Bmp signaling, and exhibit a variable dorsalized phenotype in which some embryos are similar to swr- while most embryos are less severely dorsalized. In somitabun minus embryos, which probably retain higher residual Bmp activity, it is the sensory neurons and not the interneurons that are expanded. Conversely, in severely Bmp depleted embryos, both interneurons and sensory neurons are absent and it is the most medial neurons that are expanded. These results are consistent with there being a gradient of Bmp-dependent positional information extending throughout the entire neural and non-neural ectoderm (Barth, 1999).

One hypothesis to explain how Bmp proteins establish a gradient across the entire ectoderm is that cells are responding to Bmp activity from very early stages and for extended periods of time. Thus at early blastula stages bmp2b and bmp4 are expressed throughout most of the ectoderm and over time, expression regresses from the organizer until it is restricted to non-neural ectoderm. Concommitantly, antagonists of Bmp activity progressively spread from the organizer inhibiting Bmp function. A consequence of these two events is that over time, cells near the organizer will have been exposed to a low amount of Bmp signals for a short period of time, whereas cells distant from the organizer will have been exposed to higher levels of Bmp activity for longer periods of time. If ectodermal cells can integrate the temporal duration and concentration of Bmp signals, then this provides a means by which a Bmp-dependent gradient of positional information may be established throughout the ectoderm. A similar scenario has been proposed to explain the activity of Activin as a morphogen, where relatively small differences in receptor occupancy are integrated by cells over time to promote different cell fates. If this model is correct, it raises a few interesting problems. For instance, it challenges the simple concept of neural induction in which there is a binary choice between non-neural fate and neural fate dependent upon the presence or absence of Bmp activity. This model suggests that even at the time of neural induction, cells positioned close to the organizer are likely to have encountered less Bmp activity for a shorter period of time and consequently have a more 'dorsal' identity than more laterally positioned neural cells. However, the earliest genes to be expressed in the neural plate are generally expressed throughout this tissue, whereas genes characteristic of specific DV positions are expressed only at later stages. This observation does suggest a biphasic response to Bmp activity in which the earliest neural genes are activated when Bmp activity is simply below a certain level, whereas later genes may have more refined responses to thresholds along the gradient of Bmp-dependent positional information (Barth, 1999 and references therein).

Proper dorsal-ventral patterning in the developing central nervous system requires signals from both the dorsal and ventral portions of the neural tube. Data from multiple studies have demonstrated that bone morphogenetic proteins (BMPs) and Sonic hedgehog protein are secreted factors that regulate dorsal and ventral specification, respectively, within the caudal neural tube. In the developing rostral central nervous system Sonic hedgehog protein also participates in ventral regionalization; however, the roles of BMPs in the developing brain are less clear. It was hypothesized that BMPs also play a role in dorsal specification of the vertebrate forebrain. To test this hypothesis, beads soaked in recombinant BMP5 or BMP4 were implanted into the neural tube of the chicken forebrain. Experimental embryos show a loss of the basal telencephalon that results in holoprosencephaly (a single cerebral hemisphere), cyclopia (a single midline eye), and loss of ventral midline structures. In situ hybridization using a panel of probes to genes expressed in the dorsal and ventral forebrain reveals the loss of ventral markers, although dorsal markers are maintained. Furthermore, the loss of the basal telencephalon is the result of excessive cell death and not a change in cell fates. These data provide evidence that BMP signaling participates in the dorsal-ventral patterning of the developing brain in vivo, and that disturbances in dorsal-ventral signaling result in specific malformations of the forebrain (Golden, 1999).

Inductive factors are known to direct the regional differentiation of the vertebrate central nervous system (CNS) but their role in the specification of individual neuronal cell types is less clear. The function of GDF7, a BMP family member that is expressed selectively by roof plate cells, has been studied in the generation of neuronal cell types in the dorsal spinal cord. Growth/differentiation factors (GDF) 5, 6 and 7 comprise a subgroup of factors related to known bone- and cartilage-inducing molecules: the bone morphogenetic proteins (BMPs). GDF7 can promote the differentiation in vitro of two dorsal sensory interneuron classes: D1A and D1B neurons. In Gdf7-null mutant embryos, the generation of D1A neurons is eliminated but D1B neurons and other identified dorsal interneurons are unaffected. These findings show that GDF7 is an inductive signal from the roof plate required for the specification of neuronal identity in the dorsal spinal cord and that GDF7 and other BMP family members expressed by the roof plate have non-redundant functions in vivo. More generally, these results suggest that BMP signaling may have a prominent role in the assignment of neuronal identity within the mammalian CNS (Lee, 1998).

The role of Bmp signaling in patterning neural tissue has been studied through the use of mutants in the zebrafish that disrupt three different components of a Bmp signaling pathway: swirl/bmp2b, snailhouse/bmp7 and somitabun/smad5. At postgastrulation stages, the dorsalization of neural tissue is likely mediated through the posterior tail bud expression domains of bmp2b and bmp7. Ventralization of neural tissue may occur at similar stages through Shh signaling in the dorsal organizer of the gastrula and the anterior tail bud at postgastrulation stages. Bmp signaling is essential for the establishment of the prospective neural crest and dorsal sensory Rohon-Beard neurons of the spinal cord. Moreover, Bmp signaling is necessary to limit the number of intermediate-positioned lim1+ interneurons of the spinal cord, as observed by the dramatic expansion of these prospective interneurons in many mutant embryos. The analysis also suggests a positive role for Bmp signaling in the specification of these interneurons, which is independent of Bmp2b/Swirl activity. A presumptive ventral signal, Hh signaling, acts to restrict the amount of dorsal sensory neurons and trunk neural crest. This restriction appears to occur very early in neural tissue development, likely prior to notochord or floor plate formation. A similar early role for Bmp signaling is suggested in the specification of dorsal neural cell types, since the bmp2b/swirl and bmp7/snailhouse genes are only coexpressed during gastrulation and within the tail bud, and are not found in the dorsal neural tube or overlying epidermal ectoderm. Thus, a gastrula Bmp2b/Swirl and Bmp7/Snailhouse-dependent activity gradient may not only act in the specification of the embryonic dorsoventral axis, but may also function in establishing dorsal and intermediate neuronal cell types of the spinal cord (Nguyen, 2000).

In a differential screen for downstream genes of the neural inducers, two extremely early neural genes induced by Chordin and suppressed by BMP-4 have been identified: Zic-related-1 (Zic-r1), a zinc finger factor related to the Drosophila pair-rule gene odd-paired, and Sox-2 (see Dichaete), a Sry-related HMG factor. Expression of the two genes is first detected widely in the prospective neuroectoderm at the beginning of gastrulation, following the onset of Chordin expression and preceding that of Neurogenin (Xngnr-1). Zic-r1 mRNA injection activates the proneural gene Xngnr-1, and initiates neural and neuronal differentiation in isolated animal caps and in vivo. In contrast, Sox-2 alone is not sufficient to cause neural differentiation, but can work synergistically with FGF signaling to initiate neural induction. Thus, Zic-r1 acts in the pathway bridging the neural inducer with the downstream proneural genes, while Sox-2 makes the ectoderm responsive to extracellular signals, demonstrating that the early phase of neural induction involves simultaneous activation of multiple functions (Mizuseki, 1998).

The role of the prechordal plate was analyzed in vivo in the forebrain development of chick embryos. After transplantation to uncommitted ectoderm, a prechordal plate induces an ectopic, dorsoventrally patterned, forebrain-like vesicle. Grafting laterally under the anterior neural plate causes ventralization of the lateral side of the forebrain, as indicated by a second expression domain of the homeobox gene NKX2.1 (Drosophila homolog: Vnd). Such a lateral ventralization cannot be induced by the secreted factor Sonic Hedgehog alone, as this is only able to distort the ventral forebrain medially. Removal of the prechordal plate does not reduce the rostrocaudal extent of the anterior neural tube, but leads to significant narrowing and cyclopia. Excision of the head process results in the caudal expansion of NKX2.1 expression in the ventral part of the anterior neural tube, while PAX6 expression in the dorsal part remains unchanged. It is suggested that there are three essential steps in early forebrain patterning, which culminate in the ventralization of the forebrain: (1) anterior neuralization occurs at the primitive streak stage, when BMP-4-antagonizing factors emanate from the node and spread in a planar fashion to induce anterior neural ectoderm; (2) the anterior translocation of organizer-derived cells shifts the source of neuralizing factors anteriorly, where the relative concentration of BMP-4-antagonists is thus elevated, and the medial part of the prospective forebrain becomes competent to respond to ventralizing factors, and (3) the forebrain anlage is ventralized by signals including Sonic Hedgehog, thereby creating a new identity, the prospective hypothalamus, which splits the eye anlage into two lateral domains (Pera, 1997).

Neocortical neurons begin to differentiate soon after they are generated by mitoses at the surface of the ventricular zone (VZ). Evidence is provided that bone morphogenetic protein (BMP) triggers neuronal differentiation of neocortical precursors within the VZ. In cultures of dissociated neocortical neuroepithelial cells, BMPs increase the number of MAP-2- and TUJ1-positive cells within 24 hr of treatment. In explant cultures, BMP-4 treatment leads to an increase in the number of TUJ1-positive cells within the ventricular zone. Furthermore, truncated, dominant-negative, BMP type I receptor, introduced into neocortical precursors by retrovirus-mediated gene transfer, blocks neurite elaboration and migration out of the VZ. Finally, immunocytochemistry indicates that BMP protein is present at the VZ surface. Together, these results indicate that BMP protein is present within the VZ, that BMP is capable of promoting neuronal differentiation, and that signaling through BMP receptors triggers neuronal precursors to differentiate and migrate out of the VZ (Li, 1998).

Members of the bone morphogenetic protein (BMP) family have been implicated in multiple aspects of neural development in both the CNS and peripheral nervous system. BMP ligands and receptors, as well as the BMP antagonist noggin, are expressed in the developing cerebral cortex, making the BMPs likely candidates for regulating cortical development. To define the role of these factors in the developing cerebral cortex, the effects of BMP2 and BMP4 on cortical cells were examined in vitro. Cells were cultured from embryonic day 13 (E13) and E16 rat cerebral cortex in the absence or presence of different concentrations of fibroblast growth factor 2, a known regulator of cortical cell proliferation and differentiation. At E13, the BMPs promote cell death and inhibit proliferation of cortical ventricular zone cells, resulting in the generation of fewer neurons and no glia. At E16, the effects of the BMPs are more complex. Concentrations of BMP2 in the range of 1-10 ng/ml promote neuronal and astroglial differentiation and inhibit oligodendroglial differentiation, whereas 100 ng/ml BMP2 promotes cell death and inhibits proliferation. Addition of the BMP antagonist noggin promotes oligodendrogliogenesis in vitro, demonstrating that endogenous BMP signaling influences the differentiation of cortical cells in vitro. The distribution of BMP2 and noggin within the developing cortex suggests that local concentrations of ligands and antagonists define gradients of BMP signaling during corticogenesis. Together, these results support the hypothesis that the BMPs and their antagonist noggin co-regulate cortical cell fate and morphogenesis (Mabie, 1999).

In the vertebrate spinal cord, oligodendrocytes originate from a restricted region of the ventral neuroepithelium. This ventral localization of oligodendrocyte precursors (OLPs) depends on the inductive influence of Sonic hedgehog (Shh) secreted by ventral midline cells. Whether the ventral restriction of OLP specification might also depend on inhibiting signals mediated by bone morphogenetic proteins (BMPs) was investigated. BMPs invariably and markedly inhibit oligodendrocyte development in ventral neural tissue both in vitro and in vivo. Conversely, in vivo ablation of the dorsal most part of the chick spinal cord or inactivation of BMP signaling using grafts of noggin-producing cells promotes the appearance of neuroepithelial OLPs dorsal to their normal domain of emergence, showing that endogenous BMPs contribute to the inhibition of oligodendrocyte development in the spinal cord. BMPs are able to oppose the Shh-mediated induction of OLPs in spinal cord neuroepithelial explants dissected before oligodendrocyte induction, suggesting that BMPs may repress OLP specification by interfering with Shh signaling in vivo. Strikingly, among the transcription factors involved in OLP specification, BMP treatment strongly inhibits the expression of Olig2 but not of Nkx2.2, suggesting that BMP-mediated inhibition of oligodendrogenesis is controlled through the repression of the former transcription factor. Altogether, the data show that oligodendrogenesis is not only regulated by ventral inductive signals such as Shh, but also by dorsal inhibiting signals including BMP factors. They suggest that the dorsoventral position of OLPs depends on a tightly regulated balance between Shh and BMP activities (Mekki-Dauriac, 2002).

During differentiation of the embryonic anterior pituitary, distinct hormone cell types are generated in a precise temporal and spatial order from an apparently homogenous ectodermal primordium. The anterior pituitary derives from Rathke's pouch (RP), a specialized region of the oral roof ectoderm. The posterior pituitary derives from the infundibulum (INF) an evagination of the ventral diencephalon. Evidence is provided that in RP, the coordinate control of progenitor cell identity, proliferation and differentiation is imposed by spatial and temporal restrictions in FGF- and BMP-mediated signals. These signals derive from adjacent neural and mesenchymal signaling centers: the infundibulum and ventral juxtapituitary mesenchyme (VJM), respectively. The infundibulum appears to have a dual signaling function, serving initially as a source of BMP4 and subsequently of FGF8. The onset of FGF8 expression in the INF coincides with that of Lhx3 expression in RP. The ability of the INF over the period E10.5 to E12.5 to extinguish Isl1 (in the dorsal aspect of the RP) and promote Lhx3 in the same region corresponds more closely to the temporal expression of FGF8 than of BMP4. FGF8 can mimic the ability of the INF to repress Isl1 and maintain Lhx3 expression in explants. In vitro, FGFs promote the proliferation of progenitor cells, prevent their exit from the cell cycle and contribute to the specification of progenitor cell identity. Late FGF8 signaling controls corticotroph differentiation in the dorsal Lhx3+, Isl1- domain. Maintained FGF8 signaling from the INF expands still further the dorsal corticotroph prrogenitor population; as a consequence, the most ventral of these progenitors become located beyond range of FGF signaling. Since these progenitors are also beyond range of, or by this time refractory to, BMP2/7 signals (derived from the VJM), they progress to an ACTH+ definitive corticotroph state (Ericson, 1998).

The ventral domain of RP serves as the origin of thyrotrophs, defined by expression of the alpha-glycoprotein and thyroid stimulating hormone beta subunits. The continued proliferation of progenitor cells in the dorsal domain of RP (stimulated by FGF8 signals derived from the INF) results in the progressive ventral displacement of thyrotroph progenitors such that they come to be located beyond the range of FGF8 signaling. The ventral juxtapituitary mesenchyme appears to serve as a later source of BMP2 and BMP7. BMPs have no apparent effect on cell proliferation but instead appear to act with FGFs to control the initial selection of thyrotroph and corticotroph progenitor identity. BMPs promote prospective thyrotroph differentiation and suppress corticotroph differentiation. BMPs expressed by the VJM promote Isl1 expression in the ventral domain of RP. Ultimately cells in the ventral domain begin to express TSHbeta and alpha-glycoprotein, having become established as definitive thyrotrophs (Ericson, 1998).

Distinct neuronal cell types are generated at characteristic times and positions in the dorsal horn of the spinal cord. One subset of neurons, termed D1, is generated from stage 19 close to the roof plate, and expresses LIM homeodomain protein LH2. A second class, termed D2 neurons, express Isl1. The identity and pattern of generation of dorsal neurons depend initially on BMP-mediated signals that derive from the epidermal ectoderm and induce dorsal midline cells of the roof plate. BMP4 and and BMP7 mimic the ability of the epidermal ectoderm to induce BMP4. Roof plate cells provide a secondary source of TGFbeta-related signals that are required for the generation of distinct classes of dorsal interneurons. BMP4 and DSL1 are known to be expressed by roof plate cells. These inductive interactions involve both qualitative and quantitative differences in signaling by TGFbeta-related factors and temporal changes in the response of neural progenitor cells (Liem, 1997).

Bone morphogenetic protein-7 (BMP-7) is a member of the 60A branch of the BMP family, less closely related to Dpp than is BMP2 or BMP-7. The expression pattern of BMP-7 in the hindbrain region of the headfold and early somite stage of the developing mouse embryo suggests a role for BMP-7 in the patterning of this part of the cranial CNS. In chick embryos it is thought that BMP-7 is one of the secreted molecules that mediates the dorsalizing influence of surface ectoderm on the neural tube. Mouse surface ectoderm has been shown to have a similar dorsalizing effect. While BMP-7 is expressed in the surface ectoderm of mouse embryos at the appropriate time to dorsalize the neural tube, at early stages of hindbrain development BMP-7 transcripts are present in paraxial and ventral tissues, within and surrounding the hindbrain neurectoderm; only later does expression become restricted to a dorsal domain. To determine more directly the effect that BMP-7 may have on the developing hindbrain, COS cells expressing BMP-7 were grafted into the ventrolateral mesoderm abutting the neurectoderm in order to prolong BMP-7 expression in the vicinity of ventral hindbrain. Three distinct actvities of BMP-7 are apparent. (1) BMP-7 can promote dorsal characteristics in the neural tube. (2)BMP-7 can also attenuate the expression of sonic hedgehog (Shh) in the floorplate without affecting Shh expression in the notochord, and (3) ectopic BMP-7 appears to promote growth of the neurectoderm. It seems that BMP-7 is involved in timing mechanisms necessary for the coordination of hindbrain dorsoventral patterning (Arkell, 1997).

Embryonic patterning in vertebrates is dependent on the balance of inductive signals and their specific antagonists. Noggin, which encodes a bone morphogenetic protein (BMP) antagonist expressed in the node, notochord, and dorsal somite, is required for normal mouse development. Noggin binds several BMPs with very high affinities, with a marked preference for BMP2 and BMP4 over BMP7. Although Noggin has been implicated in neural induction, examination of null mutants in the mouse indicates that Noggin is not essential for this process. However, Noggin is required for subsequent growth and patterning of the neural tube. Early BMP-dependent dorsal cell fates, such as the roof plate and neural crest, form in the absence of Noggin. However, there is a progressive loss of early, Sonic hedgehog (Shh)-dependent ventral cell fates despite the normal expression of Shh in the notochord. Somite differentiation is deficient in both muscle and sclerotomal precursors. Addition of BMP2 or BMP4 to paraxial mesoderm explants blocks Shh-mediated induction of Pax-1, a sclerotomal marker, whereas addition of Noggin is sufficient to induce Pax-1. Noggin and Shh induce Pax-1 synergistically. Use of protein kinase A stimulators blocks Shh-mediated induction of Pax-1, but not induction by Noggin, suggesting that induction is mediated by different pathways. Together these data demonstrate that inhibition of BMP signaling by axially secreted Noggin is an important requirement for normal patterning of the vertebrate neural tube and somite (McMahon, 1998).

The molecular interactions underlying neural crest formation in Xenopus have been investigated. Neural crest induction requires a suppression of BMP-mediated epidermal fate. A simple model for induction of the neural crest, a cell type that arises at the junction between neural and non-neural ectoderm, would be that neural crest is specified at levels of BMP signaling intermediate to those that induce neural plate and epidermis. Using chordin overexpression to antagonize endogenous BMP signaling in whole embryos and explants, it is demonstrated that such inhibition alone is insufficient to account for neural crest induction in vivo. However, chordin-induced neural plate tissue can be induced to adopt neural crest fates by members of the FGF and Wnt families, growth factors that have previously been shown to posteriorize induced neural tissue. Overexpression of a dominant negative XWnt-8 inhibits the expression of neural crest markers, demonstrating the necessity for a Wnt signal during neural crest induction in vivo. The requirement for Wnt signaling during neural crest induction is shown to be direct, whereas FGF-mediated neural crest induction may be mediated by Wnt signals. Overexpression of the zinc finger transcription factor Slug, one of the earliest markers of neural crest formation, is insufficient for neural crest induction. Slug-expressing ectoderm will generate neural crest in the presence of Wnt or FGF-like signals, however, bypassing the need for BMP inhibition in this process. A two-step model for neural crest induction is proposed (LaBonne, 1998).

Bone Morphogenetic Protein-4 (BMP-4) is a potent epidermal inducer and inhibitor of neural fate. Differential screening has been used to identify genes involved in epidermal induction downstream of BMP-4. A novel translational mechanism regulates the division of the vertebrate ectoderm into regions of neural and epidermal fate. In dissociated Xenopus ectoderm, addition of ectopic BMP-4 leads to an increase in the expression of translation initiation factor 4AIII (eIF-4AIII), a divergent member of the eIF-4A gene family until now characterized only in plants. In the gastrula embryo, Xenopus eIF-4AIII (XeIF-4AIII) expression is elevated in the ventral ectoderm, a site of active BMP signal transduction. Moreover, overexpression of XeIF-4AIII induces epidermis in dissociated cells that would otherwise adopt a neural fate, mimicking the effects of BMP-4. Epidermal induction by XeIF-4AIII requires both an active BMP signaling pathway and an extracellular intermediate. These results suggest that XeIF-4AIII can regulate changes in cell fate through selective mRNA translation. It is proposed that BMPs and XeIF-4AIII interact through a positive feedback loop in the ventral ectoderm of the vertebrate gastrula (Weinstein, 1997).

The cell adhesion molecule L1 regulates axonal guidance and fasciculation during development. The regulatory region of the L1 gene has been identified and shown to be sufficient for establishing the neural pattern of L1 expression in transgenic mice. A DNA element identified within this region, called the HPD, contains binding motifs for both homeodomain and Pax proteins and responds to signals from bone morphogenetic proteins (BMPs). An ATTA sequence within the core of the HPD was required for binding to the homeodomain protein Barx2 while a separate paired domain recognition motif is necessary for binding to Pax-6. In cellular transfection experiments, L1-luciferase reporter constructs containing the HPD are activated an average of 4-fold by Pax-6 in N2A cells and 5-fold by BMP-2 and BMP-4 in Ng108 cells. Both of these responses are eliminated on deletion of the HPD from L1 constructs. In transgenic mice, deletion of the HPD from an L1-lacZ reporter results in a loss of beta-galactosidase expression in the telencephalon and mesencephalon. Collectively, these experiments indicate that the HPD regulates L1 expression in neural tissues via homeodomain and Pax proteins and is likely to be a target of BMP signaling during development(Meech, 1999).

The vertebrate central nervous system (CNS) contains a small group (~24,000 in human, ~3,200 in rodent, and ~7-10 in zebrafish) of evolutionary conserved noradrenergic (NA) neurons known as the locus coeruleus (LC). These neurons reside in the ventro-lateral region of the first hindbrain rhombomere and project to regions throughout the CNS. Their degeneration is associated with Parkinson's and Alzheimer's disease, whereas their abnormal function is thought to play a role in depression, sleep disorders, and schizophrenia. The zebrafish mutation soulless, in which the development of locus coeruleus noradrenergic neurons fails to occur, disrupts the homeodomain protein Phox2a. Phox2a is not only necessary but also sufficient to induce Phox2b+ dopamine-beta-hydroxylase+ and tyrosine hydroxylase+ NA neurons in ectopic locations. Phox2a is first detected in LC progenitors in the dorsal anterior hindbrain, and its expression there is dependent on FGF8 from the mid/hindbrain boundary and on optimal concentrations of BMP signal from the epidermal ectoderm/future dorsal neural plate junction. These findings suggest that Phox2a coordinates the specification of LC in part through the induction of Phox2b and in response to cooperating signals that operate along the mediolateral and anteroposterior axes of the neural plate (Guo, 1999).

In olfactory epithelium (OE) cultures, bone morphogenetic proteins (BMPs) can strongly inhibit neurogenesis. Evidence is presented that BMPs also promote, and indeed are required, for OE neurogenesis. Addition of the BMP antagonist noggin inhibits neurogenesis in OE-stromal cell co-cultures. Bmp2, Bmp4 and Bmp7 are expressed by OE stroma, and low concentrations of BMP4 (below the threshold for inhibition of neurogenesis) stimulated neurogenesis; BMP7 does not exhibit a stimulatory effect at any concentration tested. Stromal cell conditioned medium also stimulates neurogenesis; part of this effect is due to the presence within it of a noggin-binding factor or factors. Studies of the pro-neurogenic effect of BMP4 indicate that it does not increase progenitor cell proliferation, but rather promotes survival of newly generated olfactory receptor neurons. These findings indicate that BMPs exert both positive and negative effects on neurogenesis, depending on ligand identity, ligand concentration and the particular cell in the lineage that is responding. In addition, they reveal the presence of a factor or factors, produced by OE stroma, that can synergize with BMP4 to stimulate OE neurogenesis (Shou, 2000).

The OE is one of the few neural tissues in which both neurogenesis and neuronal regeneration persist in adult animals, and these processes are thought to be regulated by cell-cell interactions. Under normal conditions, proliferation of neuronal progenitors within the OE proceeds at a low basal rate. However, when ORNs are induced to die, e.g. by ablation of their synaptic target tissue in the olfactory bulb, neurogenesis is induced and progenitor cells proliferate rapidly. This high rate of induced neurogenesis is maintained until new ORNs are generated and the epithelium is restored. Such observations have led to the hypothesis that ORNs normally produce a signal that feeds back to inhibit neurogenesis by their own progenitors. When ORNs are lost, progenitors would be released from feedback inhibition and able to proliferate. The results reported here suggest a way in which BMP4 could act as the signaling molecule mediating this feedback inhibition: Bmp4 is expressed within the neuronal layers of the OE, strongly suggesting that ORNs produce BMP4. If this is the case, when ORNs are induced to die, the overall level of BMP4 to which progenitors are exposed would then decrease, since the number of BMP4-producing cells in the OE would be reduced. This decrease in BMP4 should permit an increase in progenitor cell proliferation and favor production of new ORNs. These newly generated ORNs, in turn, would begin to produce BMP4, which would serve both to support their own survival, and, as ORN numbers (and thus BMP4 levels) increase, to suppress further progenitor cell proliferation and thus prevent overgrowth of the OE. In this way, the opposing actions of BMP4 on OE neuronal progenitors and ORNs should act in concert to maintain proper neuron number in the epithelium. This suggestion, that BMP4-mediated regulation of neurogenesis constitutes an autoregulatory pathway in vivo, is intriguing in view of the fact that in most of the mammalian nervous system, neurogenesis gradually slows during development as increasing numbers of differentiated neurons are produced by progenitors residing in germinative neuroepithelia. These observations in the OE system suggest that this widespread phenomenon may be explained, at least in part, by the accumulation of neuron-derived factors (such as BMP4) that have dual actions: inhibiting proliferation of neuronal progenitors, but promoting the survival of neurons themselves (Shou, 2000).

In vertebrates, BMP signaling before gastrulation suppresses neural development. Later in development, BMP signaling specifies a dorsal and ventral fate in the forebrain and dorsal fate in the spinal cord. It is therefore possible that a change in the competence of the ectoderm to respond to BMP signaling occurs at some point in development. Exposure of the anterior neural plate to BMP4 before gastrulation causes suppression of all neural markers tested. To determine the effects of BMP4 after gastrulation, BMP4 was misexpressed using a Pax-6 promoter fragment in transgenic frog embryos and beads soaked in BMP4 were implanted in the anterior neural plate. Suppression of most anterior neural markers was observed. It is concluded that most neural genes continue to require suppression of BMP signaling into the neurula stages. Additionally, BMP4 and BMP7 are abundantly expressed in the prechordal mesoderm of the neurula stage embryo. This poses the paradox of how the expression of most neural genes is maintained if they can be inhibited by BMP signaling. At least one gene in the anterior neural plate suppresses the response of the ectoderm to BMP signaling. It is proposed that the suppressive effect of BMP signaling on the expression of neural genes coupled with localized suppressors of BMP signaling result in the fine-tuning of gene expression in the anterior neural plate (Hartley, 2001).

Synaptotagmin I and neurexin I mRNAs, coding for proteins involved in neurotransmitter secretion, become detectable in primary sympathetic ganglia shortly after initial induction of the noradrenergic transmitter phenotype. To test whether the induction of these more general neuronal genes is mediated by signals known to initiate noradrenergic differentiation in a neuronal subpopulation, their expression was examined in noradrenergic neurons induced by ectopic overexpression of growth and transcription factors. Overexpression of BMP4 or Phox2a in vivo results in synaptotagmin I and neurexin I expression in ectopically located noradrenergic cells. In vitro, BMP4 initiates synaptotagmin I and neurexin I expression in addition to tyrosine hydroxylase induction. Thus, the induction of synaptotagmin I and neurexin I, which are expressed in a large number of different neuron populations, can be accomplished by growth and transcription factors available only to a subset of neurons. These findings suggest that the initial expression of proteins involved in neurotransmitter secretion is regulated by different signals in different neuron populations (Patzke, 2001).

In the spinal neural tube, populations of neuronal precursors that express a unique combination of transcription factors give rise to specific classes of neurons at precise locations along the dorsoventral axis. Understanding the patterning mechanisms that generate restricted gene expression along the dorsoventral axis is therefore crucial to understanding the creation of diverse neural cell types. Bone morphogenetic proteins (BMPs) and other transforming growth factor ß proteins are expressed by the dorsal-most cells of the neural tube (the roofplate) and surrounding tissues, and evidence indicates that they play a role in assigning cell identity. The level of BMP signaling has been manipulated in the chicken neural tube to show that BMPs provide patterning information to both dorsal and intermediate cells. BMP regulation of the expression boundaries of the homeobox proteins Pax6, Dbx2 and Msx1 generate precursor populations with distinct developmental potentials. Within the resulting populations, thresholds of BMP act to set expression domain boundaries of developmental regulators of the homeobox and basic helix-loop-helix (bHLH) families, ultimately leading to the generation of a diversity of differentiated neural cell types. This evidence strongly suggests that BMPs are the key regulators of dorsal cell identity in the spinal neural tube (Timmer, 2002).

In addition to their role in promoting the formation of an Msx1-expressing dorsal progenitor pool, BMPs also act to subdivide this domain into discrete cell populations. Several of the cell types generated by this region depend upon the expression of bHLH proteins that have been found to be regulated by BMPs. High levels of BMP signaling activity promote the expression of the most dorsal bHLH protein, Cath1. The expanded domain of Cath1 expression, however, remains restricted to the dorsal neural tube. The broad expression of Cath1 correlates with the repression of other regulators expressed in this region, such as the neurogenins and Cash1. As with Cath1, this regulation is specific to dorsal neural cells; neurogenin expression in ventral cells appears to be unaltered. The restriction of these responses to dorsal cells is consistent with this region having been previously assigned a distinct developmental identity and potential. It is also consistent with findings that the dorsal and ventral regulatory sequences of the neurogenins are physically separated (Timmer, 2002).

The data indicate that thresholds of BMP signaling set the expression boundaries of dorsal regulators. High levels of BMP signaling repress Cash1 in its most dorsal domain, although expression is retained in more ventral cells of this domain. In experiments where the roofplate is genetically ablated and expression of its BMPs abolished, Cash1 expression expands dorsally. Thus, it appears that BMP signaling sets the dorsal border of Cash1 expression at a precise level of activity, while expression in more ventral cells is independent of BMP activity. By contrast, dorsal Ngn1 expression is absent both in embryos with high levels of BMP activity and those in which the roofplate is absent and BMPs are not expressed. This suggests that the dorsal cells that express Ngn1 are formed within a limited range of BMP activity. This suggestion has been confirmed by showing that Ngn1 expression is broadly activated in the dorsal neural tube by low levels of BMP signaling. These results indicate that the BMPs collectively act to regulate genes along a gradient of activity, possibly behaving as morphogens to pattern cells within the dorsal neural tube. This function would be consistent with other descriptions of BMPs and other TGFß superfamily members acting as morphogens during embryonic development. The recent finding that the bHLH genes expressed in this region inter-regulate, indicates the BMP activity may only have to set the boundary of a limited number of gene(s) in order to generate several distinct cell populations (Timmer, 2002).

The regulation of the dorsally expressed genes by BMP signaling has significant consequences for the generation of differentiated dorsal interneurons. The expanded expression of Cath1 drives the generation of LH2A/B+ cells throughout the dorsal third of the neural tube. The expansion of Lh2A/B+ interneurons appears to come at the expense of other cell fates, as the generation of other classes of dorsal interneurons, marked by Isl1 or Lim1/2, is reduced or eliminated. Thus, the presence of high levels of BMP signaling throughout the dorsal third of the neural tube appears to generate a uniform pool of progenitors and a single type of differentiated neuron in this region. Small increases in BMP signaling in dorsal cells, however, can expand the generation of other cell types in this region. This reinforces the suggestion that a gradient of BMP signaling activity is essential for the generation of a diversity of dorsal interneurons (Timmer, 2002).

The roles of the organizer factors chordin and noggin, which are dedicated antagonists of the bone morphogenetic proteins (BMPs), were investigated in formation of the mammalian head. The mouse chordin and noggin genes (Chrd and Nog) are expressed in the organizer (the node) and its mesendodermal derivatives, including the prechordal plate, an organizing center for rostral development. They are also expressed at lower levels in and around the anterior neural ridge, another rostral organizing center. To elucidate roles of Chrd and Nog that are masked by the severe phenotype and early lethality of the double null, embryos of the genotype Chrd^-/-;Nog^+/- were characterized. These animals display partially penetrant neonatal lethality, with defects restricted to the head. The variable phenotypes include cyclopia, holoprosencephaly, and rostral truncations of the brain and craniofacial skeleton. In situ hybridization reveals a loss of SHH expression and signaling by the prechordal plate, and a decrease in FGF8 expression and signaling by the anterior neural ridge at the five-somite stage. Defective Chrd^-/-;Nog^+/- embryos exhibit reduced cell proliferation in the rostral neuroepithelium at 10 somites, followed by increased cell death 1 day later. Because these phenotypes result from reduced levels of BMP antagonists, it is hypothesized that they are due to increased BMP activity. Ectopic application of BMP2 to wild-type cephalic explants results in decreased FGF8 and SHH expression in rostral tissue, suggesting that the decreased expression of FGF8 and SHH observed in vivo is due to ectopic BMP activity. Cephalic explants isolated from Chrd;Nog double mutant embryos show an increased sensitivity to ectopic BMP protein, further supporting the hypothesis that these mutants are deficient in BMP antagonism. These results indicate that the BMP antagonists chordin and noggin promote the inductive and trophic activities of rostral organizing centers in early development of the mammalian head (Anderson, 2002).

Chick brain factor 1 (CBF1), a nasal retina-specific winged-helix transcription factor, is known to prescribe the nasal specificity that leads to the formation of the precise retinotectal map, especially along the anteroposterior (AP) axis. However, its downstream topographic genes and the molecular mechanisms by which CBF1 controls the expression of them have not been elucidated. Misexpression of CBF1 represses the expression of EphA3 and CBF2, and induces that of SOHo1, GH6, ephrin A2 and ephrin A5. CBF1 controls ephrin A5 by a DNA binding-dependent mechanism, ephrin A2 by a DNA binding-independent mechanism, and CBF2, SOHo1, GH6 and EphA3 by dual mechanisms. BMP2 expression begins double-gradiently (varying in both naso-temporal and ventral-dorsal axes) in the retina from E5 in a complementary pattern to Ventroptin expression. Ventroptin antagonizes BMP2 as well as BMP4. CBF1 interferes in BMP2 signaling and thereby induces expression of ephrin A2. These data suggest that CBF1 is located at the top of the gene cascade for the regional specification along the nasotemporal (NT) axis in the retina and distinct BMP signals play pivotal roles in the topographic projection along both axes (Takahashi, 2003).

The role of target-derived BMP signaling in development of sensory ganglia and the sensory innervation of the skin was examined in transgenic animals that overexpress either the BMP inhibitor noggin or BMP4 under the control of a keratin 14 (K14) promoter. Overexpression of noggin results in a significant increase in the number of neurons in the trigeminal and dorsal root ganglia. Conversely, overexpression of BMP4 results in a significant decrease in the number of dorsal root ganglion neurons. There is no significant change in proliferation of trigeminal ganglion neurons in the noggin transgenic animals, and neuron numbers do not undergo the normal developmental decrease between E12.5 and the adult, suggesting that programmed cell death is decreased in these animals. The increase in neuron numbers in the K14-noggin animals is followed by an extraordinary increase in the density of innervation in the skin and a marked change in the pattern of innervation by different types of fibers. Conversely, the density of innervation of the skin is decreased in the BMP4 overexpressing animals. Further Merkel cells and their innervation are increased in the K14-noggin mice and decreased in the K14-BMP4 mice. The changes in neuron numbers and the density of innervation are not accompanied by a change in the levels of neurotrophins in the skin. These findings indicate that the normal developmental decrease in neuron numbers in sensory ganglia depends upon BMP signaling, and that BMPs may limit both the final neuron number in sensory ganglia as well as the extent of innervation of targets. Coupled with prior observations, this suggests that BMP signaling may regulate the acquisition of dependence of neurons on neurotrophins for survival, as well as their dependence on target-derived neurotrophins for determining the density of innervation of the target (Guha, 2004).

During development of the cerebellum, sonic hedgehog (Shh) is directly responsible for the proliferation of granule cell precursors in the external germinal layer. Signals able to regulate a switch from the Shh-mediated proliferative response to one that directs differentiation of granule neurons have been sought. Bone morphogenetic proteins (BMPs) are expressed in distinct neuronal populations within the developing cerebellar cortex. Bmp2 and Bmp4 are expressed in the proliferating precursors and subsequently in differentiated granule neurones of the internal granular layer, whereas Bmp7 is expressed by Purkinje neurones. In primary cultures, Bmp2 and Bmp4, but not Bmp7, are able to prevent Shh-induced proliferation, thereby allowing granule neuron differentiation. Furthermore, Bmp2 treatment downregulates components of the Shh pathway in proliferating granule cell precursors. Smad proteins, the only known BMP receptor substrates capable of transducing the signal, are also differentially expressed in the developing cerebellum: Smad1 in the external germinal layer and Smad5 in newly differentiated granule neurons. Among them, only Smad5 is phosphorylated in vivo and in primary cultures treated with Bmp2, and overexpression of Smad5 is sufficient to induce granule cell differentiation in the presence of Shh. A model is proposed in which Bmp2-mediated Smad5 signalling suppresses the proliferative response to Shh by downregulation of the pathway, and allows granule cell precursor to enter their differentiation programme (Rios, 2004).

In the tadpole larvae of the ascidian Halocynthia roretzi, six motor neurons, Moto-A, -B, and -C (a pair of each), are localized proximal to the caudal neural tube and show distinct morphology and innervation patterns. To gain insights into early mechanisms underlying differentiation of individual motor neurons, an ascidian homologue of Islet, a LIM type homeobox gene, has been isolated. Earliest expression of Islet was detected in a pair of bilateral blastomeres on the dorsal edge of the late gastrula. At the neurula stage, this expression begins to disappear and more posterior cells start to express Islet. Compared to expression of a series of motor neuron genes, it was confirmed that early Islet-positive blastomeres are the common precursors of Moto-A and -B, and late Islet-positive cells in the posterior neural tube are the precursors of Moto-C. Overexpression of Islet induces ectopic expression of motor neuron markers, suggesting that Islet is capable of regulating motor neuron differentiation. Since early expression of Islet colocalizes with that of HrBMPb, the ascidian homologue of BMP2/4, a role for BMP in specification of the motor neuron fate was tested. Overexpression of HrBMPb leads to expansion of Lim and Islet expression toward the central area of the neural plate, and microinjection of mRNA coding for a dominant-negative BMP receptor weakens the expression of these genes. These results suggest that determination of the ascidian motor neuron fate takes place at late gastrula stage and local BMP signaling may play a role in this step (Katsuyama, 2005).

Somatosensory information from the face is transmitted to the brain by trigeminal sensory neurons. Whether neurons innervating distinct areas of the face possess molecular differences has been an open question. This study identified a set of genes differentially expressed along the dorsoventral axis of the embryonic mouse trigeminal ganglion and thus can be considered trigeminal positional identity markers. Interestingly, establishing some of the spatial patterns requires signals from the developing face. Bone morphogenetic protein 4 (BMP4) was identified as one of these target-derived factors; spatially defined retrograde BMP signaling controls the differential gene expressions in trigeminal neurons through both Smad4-independent and Smad4-dependent pathways. Mice lacking one of the BMP4-regulated transcription factors, Onecut2 (OC2), have defects in the trigeminal central projections representing the whiskers. These results provide molecular evidence for both spatial patterning and retrograde regulation of gene expression in sensory neurons during the development of the somatosensory map (Hodge, 2007).

Several different populations of interneurons in the murine cortex, including somatostatin (SST)- or parvalbumin (PV)-expressing cells, are born in the ventral ganglionic eminences during mid-gestation and then migrate tangentially to the cortex. SST is expressed by some interneuron progenitors in the cerebral cortex and in migrating populations in the ventrolateral cortex at birth. However, PV (also known as PVALB) is not expressed by interneurons until the second postnatal week after reaching the cortex, suggesting that molecular cues in the cerebral cortex might be involved in the differentiation process. BMP4 is expressed at high levels in the somatosensory cortex at the time when the PV(+) interneurons differentiate. Treatment of cortical cultures containing interneuron precursors is sufficient to generate PV(+) interneurons prematurely and inhibit SST differentiation. Furthermore, overexpression of BMP4 in vivo increases the number of interneurons expressing PV, with a reduction in the number of SST(+) interneurons. PV(+) interneurons in the cortex express BMP type I receptors and a subpopulation displays activated BMP signaling, assessed by downstream molecules including phosphorylated SMAD1/5/8. Conditional mutation of BMP type I receptors in interneuron precursors significantly reduces the number of cortical PV(+) interneurons in the adult brain. Thus, BMP4 signaling through type I receptors regulates the differentiation of two major medial ganglionic eminence-derived interneuron populations and defines their relative numbers in the cortex (Mukhopadhyay, 2009).

BMPs and the patterning of the telencephalon

To investigate the roles of Bone Morphogenetic Proteins (BMPs) in early brain development, the expression patterns of five Bmp genes belonging to the Drosophila Decapentaplegic (Bmp2 and Bmp4) and 60A subgroups (Bmp5, Bmp6 and Bmp7) were studied. Striking co-expression of these Bmps is observed within the dorsomedial telencephalon, coincident with a future site of choroid plexus development. Bmp co-expression overlaps that of Msx1 (a Drosophila Muscle segment homeobox homolog) and Hfh4 (a winged helix transcription factor - See Drosophila Forkhead), and is complementary to that of Bf1 (another winged helix transcription factor). The domain of Bmp co-expression is also associated with limited growth of the neuroectoderm, as revealed by morphological observation, reduced cell proliferation, and increased local programmed cell death. In vitro experiments using explants from the embryonic lateral telencephalic neuroectoderm reveal that exogenous BMP proteins (BMP4 and BMP2) induce expression of Msx1 and inhibit Bf1 expression, a finding consistent with their specific expression patterns in vivo. Moreover, BMP proteins locally inhibit cell proliferation and increase apoptosis in the explants. These results provide evidence that BMPs function during regional morphogenesis of the dorsal telencephalon by regulating (1) specific gene expression, (2) cell proliferation and (3) local cell death. The fact that Bf1 is essential for continued proliferation of the cells in the telencephalon suggests that BMPs and Bf1 may function antagonistically during dorsal telencephalon development (Furuta, 1997).

Pattern formation of the dorsal telencephalon is governed by a regionalization process that leads to the formation of distinct domains, including the future hippocampus and neocortex. Recent studies have implicated signaling proteins of the Wnt and Bmp gene families as well as several transcription factors, including Gli3 and the Emx homeobox genes, in the molecular control of this process. The regulatory relationships between these genes, however, remain largely unknown. Transgenic analysis was used to investigate the upstream mechanisms for regulation of Emx2 in the dorsal telencephalon. An enhancer from the mouse Emx2 gene has been identified that drives specific expression of a lacZ reporter gene in the dorsal telencephalon. This element contains binding sites for Tcf and Smad proteins, transcriptional mediators of the Wnt and Bmp signaling pathway, respectively. Mutations of these binding sites abolish telencephalic enhancer activity, while ectopic expression of these signaling pathways leads to ectopic activation of the enhancer. These results establish Emx2 as a direct transcriptional target of Wnt and Bmp signaling and provide insights into a genetic hierarchy involving Gli3, Emx2 and Bmp and Wnt genes in the control of dorsal telencephalic development (Theil, 2002).

The analysis presented here has revealed several aspects of the complexity of Emx2 regulation. Although the 4.6 kb fragment mediates reporter gene expression in the dorsal telencephalon indistinguishable from the expression pattern of the endogenous gene, enhancer activity was not observed in the early developing dorsal forebrain. This difference suggests that the spatial and temporal control of Emx2 expression might involve the use of distinct regulatory modules. A similar conclusion was obtained for the control of the segmental expression of the Epha4 gene and of the Hox genes in the hindbrain (Theil, 2002).

Several observations of this study indicate a cooperative interaction between Wnt and Bmp signaling to regulate Emx2 expression in the telencephalon. While mutations of the Tcf- and Smad-binding sites abolish Emx2 enhancer activity in the telencephalon, the single site mutations only affect specific aspects of reporter gene expression. Furthermore, in vitro binding of the Tcf/Smad factors is enhanced in the presence of both factors. Similarly, ectopic expression experiments show an increased induction of the telencephalic enhancer through both Wnt and Bmp signaling. Synergy between Tgfß and Wnt signaling to regulate developmental events has been observed in various cases and may involve direct interactions between Lef1 and Smad proteins. Since expression of Bmp family members is confined to the dorsomedial telencephalon, a cooperative effect between Wnt and Bmp signaling would mainly be restricted to development of the hippocampus and adjacent medial neocortex. Interaction between these signaling pathways therefore provides a molecular mechanism to specify the gradient of Emx2 expression along the medial/lateral axis of the telencephalon (Theil, 2002).

Within the neocortical neuroepithelium, control of regional Emx2 expression requires a functional Tcf-binding site. The similarities between the Wnt7b expression and the ß-galactosidase staining pattern of the Emx2 enhancer construct just containing the functional Tcf-binding site make this Wnt family member a good candidate for being an upstream regulator of Emx2 expression in the telencephalon. This idea is further supported by recent findings showing that Wnt7b can induce the formation of a free cytoplasmic pool of ß-catenin and can stimulate the expression of the Tcf target gene Cdx1. In addition, enhancer activity in the ventral diencephalon coincides with another prominent Wnt7b expression domain. Alternatively, control of Emx2 expression could involve other yet to be identified Wnt genes with expression in the cortical neuroepithelium. The Tcf-binding site alone, however, only confers weak lacZ expression in the telencephalon, suggesting a requirement for additional factors. Although Bmp expression and signaling is mainly confined to the dorsomedial telencephalon, mutational analysis suggests an important role for the Smad-binding site in this regulation (Theil, 2002).

While the data establish Bmps and Wnts as essential components of the molecular mechanisms governing regional Emx2 expression, several lines of evidence suggest that activation of Bmp and Wnt signaling is not sufficient for the induction of Emx2 expression during normal development. (1) Even within the neural tube, co-expression of several Wnt and Bmp genes is widespread while Emx2 transcription as well as Emx2 enhancer activity are confined to the forebrain. (2) A second regulatory element, DT2, was defined that is required for reporter gene expression in the dorsal telencephalon. While DT1 on its own is not sufficient to mediate this activity, a fusion construct consisting of just DT1 and DT2 drives lacZ expression in a pattern indistinguishable from the original enhancer construct. This data indicates that DT2 does not solely act to inhibit potential repressive elements within the Emx2 enhancer but functions as a positive regulator and synergises with DT1 in the tissue-specific regulation of Emx2. Region specific expression of the yet unknown factor(s) binding to the DT2 element might therefore be responsible for conferring forebrain specific activation of the Emx2 enhancer (Theil, 2002).

The identification of Wnts/Bmps as regulators of Emx2 expression places this homeobox gene downstream of these signaling pathways in the genetic hierarchy controlling telencephalic development. Consistent with this idea, hippocampal development is affected by both the Wnt3a and the Emx2 mutation, though to different extents. Similar to the Gli3 mutation, loss of Wnt3a function leads to a loss of the hippocampus, while it is reduced in size in the Emx2 mutant. This difference suggests the involvement of Wnt target genes other than Emx2 in the control of this developmental process, such as the Lhx5 homeobox gene. In addition, a role for Bmps in Emx2 regulation could be demonstrated by the finding that ectopic expression of Bmp4 throughout the dorsal telencephalon, as observed in Bf1 mutant mice, coincides with an expansion of the Emx2 expression domain into the ventral telencephalon. Furthermore, the unaltered expression patterns of Gli3 and Wnt genes in the Emx2 mutant telencephalon show that these genes are not regulated by Emx2 (Theil, 2002).

Pattern formation and growth must be tightly coupled during embryonic development. In vertebrates, however, little is known of the molecules that serve to link these two processes. Bone morphogenetic proteins (BMP) coordinate the acquisition of pattern information and the stimulation of proliferation in the embryonic spinal neural tube. BMP and transforming growth factor-β superfamily (TGFβ) function was blocked in the chick embryo using Noggin, a BMP antagonist, and siRNA against Smad4. BMPs/TGFβs are shown to be necessary to regulate pattern formation and the specification of neural progenitor populations in the dorsal neural tube. BMPs also serve to establish discrete expression domains of Wnt ligands, receptors, and antagonists along the dorsal-ventral axis of the neural tube. Using the extracellular domain of Frizzled 8 to block Wnt signaling and Wnt3a ligand misexpression to activate WNT signaling, it has been demonstrated that the Wnt pathway acts mitogenically to expand the populations of neuronal progenitor cells specified by BMP. Thus, BMPs, acting through WNTs, couple patterning and growth to generate dorsal neuronal fates in the appropriate proportions within the neural tube (Chesnutt, 2004).

These studies led to an integrated model for neural patterning and growth. BMP/TGFβs regulate the expression of D/V patterning genes, as well as WNT signaling components, and WNTs function as mitogenic signals regulating neural tube growth. As such, BMP/TGFβs are the key signals in the coordination of patterning and growth in the dorsal neural tube. Loss of BMP/TGFβ function alters D/V pattern, eliminates Wnt1 and Wnt3a, and reduces proliferation because of reduced WNT activity. The results utilizing activated BMPR show expanded domains of midline Wnt1 and Wnt3a expression. This coupled with the mitogenic activity of WNTs suggest that the increased proliferation in the activated BMPR-Ia transgenic mouse is likely a direct consequence of the robust expansion of Wnt1. On the ventral side of the neural tube, SHH may serve an analogous role in the coordination of patterning and growth. Indeed, Pax6, known to be under SHH control in the spinal cord, is required for expression of sFRP2 and Wnt7b. BMPs may similarly control dorsal Wnt expression boundaries through Pax7 activation and Pax6 repression. The intersection of the BMP/TGFb and WNT signaling pathways appears to coordinate not only the specification of cell fate, but also the maintenance of proper growth, as well as appropriate cell cycle exit to ensure the correct organization of the spinal cord (Chesnutt, 2004).

The cortical hem regulates the size and patterning of neocortex

The cortical hem, a source of Wingless-related (WNT) and bone morphogenetic protein (BMP) signaling in the dorsomedial telencephalon, is the embryonic organizer for the hippocampus. Whether the hem is a major regulator of cortical patterning outside the hippocampus has not been investigated. This study examined regional organization across the entire cerebral cortex in mice genetically engineered to lack the hem. Indicating that the hem regulates dorsoventral patterning in the cortical hemisphere, the neocortex, particularly dorsomedial neocortex, was reduced in size in late-stage hem-ablated embryos, whereas cortex ventrolateral to the neocortex expanded dorsally. Unexpectedly, hem ablation also perturbed regional patterning along the rostrocaudal axis of neocortex. Rostral neocortical domains identified by characteristic gene expression were expanded, and caudal domains diminished. A similar shift occurs when fibroblast growth factor (FGF) 8 is increased at the rostral telencephalic organizer, yet the FGF8 source was unchanged in hem-ablated brains. Rather hem WNT or BMP signals, or both, were found to have opposite effects to those of FGF8 in regulating transcription factors that control the size and position of neocortical areas. When the hem is ablated a necessary balance is perturbed, and cerebral cortex is rostralized. These findings reveal a much broader role for the hem in cortical development than previously recognized, and emphasize that two major signaling centers interact antagonistically to pattern cerebral cortex (Caronia-Brown, 2014).

Embryonic signaling centers expressing BMP, WNT and FGF proteins, integrated by EMX2, interact to pattern the cerebral cortex

Recent findings implicate embryonic signaling centers in patterning the mammalian cerebral cortex. Mouse in utero electroporation and mutant analysis was used to test whether cortical signaling sources interact to regulate one another. Interactions were identified between the cortical hem (part of the dorsomedial edge of each cerebral cortical hemisphere), rich in Wingless-Int (WNT) proteins and bone morphogenetic proteins (BMPs), and an anterior telencephalic source of fibroblast growth factors (FGFs). Expanding the FGF8 domain suppressed Wnt2b, Wnt3a and Wnt5a expression in the hem. Next to the hem, the hippocampus was shrunken, consistent with its dependence for growth on a hem-derived WNT signal. Maintenance of hem WNT signaling and hippocampal development thus require a constraint on the FGF8 source, which is likely to be supplied by BMP activity. When endogenous BMP signaling is inhibited by noggin, robust Fgf8 expression appears ectopically in the cortical primordium. Abnormal signaling centers were further investigated in mice lacking the transcription factor EMX2, in which FGF8 activity is increased, WNT expression is reduced, and the hippocampus is defective. Suggesting that these defects are causally related, sequestering FGF8 in Emx2 homozygous mutants substantially recovered WNT expression in the hem and partially rescued hippocampal development. Because noggin can induce Fgf8 expression, noggin and BMP signaling were examined in the Emx2 mutant. As the telencephalic vesicle closed, Nog expression expanded and BMP activity reduced, potentially leading to FGF8 upregulation. These findings point to a cross-regulation of BMP, FGF, and WNT signaling in the early telencephalon, integrated by EMX2, and required for normal cortical development (Shimogori, 2004).

The Emx2 mutant mouse line provides an informative illustration of the consequences of signaling center defects. Homozygous mutants display an expanded FGF8 domain, and predictably, given the present findings, a partial loss of WNT gene expression in the hem. Evidence has been provided that shifts in region-specific gene expression in the Emx2 mutant neocortex are in part caused by excess FGF8. Findings from the present study indicate that the expanded FGF8 source in the mutant reduces WNT signaling from the cortical hem, which in turn could contribute to defective development of the hippocampus (Shimogori, 2004).

Emx2 is expressed broadly in the cortical primordium, but its loss does not lead to a broad expansion of Fgf8 expression. Instead, the normally medial and anterior FGF8 domain is enlarged laterally and posteriorly, but retains clear boundaries. Findings from the present study suggest a partial explanation. A likely cause of the expanded FGF8 domain in the Emx2 mutant is early overexpression of noggin at the telencephalic midline, decreasing local BMP activity. BMP inhibition of Fgf8 expression is thereby relieved close to the midline, but not at a distance. Remaining BMP activity may contain further lateral spread of Fgf8 expression (Shimogori, 2004).

It is suggested that cortically expressed EMX2 influences signaling centers by direct gene regulation in the cortical primordium. However, an indirect influence by EMX2 function outside the cortical primordium remains a formal possibility. Emx2 expression appears at E8-8.5 in rostral brain, and continues in both the cortical and subcortical forebrain, where EMX2 has diverse roles in development. These complexities challenge easy interpretation of specific defects in the Emx2 mutant. For example, a misrouting of thalamocortical axons, first ascribed to the absence of EMX2 in the neocortex, may be partially due to loss of gene function in the ventral telencephalon where the thalamocortical pathway begins (Shimogori, 2004).

Ultimately, the timing and sites of Emx2 expression that are crucial to particular aspects of development will be resolved by appropriate conditional deletions, or regional misexpression, of the gene. A recent advance has been the generation of a mouse that overexpresses Emx2 under the control of the nestin promotor. FGF8 levels appear unaffected, perhaps because Emx2 is overexpressed too late, yet area boundaries are shifted. These findings, together with the current ones, indicate a primary effect of EMX2 on cortical patterning, and a secondary effect via two signaling sources (Shimogori, 2004).

It is proposed that early in telencephalic development, EMX2 acts directly or indirectly on noggin to derepress BMP activity. BMP activity constrains expansion of the anterior FGF8 source, and keeps the cortical hem clear of FGF8, protecting local WNT gene expression. Meanwhile, normal levels of midline noggin allow the FGF8 source to be established and maintained. Effectively completing a negative feedback loop, FGF8 downregulates Emx2 expression. These interactions help to ensure FGF and WNT/BMP sources of appropriate size, position and duration to regulate cortical patterning and growth (Shimogori, 2004).

BMPs and axon guidance

Bone morphogenic proteins (BMPs) are involved in axon pathfinding, but how they guide growth cones remains elusive. This study reports that a BMP7 gradient elicits bidirectional turning responses from nerve growth cones by acting through LIM kinase (LIMK) and Slingshot (SSH) phosphatase to regulate actin-depolymerizing factor (ADF)/cofilin-mediated actin dynamics. Xenopus laevis growth cones from 4-8-h cultured neurons are attracted to BMP7 gradients but become repelled by BMP7 after overnight culture. The attraction and repulsion are mediated by LIMK and SSH, respectively, which oppositely regulate the phosphorylation-dependent asymmetric activity of ADF/cofilin to control the actin dynamics and growth cone steering. The attraction to repulsion switching requires the expression of a transient receptor potential (TRP) channel TRPC1 and involves Ca2+ signaling through calcineurin phosphatase for SSH activation and growth cone repulsion. Together, this study shows that spatial regulation of ADF/cofilin activity controls the directional responses of the growth cone to BMP7, and Ca2+ influx through TRPC tilts the LIMK-SSH balance toward SSH-mediated repulsion (Wen, 2007).

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