The development of the reproductive system in C. elegans is a well-established model system for patterning and organogenesis. Mutation in the cog-1 gene cause novel phenotypes in late patterning in vulval lineages, establishment of the vulva-uterine connection, development and function of the spermathecal-uterine junction, and the development of vas deferens-proctodeal connection in the male. cog-1 was positionally cloned and found to encodes a homeobox protein most similar to the mammalian GTX and Nkx6.1 proteins. Analysis of cog-1 transcripts revealed that cog-1 is likely a complex locus with two promoters. Two mutant alleles of cog-1 differentially affect alternative transcripts and cause different phenotypes, suggesting that the two forms of cog-1 have distinct functions in C. elegans (Palmer, 2002).
The molecular mechanisms of differential pattern formation along the left/right (L/R) axis in the nervous system are poorly understood. The nervous system of the nematode C. elegans displays several examples of L/R asymmetry, including the directional asymmetry displayed by the two ASE taste receptor neurons, ASE left (ASEL) and ASE right (ASER). Although bilaterally symmetric in regard to all known morphological criteria, these two neurons display distinct chemosensory capacities that correlate with the L/R asymmetric expression of three putative sensory receptor genes, gcy-5, expressed only in ASER, and gcy-6 and gcy-7, expressed only in ASEL. In order to understand the genetic basis of L/R asymmetry establishment, a screen was performed for mutants in which patterns of asymmetric gcy gene expression are disrupted; a cascade of several symmetrically and asymmetrically expressed transcription factors was identified; these factors are sequentially required to restrict gcy gene expression to either the left or right ASE cell. These factors include the zinc finger transcription factor che-1; the homeobox genes cog-1, ceh-36, and lim-6; and the transcriptional cofactors unc-37/Groucho and lin-49. Specific features of this regulatory hierarchy are sequentially acting repressive interactions and the finely balanced activity of antagonizing positive and negative regulatory factors. A key trigger for asymmetry is the L/R differential expression of the Nkx6-type COG-1 homeodomain protein. These studies have thus identified transcriptional mediators of a putative L/R-asymmetric signaling event and suggest that vertebrate homologs of these proteins may have similar functions in regulating vertebrate brain asymmetries (Chang, 2003).
How left/right functional asymmetry is layered on top of an anatomically symmetrical nervous system is poorly understood. In the nematode C. elegans, two morphologically bilateral taste receptor neurons, ASE left (ASEL) and ASE right (ASER), display a left/right asymmetrical expression pattern of putative chemoreceptor genes that correlates with a diversification of chemosensory specificities. A previously undefined microRNA termed lsy-6 controls this neuronal left/right asymmetry of chemosensory receptor expression. lsy-6 mutants that were retrieved from a genetic screen for defects in neuronal left/right asymmetry display a loss of the ASEL-specific chemoreceptor expression profile with a concomitant gain of the ASER-specific profile. A lsy-6 reporter gene construct is expressed in less than ten neurons including ASEL, but not ASER. lsy-6 exerts its effects on ASEL through repression of cog-1, an Nkx-type homeobox gene, which contains a lsy-6 complementary site in its 3' untranslated region and that has been shown to control ASE-specific chemoreceptor expression profiles. lsy-6 is the first microRNA identified with a role in neuronal patterning, providing new insights into left/right axis formation (Johnston, 2003).
To elucidate the evolutionary origin of nervous system centralization, the molecular architecture of the trunk nervous system was investigated in the annelid Platynereis dumerilii. Annelids belong to Bilateria, an evolutionary lineage of bilateral animals that also includes vertebrates and insects. Comparing nervous system development in annelids to that of other bilaterians could provide valuable information about the common ancestor of all Bilateria. The Platynereis neuroectoderm is subdivided into longitudinal progenitor domains by partially overlapping expression regions of nk and pax genes. These domains match corresponding domains in the vertebrate neural tube and give rise to conserved neural cell types. As in vertebrates, neural patterning genes are sensitive to Bmp signaling. These data indicate that this mediolateral architecture was present in the last common bilaterian ancestor and thus support a common origin of nervous system centralization in Bilateria (Denes, 2007).
Given the obvious paucity of information from the fossil record, the main strategy to elucidate CNS evolution is to compare nervous system development in extant forms. This comparative study of mediolateral neural patterning and neuron-type distribution in the developing trunk CNS of the annelid Platynereis revealed an unexpected degree of similarity to the mediolateral architecture of the developing vertebrate neural tube (Denes, 2007).
Three similarities are described. (1) The Platynereis and vertebrate neuroepithelium are similarly subdivided (from medial to lateral) into a sim+ midline and four longitudinal CNS progenitor domains (nk2.2+/nk6+, pax6+/nk6+, pax6+/pax3/7+, and msx+/pax3/7+), laterally bounded by an msx+, dlx+ territory. This strongly indicates a common evolutionary origin from an equally complex ancestral pattern. It is highly unlikely that precisely this mediolateral order and overlap in expression of orthologous genes in the CNS neuroectoderm should evolve twice independently. One can also discount the possibility that these genes are necessarily linked and thus co-opted as a package because they also act independently of each other in other developmental contexts (nk2.2 in endoderm development; pax6 in eye development, pax3/7 in segmentation, and msx in muscle development). Following similar reasoning, the complex conserved topography of gene expression along the anteroposterior axis in the enteropneust and vertebrate head is considered homologous (Denes, 2007).
(2) Evidence was found for conserved neuron types emerging from corresponding domains in Platynereis and in vertebrates. Serotonergic neurons involved in locomotor control form from the medial nk2.2+/nk6+ domain. A conserved population of hb9+ cholinergic somatic motoneurons emerges from the adjacent pax6+/nk6+ domain. Neurons expressing interneuron markers are found at the same level and more laterally, and single cells positive for sensory marker genes populate the lateral dlx+ domain. Notably, characterization of neuron types in the developing Platynereis nervous system is yet incomplete so that the full extent of conservation in neuron type distribution remains to be determined (Denes, 2007).
(3) Bmp signaling is similarly involved in the dose-dependent control of the neural genes. The finding that exogenous Bmp4 protein differentially regulates neural patterning genes in Platynereis nervous system development corroborates recent evidence that Bmps play an ancestral role in the mediolateral patterning of the bilaterian CNS neuroectoderm. Also, the strong upregulation of Pdu-atonal in the larval ectoderm goes in concert with Drosophila data that indicate that Dpp signaling positively regulates atonal expression in the lateral PNS anlage, and it supports the view that Bmp signaling also plays a conserved role in the specification of peripheral sensory neurons. Conservation of the molecular mediolateral CNS architecture concomitant with its sensitivity to Bmp signaling indicates that the developmental link between Bmp signaling and nervous system centralization predates Bilateria (Denes, 2007).
Taken together, these data make a very strong case that the complex molecular mediolateral architecture of the developing trunk CNS, as shared between Platynereis and vertebrates, was already present in their last common ancestor, Urbilateria. The concept of bilaterian nervous system centralization implies that neuron types concentrate on one side of the trunk, as is the case in vertebrates and many invertebrates including Platynereis, where they segregate and become spatially organized (as opposed to a diffuse nerve net). The data reveal that a large part of the spatial organization of the annelid and vertebrate CNS was already present in their last common ancestor, which implies that Urbilateria had already possessed a CNS (Denes, 2007).
Evolutionary conservation of the molecular mediolateral architecture as shared between Platynereis and vertebrates would imply that it was initially present also in the evolutionary lines leading to Drosophila, the nematode Caenorhabditis, and the enteropneust Saccoglossus. Yet it is clear from the available data that these animals are missing or have modified at least part of this pattern, although the extent of conservation may actually be larger than is currently apparent. For example, nk2.2/vnd and pax6 expression were costained in the fly, and a complementary pattern was found at germ-band-extended stage, reminiscent of the Platynereis and vertebrate situation. Strikingly, however, there is no trace so far of the conserved mediolateral architecture in the nematode Caenorhabditis and hardly any in the enteropneust Saccoglossus. How did this come about? Fly and nematode exhibit very fast development, making it plausible that they have (partially) omitted the transitory formation of longitudinal progenitor domains to speed up neurodevelopment. For the enteropneust, however, the situation is less clear. Why is the pattern absent in an animal that otherwise shows strong evolutionary conservation? One possible explanation is that the enteropneust trunk has lost part of its neuroarchitecture due to an evolutionary change in locomotion. While annelids and vertebrates propel themselves through trunk musculature (and associated trunk CNS), the enteropneust body is mainly drawn forward by means of the contraction of the longitudinal muscles in their anterior proboscis and collar. Possibly, enteropneusts have partially reduced their locomotor trunk musculature concomitant with motor parts of the CNS (while the peripheral sensory neurons prevailed in 'diffuse' arrangement). In line with this, expression of the conserved somatic motoneuron marker hb9/mnx is mostly absent from the Saccoglossus trunk ectoderm except for few patches. A more detailed understanding of enteropneust nervous system organization, neuron type distribution, and locomotion will help with resolving this issue (Denes, 2007).
An overall conservation of mediolateral CNS neuroarchitecture as proposed in this study does not imply that everything is similar. It is clear that the lines of evolution leading to annelids and vertebrates diverged for more than 600 million years, and numerous smaller or larger modifications of the ancestral pattern must have accumulated in both lines. The common-ground pattern as elucidated in this study helps in identifying these changes. For example, annelid and vertebrate differ in the deployment of gsx and dbx orthologs. While mouse gsh and dbx genes act early to specify interneuron progenitor domains in the dorsal neural tube, it was found the Platynereis gsx and dbx genes expressed at differentiation stages only. Adding to this, Pdu-gsx is expressed at a different mediolateral position in the nk2.2+ domain, and Pdu-dbx expression is much more restricted than that of its vertebrate counterparts (though the overall mediolateral coordinates correspond). It is hypothesized that these differences relate to the emergence of new interneuron domains (gsx+; dbx+) inside of the ancestral pax6+/pax3/7+ domain in the dorsal vertebrate neural tube. For this, it is conceivable that genes were recruited that had been active already in the differentiation of the diversifying interneuron populations. It is worth mentioning that the role of gsx in neuronal development also varies among vertebrates (Denes, 2007).
Homology of the vertebrate and Platynereis mediolateral molecular architecture is inevitably linked to the notion of dorsoventral axis inversion during early chordate evolution. In his 1875 essay on the origin of vertebrates Anton Dohrn discusses the resemblances between vertebrates and annelids and states that 'what stands most in the way of such a comparison has been the viewpoint that the nervous system of [annelids] is located in the venter, but that of vertebrates in the dorsum. Hence the one is called the ventral nerve cord, the other the dorsal nerve cord. Had we not possessed the terms dorsal and ventral, then the comparison would have been much easier.' How did the relocation of the trunk CNS from ventral to dorsal come about? Anton Dohrn proposed that vertebrate ancestors inverted their entire body dorsoventrally so that the former belly became the new back. This would not necessarily involve a sudden major shift in the lifestyle of an ancestor, as argued by critics of DV axis inversion. One can also imagine that an inversion involved transitional forms, with hemisessile or burrowing lifestyle and changing orientation toward the substrate. These animals had gill slits and lived as filter feeders. Only when early vertebrates left the substrate and acquired a free-swimming lifestyle would their new belly-up orientation have been fixed such that their CNS was then dorsal. Dohrn believed that the foremost gill slits then formed a new mouth on the new ventral body side. More than 130 years later, the molecular data on annelid neurodevelopment corroborate the key aspect of Dohrn's annelid theory, which is the homology of the annelid and vertebrate trunk CNS (Denes, 2007).
The isolation, sequence and developmental expression in the central nervous system of several members of the chicken and mouse Nkx gene family is reported. These are among the earliest genes to be regionally expressed in the neural plate; they are expressed just above the axial mesendoderm (prechordal mesendoderm and notochord). Each Nkx gene has a distinct spatial pattern of expression along the anterior-posterior axis of the ventral central nervous system: Nkx-2. 2 is expressed along the entire axis, whereas Nkx-2.1 is restricted to the forebrain, and Nkx-6.1 and Nkx-6.2 are largely excluded from the forebrain. They are also expressed in distinct patterns along the dorsal-ventral axis. These genes are expressed in both the ventricular and mantle zones; in the mantle zone, Nkx-6.1 is co-expressed with Islet-1 in a subset of motor neurons. Like other Nkx genes, expression of Nkx-6.1 is induced by the axial mesendoderm and by sonic hedgehog protein. BMP-7 represses Nkx-6.1 expression. While the notochord can induce Nkx-6.1 expression in the anterior neural plate, sonic hedgehog protein does not, suggesting that the notochord produces additional molecules that can regulate ventral patterning (Qiu, 1998).
Distinct classes of neurons are generated at defined positions in the ventral neural tube in response to a gradient of Sonic Hedgehog (Shh) activity. A set of homeodomain transcription factors expressed by neural progenitors act as intermediaries in Shh-dependent neural patterning. These homeodomain factors fall into two classes: class I proteins are repressed by Shh and class II proteins require Shh signaling for their expression. The profile of class I and class II protein expression defines five progenitor domains, each of which generates a distinct class of postmitotic neurons. Cross-repressive interactions between class I and class II proteins appear to refine and maintain these progenitor domains. The combinatorial expression of three of these proteins (Nkx6.1, Nkx2.2, and Irx3) specifies the identity of three classes of neurons generated in the ventral third of the neural tube (Briscoe, 2000).
There is growing evidence that sonic hedgehog (Shh) signaling regulates ventral neuronal fate in the vertebrate central nervous system through Nkx-class homeodomain proteins. The patterns of neurogenesis in mice carrying a targeted mutation in Nkx6.1 have been examined. These mutants show a dorsal-to-ventral switch in the identity of progenitors and in the fate of postmitotic neurons. At many axial levels there is a complete block in the generation of V2 interneurons and motor neurons and a compensatory ventral expansion in the domain of generation of V1 neurons, demonstrating the essential functions of Nkx6.1 in regional patterning and neuronal fate determination (Sander, 2000a).
To define the role of Nkx6.1 in neural development, patterns of neurogenesis were compared in the embryonic spinal cord and hindbrain of wild-type mice and mice lacking Nkx6.1. In wild-type embryos, neural expression of Nkx6.1 is first detected at spinal cord and caudal hindbrain levels at about embryonic day 8.5 (E8.5; Qiu, 1998), and by E9.5 the gene is expressed throughout the ventral third of the neural tube. The expression of Nkx6.1 persists until at least E12.5. Nkx6.1 expression was also detected in mesodermal cells flanking the ventral spinal cord. To define more precisely the domain of expression of Nkx6.1, its expression was compared with that of 10 homeobox genes (Pax3, Pax7, Gsh1, Gsh2, Irx3, Pax6, Dbx1, Dbx1, Dbx2, and Nkx2.9>) that have been shown to define discrete progenitor cell domains along the dorsoventral axis of the ventral neural tube (Sander, 2000a).
This analysis revealed that the dorsal boundary of Nkx6.1 expression is positioned ventral to the boundaries of four genes expressed in dorsal progenitor cells: Pax3, Pax7, Gsh1, and Gsh2. Within the ventral neural tube, the dorsal boundary of Nkx6.1 expression is positioned ventral to the domain of Dbx1 expression and close to the ventral boundary of Dbx2 expression. The domain of Pax6 expression extends ventrally into the domain of Nkx6.1 expression, whereas the expression of Nkx2.2 and Nkx2.9 overlaps with the ventral-most domain of Nkx6.1 expression (Sander, 2000a).
To address the function of Nkx6.1 in neural development, progenitor cell identity and the pattern of neuronal differentiation were examined in Nkx6.1 null mutant mice. A striking change was detected in the profile of expression of three homeobox genes (Dbx2, Gsh1, and Gsh2) in Nkx6.1 mutants. The domains of expression of Dbx2, Gsh1, and Gsh2 each expand into the ventral neural tube. At E10.5, Dbx2 is expressed at high levels by progenitor cells adjacent to the floor plate, but at this stage ectopic Dbx2 expression is detected only at low levels in regions of the neural tube that generate motor neurons. By E12.5, however, the ectopic ventral expression of Dbx2 has become more uniform and now clearly includes the region of motor neuron and V2 neuron generation. Similarly, in Nkx6.1 mutants, both Gsh1 and Gsh2 are ectopically expressed in a ventral domain of the neural tube and also in adjacent paraxial mesodermal cells (Sander, 2000a).
The ventral limit of Pax6 expression is unaltered in Nkx6.1 mutants, although the most ventrally located cells within this progenitor domain express a higher level of Pax6 protein than those in wild-type embryos. No change was detected in the patterns of expression of Pax3, Pax7, Dbx1, Irx3, Nkx2.2, or Nkx2.9 in Nkx6.1 mutant embryos. Importantly, the level of Shh expression by floor plate cells is unaltered in Nkx6.1 mutants. Thus, the loss of Nkx6.1 function deregulates the patterns of expression of a selected subset of homeobox genes in ventral progenitor cells without an obvious effect on Shh levels. The role of Shh in excluding Dbx2 from the most ventral region of the neural tube appears therefore to be mediated through the induction of Nkx6.1 expression. Consistent with this view, ectopic expression of Nkx6.1 represses Dbx2 expression in chick neural tube (Briscoe, 2000). The detection of sites of ectopic Gsh1/2 expression in the paraxial mesoderm as well as the ventral neural tube, both sites of Nkx6.1 expression, suggests that Nkx6.1 has a general role in restricting Gsh1/2 expression. The signals that promote ventral Gsh1/2 expression in Nkx6.1 mutants remain unclear but could involve factors other than Shh that are secreted by the notochord (Sander, 2000a).
The domain of expression of Nkx6.1 within the ventral neural tube of wild-type embryos encompasses the progenitors of three main neuronal classes: V2, MN, and V3 interneurons. Whether the generation of any of these neuronal classes is impaired in Nkx6.1 mutants was examined, focusing first on the generation of motor neurons. In Nkx6.1 mutant embryos there is a marked reduction in the number of spinal motor neurons, as assessed by expression of the homeodomain proteins Lhx3, Isl1/2, and HB9 and by expression of the gene encoding the transmitter synthetic enzyme choline acetyltransferase. In addition, few if any axons were observed to emerge from the ventral spinal. The incidence of motor neuron loss, however, varied along the rostrocaudal axis of the spinal cord. Few if any motor neurons were detected at caudal cervical and upper thoracic levels of Nkx6.1 mutants analyzed at E11-E12.5, whereas motor neuron number was reduced only by 50%-75% at more caudal levels. At all axial levels, the initial reduction in motor neuron number persisted at both E12.5 and p0, indicating that the loss of Nkx6.1 activity does not simply delay motor neuron generation. Moreover, no increase was detected in the incidence of TUNEL+ cells in Nkx6.1 mutants, providing evidence that the depletion of motor neurons does not result solely from apoptotic death (Sander, 2000a).
The persistence of some spinal motor neurons in Nkx6.1 mutants raises the possibility that the generation of particular subclasses of motor neurons is selectively impaired. To address this issue, the expression of markers of distinct subtypes of motor neurons was monitored at both spinal and hindbrain levels of Nkx6.1 mutant embryos. At spinal levels, the extent of the reduction in the generation of motor neurons that populate the median (MMC) and lateral (LMC) motor columns was similar in Nkx6.1 mutants as assessed by the number of motor neurons that coexpressed Isl1/2 and Lhx3 (defining MMC neurons) and by the expression of Raldh2 (defining LMC neurons). In addition, the generation of autonomic visceral motor neurons was reduced to an extent similar to that of somatic motor neurons at thoracic levels of the spinal cord of E12.5 embryos. Thus, the loss of Nkx6.1 activity depletes the major subclasses of spinal motor neurons to a similar extent (Sander, 2000a).
At hindbrain levels, Nkx6.1 is expressed by the progenitors of both somatic and visceral motor neurons. Therefore whether the loss of Nkx6.1 might selectively affect subsets of cranial motor neurons was examined. A virtually complete loss in the generation of hypoglossal and abducens somatic motor neurons was detected in Nkx6.1 mutants, as assessed by the absence of dorsally generated HB9+ motor neurons. In contrast, there was no change in the initial generation of any of the cranial visceral motor neuron populations, assessed by coexpression of Isl1 and Phox2a within ventrally generated motor neurons. Moreover, at rostral cervical levels, the generation of spinal accessory motor neurons was also preserved in Nkx6.1 mutants. Thus, in the hindbrain the loss of Nkx6.1 activity selectively eliminates the generation of somatic motor neurons, while leaving visceral motor neurons intact. Cranial visceral motor neurons, unlike spinal visceral motor neurons, derive from progenitors that express the related Nkx genes Nkx2.2 and Nkx2.9. The preservation of cranial visceral motor neurons in Nkx6.1 mutant embryos may therefore reflect the dominant activities of Nkx2.2 and Nkx2.9 within these progenitor cells (Sander, 2000a).
Whether the generation of ventral interneurons is affected by the loss of Nkx6.1 activity was examined. V2 and V3 interneurons are defined, respectively, by expression of Chx10 and Sim1. A severe loss of Chx10 V2 neurons was detected in Nkx6.1 mutants at spinal cord levels, although at hindbrain levels of Nkx6.1, mutants ~50% of V2 neurons persist. In contrast, there is no change in the generation of Sim1 V3 interneurons at any axial level of Nkx6.1 mutants. Thus, the elimination of Nkx6.1 activity affects the generation of only one of the two major classes of ventral interneurons that derive from the Nkx6.1 progenitor cell domain (Sander, 2000a).
Evx1+, Pax2+ V1 interneurons derive from progenitor cells located dorsal to the Nkx6.1 progenitor domain within a domain that expresses Dbx2 but not Dbx1. Because Dbx2 expression undergoes a marked ventral expansion in Nkx6.1 mutants, whether there might be a corresponding expansion in the domain of generation of V1 neurons was examined. In Nkx6.1 mutants, the region that normally gives rise to V2 neurons and motor neurons now also generates V1 neurons, as assessed by the ventral shift in expression of the En1 and Pax2 homeodomain proteins. Consistent with this, there was a two- to three-fold increase in the total number of V1 neurons generated in Nkx6.1 mutants. In contrast, the domain of generation of Evx1/2 V0 neurons, which derive from the Dbx1 progenitor domain, was unchanged in Nkx6.1 mutants. Thus, the ventral expansion in Dbx2 expression is accompanied by a selective switch in interneuronal fates from V2 neurons to V1 neurons. In addition, some neurons within the ventral spinal cord of Nkx6.1 mutants coexpress the V1 marker En1 and the V2 marker Lhx3. The coexpression of these markers is rarely if ever observed in single neurons in wild-type embryos. Thus, within individual neurons in Nkx6.1 mutants, the ectopic program of V1 neurogenesis appears to be initiated in parallel with a residual, albeit transient, program of V2 neuron generation. This result complements observations in Hb9 mutant mice, in which the programs of V2 neuron and motor neuron generation coincide transiently within individual neurons (Sander, 2000a).
Taken together, these findings reveal an essential role for the Nkx6.1 homeobox gene in the specification of regional pattern and neuronal fate in the ventral half of the mammalian CNS. Within the broad ventral domain within which Nkx6.1 is expressed, its activity is required to promote MN and V2 interneuron generation and to restrict the generation of V1 interneurons. The idea is favored that the loss of MN and V2 neurons is a direct consequence of the loss of Nkx6.1 activity, since the depletion of these two neuronal subtypes is evident at stages when only low levels of Dbx2 are expressed ectopically in most regions of the ventral neural tube. Nonetheless, the possiblity that low levels of ectopic ventral Dbx2 expression could contribute to the block in motor neuron generation cannot be excluded. Consistent with this view, the ectopic expression of Nkx6.1 is able to induce both motor neurons and V2 neurons in chick neural tube. V3 interneurons and cranial visceral motor neurons derive from a set of Nkx6.1 progenitors that also express Nkx2.2 and Nkx2.9 (Briscoe, 1999). The generation of these two neuronal subtypes is unaffected by the loss of Nkx6.1 activity, suggesting that the actions of Nkx2.2 and Nkx2.9 dominate over that of Nkx6.1 within these progenitors. The persistence of some spinal motor neurons and V2 neurons in Nkx6.1 mutants could reflect the existence of a functional homolog within the caudal neural tube (Sander, 2000a).
The role of Nkx6.1 revealed in these studies suggests a model in which the spatially restricted expression of Nkx genes within the ventral neural tube has a pivotal role in defining the identity of ventral cell types induced in response to graded Shh signaling. Strikingly, in Drosophila, the Nkx gene NK2 has been shown to have an equivalent role in specifying neuronal fates in the ventral nerve cord. Moreover, the ability of Nkx6.1 to function as a repressor of the dorsally expressed Gsh1/2 homeobox genes parallels the ability of Drosophila NK2 to repress Ind, a Gsh1/2-like homeobox gene. Thus, the evolutionary origin of regional pattern along the dorsoventral axis of the central nervous system may predate the divergence of invertebrate and vertebrate organisms (Sander, 2000a).
Specification of neuronal fate in the vertebrate central nervous system depends on the profile of transcription factor expression by neural progenitor cells, but the precise roles of such factors in neurogenesis remain poorly characterized. Two closely related transcriptional repressors, Nkx6.2 and Nkx6.1, are expressed by progenitors in overlapping domains of the ventral spinal cord. Tenetic evidence that differences in the level of repressor activity of these homeodomain proteins underlies the diversification of interneuron subtypes, and provides a fail-safe mechanism during motor neuron generation. A reduction in Nkx6 activity further permits V0 neurons to be generated from progenitors that lack homeodomain proteins normally required for their generation, providing direct evidence for a model in which progenitor homeodomain proteins direct specific cell fates by actively suppressing the expression of transcription factors that direct alternative fates (Vallstedt, 2001).
The Nkx homeobox genes are expressed in a variety of developing tissues and have been implicated in controlling tissue patterning and cell differentiation. Expression of Nkx6.2 (Gtx) was previously observed in the embryonic neural tube, testis, and differentiating oligodendrocytes. To investigate the role of Nkx6.2 in the control of cell specification and differentiation, mice with null mutations in Nkx6.2 were generated using the standard gene targeting approach. Null mutant mice are viable and fertile without apparent histological and immunohistochemical changes in the central nervous systems and testis. The absence of detectable phenotypes suggests a redundant function of Nkx6.2 in mouse development (Cai, 2001).
During early neural development, the Nkx6.1 homeodomain neural progenitor gene is specifically expressed in the ventral neural tube, and its activity is required for motoneuron generation in the spinal cord. Nkx6.1 also controls oligodendrocyte development in the developing spinal cord, possibly by regulating Olig gene expression in the ventral neuroepithelium. In Nkx6.1 mutant spinal cords, expression of Olig2 in the motoneuron progenitor domain is diminished, and the generation and differentiation of oligodendrocytes are significantly delayed and reduced. The regulation of Olig gene expression by Nkx6.1 is stage dependent; ectopic expression of Nkx6.1 in embryonic chicken spinal cord results in an induction of Olig2 expression at early stages, but an inhibition at later stages. Moreover, the regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 also appears to be region specific. In the hindbrain, unlike in the spinal cord, Olig1 and Olig2 can be expressed both inside and outside the Nkx6.1-expressing domains and oligodendrogenesis in this region is not dependent on Nkx6.1 activity (Liu, 2003).
Although many studies have focused on the mechanisms of motoneuron specification, little is known about the factors that control the subsequent development of postmitotic motoneurons. The transcription factor Nkx6.1 is required for the early specification of somatic motoneuron progenitors in the spinal cord. The present analysis of hindbrain motoneuron development in Nkx6.1-deficient mouse embryos reveals that the early specification of branchio-motoneurons is independent of Nkx6.1 function, but that it is required for their subsequent development. In Nkx6.1 mutant mice, defects are observed in the migration, as well as in the axon projections of branchio-motoneurons. A detailed analysis of the migratory defect in facial branchio-motoneurons reveals ectopic expression of the cell surface receptors Ret and Unc5h3 in premigratory neurons, but no changes in the rhombomeric environment. Taken together, these findings demonstrate a requirement for Nkx6.1 in the development of postmitotic motoneurons, and suggest a cell-autonomous function in the control of branchio-motoneuron migration (Müller, 2003).
The pattern of neuronal specification in the ventral neural tube is controlled by homeodomain transcription factors expressed by neural progenitor cells, but no general logic has emerged to explain how these proteins determine neuronal fate. Most of these homeodomain proteins possess a conserved eh1 motif that mediates the recruitment of Gro/TLE corepressors. The eh1 motif underlies the function of these proteins as repressors during neural patterning in vivo. Inhibition of Gro/TLE-mediated repression in vivo results in a deregulation of cell pattern in the neural tube. These results imply that the pattern of neurogenesis in the neural tube is achieved through the spatially controlled repression of transcriptional repressors -- a derepression strategy of neuronal fate specification (Muhr, 2001).
Graded inductive signals specify cell fates in a position-dependent manner in the neural tube. Within the ventral neural tube, the identities of neural progenitor cells are assigned initially by the actions of Sonic hedgehog (Shh). Graded Shh signaling establishes distinct ventral progenitor domains by regulating the spatial pattern of expression of a set of homeodomain (HD) proteins that comprise members of the Pax, Nkx, Dbx, and Irx families. These HD proteins can be subdivided into class I and class II proteins based on their differential regulation by Shh signaling. The class I proteins are expressed by neural progenitor cells in the absence of Shh signaling, and their expression is repressed by Shh. In contrast, the expression of the class II proteins depends on exposure to Shh (Muhr, 2001 and references therein).
How do these HD proteins specify neuronal fate. The establishment of progenitor cell identity appears to involve cross-regulatory interactions between complementary pairs of class I and class II HD proteins that share a common boundary. These interactions define the spatial extent of individual progenitor domains and establish sharp boundaries between adjacent domains, thus ensuring that cells within individual domains express distinct combinations of HD proteins. The profile of class I and class II HD protein expression within a progenitor cell appears to direct neuronal fate. Most strikingly, several of these progenitor HD proteins have the ability to induce the ectopic generation of neuronal subtypes when misexpressed outside the confines of their normal progenitor domains. The inductive activities of these progenitor HD proteins involve the activation of expression of downstream transcription factors that serve intermediary roles in the determination of neuronal fate. In addition, gene targeting studies in mice have established the essential role of many of these class I and class II proteins in the specification of ventral neuronal identity (Muhr, 2001 and references therein).
Eight of the ten progenitor HD proteins implicated in ventral neural patterning share a motif related to the core eh1 region of the Engrailed repressor (EnR) domain. This motif mediates in vitro interactions of class I and class II HD proteins with Groucho-TLE (Gro/TLE) corepressors, and underlies the function of these proteins as repressors in neural patterning in vivo. Disruption of Gro/TLE function in neural cells in vivo leads to an impairment of ventral patterning. Three conclusions have been reached: (1) there is a common mechanism of action of the class I and class II progenitor HD proteins involved in ventral patterning; (2) Gro/TLE corepressors play a role in patterning the ventral neural tube; (3) the spatial pattern of neurogenesis in the ventral neural tube is achieved through the repression of repressors (Muhr, 2001).
To identify functional domains that mediate the neural patterning activity of the Nkx proteins, a focus was placed on a conserved ~10 amino acid motif, termed the TN, or NK decapeptide, domain. Nkx2.2, Nkx2.9, Nkx6.1, Nkx6.2 and Drosophila Ventral nervous system defective (Vnd) each possess a TN domain. This domain shows sequence similarity to the core region of the engrailed homology-1 (eh1) domain present in Engrailed (En), a transcriptional repressor. The eh1 motif interacts with Gro/TLE corepressors, and Gro/TLE proteins can bind to certain Nkx class proteins (Muhr, 2001).
The idea that Gro/TLE corepressors mediate the neural patterning activity of progenitor HD proteins currently rests on three lines of evidence: (1) the presence of an eh1 domain in class I and class II proteins underlies their Gro/TLE binding activity in vitro, and is required for their repressor functions in vivo; (2) Gro/TLE genes are expressed in the ventral neural tube at the time that neural pattern is established; (3) Grg5, a protein that inhibits Gro/TLE repressor function, deregulates the pattern of progenitor HD protein expression and blocks ectopic neuronal specification in vivo (Muhr, 2001).
The dorsal expansion in the domains of expression of the class II proteins Nkx6.1 and Nkx2.2 observed after Grg5 expression provides evidence that Gro/TLE function is required normally to establish the p1/p2 and pMN/p3 progenitor domain boundaries (MN refering to motor neuron). Expression of Grg5 also disrupted the normal mutual exclusion in the domains of expression of the class I/class II protein pairs Dbx2/Nkx6.1 and Pax6/Nkx2.2. Thus, a reduction in Gro/TLE activity blocks the ability of class II proteins to repress class I protein expression. However, there is not a ventral expansion in the domains of expression of the class I proteins Dbx2 and Pax6. One possible explanation for this asymmetry in HD protein deregulation is that a higher level of Gro/TLE activity is required for the repressor activity of the class I proteins than for the class II proteins. In addition, the detection of higher levels of ventral Gro/TLE gene expression than dorsal implies that expression of Grg5 will be more effective in reducing the net level of Gro/TLE activity in dorsal than ventral regions of the neural tube, favoring a dorsal expansions in progenitor HD protein expression. The early onset of Nkx6.1 expression, together with a higher level of ventral Gro/TLE gene expression, may also explain why Grg5 expression blocks the Nkx6.1-mediated induction of ectopic MNs, but is not able to inhibit the generation of MNs within the pMN domain. The findings with Grg5 support an essential role for Gro/TLE proteins in neural patterning, but there is still a need to define changes in neuronal fate that occur after elimination of the Gro/TLE proteins themselves (Muhr, 2001).
Pax6, in contrast to most other class I proteins, lacks an eh1 domain and functions as an activator. Nevertheless, Pax6 represses Nkx2.2 expression in vivo, implying that its function at the pMN-p3 boundary is achieved through an intermediary repressor. The finding that the domain of Nkx2.2 expression expands dorsally upon Grg5 overexpression implies that this intermediary repressor itself functions in a Gro/TLE-dependent manner. Taken together, these observations suggest that the establishment and maintenance of ventral progenitor domains -- whether achieved by direct repression or by activation of intermediary repressors -- depend on the activity of Gro/TLE corepressors (Muhr, 2001).
The finding that the activity of progenitor HD proteins depends on Gro/TLE-mediated repression provides several insights into the strategies used to establish neuronal diversity in the central nervous system. Focus is placed here on how transcriptional repression mediates the functions of the class II repressor proteins Nkx6.1 and Nkx2.2, although similar arguments apply for many of the class I proteins. The class II proteins Nkx6.1 and Nkx2.2 are required for the generation of MNs and V3 neurons, respectively. These activities appear to be achieved through the expression of downstream determinants of neuronal subtype identity. For example, within the pMN domain, Nkx6.1 promotes the expression of MNR2, a dedicated MN determinant. Nkx6.1 functions as a repressor during the specification of MNs in dorsal regions of the neural tube, favoring the idea that Nkx6.1 controls the expression of MNR2 within the pMN domain itself through its role as a repressor of class I proteins, although this remains to be established. In this view, the loss of MNs in Nkx6.1 mutant mice results from the ectopic ventral expression of class I proteins rather than from the loss of an Nkx6.1 activator function (Muhr, 2001).
How do Nkx6.1 and Nkx2.2 induce MNs and V3 neurons along the entire dorsoventral axis of the neural tube? In ventral progenitor cells, the inductive activities of Nkx6.1 and Nkx2.2 appear to depend on their ability to act as repressors of their complementary class I proteins, Dbx2 and Pax6. But in the dorsal neural tube, progenitor cells lack expression of many of the ventral class I repressor proteins. Thus, dorsal neural progenitors must also express repressors of MN and V3 neuronal differentiation -- repressors that are themselves subject to repression by Nkx6.1 or Nkx2.2. The identity of the dorsal repressors of MN and V3 neuron generation is not known, but the Gsh1/2 HD proteins are plausible candidates as suppressors of MN specification. Both Gsh proteins possess an eh1 motif (see Supplemental table) and are normally restricted to the dorsal neural tube, but are ectopically expressed ventrally in mouse Nkx6.1 mutants (Muhr, 2001).
The class II proteins also inhibit alternative neuronal fates within their normal domains of expression. Within the pMN and p2 domains, the expression of Nkx6.1 prevents V1 interneuron generation, and within the p3 domain, Nkx2.2 expression prevents MN generation. Thus, the Nkx proteins promote certain neuronal fates and block others, even though both activities are mediated primarily through repression. This reliance on repression distinguishes the function of Nkx proteins in neural fate specification from that of many other transcription factors whose roles in the selection of cell fates appears to reflect a combination of activator and repressor functions. The expression of Nkx2.2 and Nkx6.1 persists in certain post-mitotic neurons, and thus it remains possible that putative activator functions of these proteins are relevant for aspects of neuronal differentiation other than those examined in this study. Indeed, in other regions of the developing nervous system, the Phox2 HD proteins have been shown to function as activators of neuronal differentiation genes (Muhr, 2001).
How is neuronal fate decided when two repressor HD proteins are coexpressed within individual neural progenitor cells? Within the p3 domain, cells coexpress Nkx6.1 and Nkx2.2, yet the activity of Nkx2.2 is dominant, and progenitors generate V3 neurons rather than MNs. One conceivable reason for this is that Nkx2.2 has a higher affinity than Nkx6.1 for Gro/TLE proteins and thus sequesters available Gro/TLE corepressor activity, preventing Nkx6.1 function. Against this idea, Dbx2 is ectopically expressed in p3 domain progenitors in Nkx6.1 mutants, indicating that Nkx6.1 still functions as a repressor in this domain. A second and more plausible explanation is that Nkx2.2 blocks MN generation in p3 progenitors at a step downstream of progenitor HD proteins by repressing the expression of MN subtype determinants. Thus, instances of coexpression of class I and/or class II repressor proteins within progenitor cells may reflect the selection of neuronal fate through repression at the level of downstream neuronal subtype determinants rather than at the level of progenitor HD proteins (Muhr, 2001).
Taken together, these findings favor a model in which the pattern of neuronal specification is achieved primarily through the selectivity of repressor interactions with cis-acting DNA sequences present in the regulatory regions of different progenitor HD proteins and neuronal subtype determinants. This model requires that repressor HD proteins with distinct activities in neuronal specification recognize distinct DNA target sequences. In support of this idea, the class II proteins Nkx2.2 and Nkx6.1 have different patterning activities in the neural tube, possess divergent HDs, and recognize distinct target DNA sequences. In addition, the finding that hybrid class I and class II proteins consisting solely of the HD fused to the EnR or TN domain mimic the activity of the full-length proteins indicates that the distinct activities of class II and class I repressor proteins in neural patterning are likely to reside in the specificity of DNA recognition encoded in the HD (Muhr, 2001).
The finding that class II proteins and most class I proteins function as repressors leaves unresolved the issue of the role of transcription factors that activate the expression of neuronal subtype determinants. The results imply that progenitor cells arrayed along the entire dorsoventral axis of the neural tube possess a latent potential for activation of expression of all neuronal subtype determinants. In an extreme view, these subtype determinants may be activated by a single common activator protein that is expressed in a uniform manner along the entire dorsoventral axis of the neural tube. The ability of such an activator to induce different subtype determinant genes would then be constrained by the repertoire of cis-acting binding sites for class I and class II HD protein repressors present in their regulatory regions. This view argues that the specificity of neuronal subtype generation emerges largely from the patterned expression of repressors (Muhr, 2001).
In principle, it is possible to consider an alternative view in which distinct activator proteins are expressed within individual progenitor domains, with these activators operating upstream of but in a linear pathway with neuronal subtype determinants such as MNR2. In this view, the patterns of expression of these upstream activators would themselves need to be defined by the repressor activities of the class I and class II HD proteins. But the question of what activates the domain-restricted expression of these upstream activators immediately resurfaces. Thus, at its root, the activation of subtype determinants along the dorsoventral axis of the neural tube is likely to be a spatially unrestricted process. Clarification of this issue will require the identification of proteins that activate the expression of neuronal subtype determinants (Muhr, 2001).
In this context, it is intriguing that several basic helix-loop-helix (bHLH) transcriptional factors are expressed in discrete domains along the dorsoventral axis of the neural tube. Some of these genes transgress progenitor domain boundaries, whereas others are restricted to individual progenitor domains. Studies of bHLH protein function in vertebrates have begun to suggest that these proteins can influence neuronal subtype identity, in addition to their more general roles in neurogenesis. Determining whether and how the activity of bHLH proteins is integrated with progenitor HD protein-mediated repression during the specification of neuronal fate may help in the further dissection of mechanisms of ventral neuronal patterning (Muhr, 2001).
This analysis of the function of progenitor HD proteins has focused on neuronal specification along the dorsoventral axis of the neural tube. There are also clear restrictions in the potential for neuronal generation along the rostrocaudal axis of the neural tube. It is noteworthy that many HD proteins implicated in rostrocaudal neural patterningóincluding other Pax and Nkx proteins, and the Gsh, Msx, Gbx, and Tlx proteinsóalso possess eh1-like domains. Indeed, in a sample of 165 vertebrate HD proteins, many expressed by neural cells, ~36% were found to possess an eh1 domain (see Supplemental table). Gro/TLE-dependent repression may, therefore, have a more pervasive role in establishing precise spatial patterns of neuronal generation along both major axes of neural tube development. In addition, since homologs of the Nkx, Msx, and Gsh proteins control neuronal patterning along the dorsoventral axis of the Drosophila CNS, these results suggest that Gro/TLE-mediated corepression may be an evolutionarily conserved step in CNS patterning (Muhr, 2001).
The genetic program that underlies the generation of visceral motoneurons in the developing hindbrain remains poorly defined. The roles of Nkx6 and Nkx2 (Drosophila homolog: Vnd) class homeodomain proteins in this process were examined; evidence is provided that these proteins mediate complementary roles in the specification of visceral motoneuron fate. The expression of Nkx2.2 in hindbrain progenitor cells is sufficient to mediate the activation of Phox2b, a homeodomain protein required for the generation of hindbrain visceral motoneurons. The redundant activities of Nkx6.1 and Nkx6.2, in turn, are dispensable for visceral motoneuron generation but are necessary to prevent these cells from adopting a parallel program of interneuron differentiation. The expression of Nkx6.1 and Nkx6.2 is further maintained in differentiating visceral motoneurons, and consistent with this the migration and axonal projection properties of visceral motoneurons are impaired in mice lacking Nkx6.1 and/or Nkx6.2 function. This analysis provides insight also into the role of Nkx6 proteins in the generation of somatic motoneurons. Studies in the spinal cord have shown that Nkx6.1 and Nkx6.2 are required for the generation of somatic motoneurons, and that the loss of motoneurons at this level correlates with the extinguished expression of the motoneuron determinant Olig2. Unexpectedly, it has been found that the initial expression of Olig2 is left intact in the caudal hindbrain of Nkx6.1/Nkx6.2 compound mutants, and despite this, all somatic motoneurons are missing. These data argue against models in which Nkx6 proteins and Olig2 operate in a linear pathway, and instead indicate a parallel requirement for these proteins in the progression of somatic motoneuron differentiation. Thus, both visceral and somatic motoneuron differentiation appear to rely on the combined activity of cell intrinsic determinants, rather than on a single key determinant of neuronal cell fate (Pattyn, 2003).
The current analysis provides new insight also into the role of Nkx6 and Olig proteins in the generation of sMNs. Olig2 has a dual role in sMN fate determination; it suppresses the expression of Irx3 in sMN progenitors, and also promotes cell-cycle exit and neuronal differentiation by derepression of the pro-neural bHLH protein Ngn2 in the sMN progenitor domain. Nkx6 proteins are required for the expression of Olig2 in the spinal cord, and there is a similar deficit of sMNs in Nkx6 mutants, Olig2 mutants and Olig1/2 compound mutants. Because forced expression of Nkx6.1 in the chick spinal cord results in the ectopic activation of Olig2 expression and the expression of Nkx6.1 is left unaffected in Olig mutants, a model in which Olig2 acts downstream of Nkx6 proteins in the sMN pathway has been proposed. In contrast to spinal cord levels, the initial phase of Olig2 expression is unaffected in the caudal hindbrain in Nkx6 mutants, and neither the expression of Irx3 nor Nkx2.2 have encroached into the sMN progenitor domain at this stage. Despite this, all sMNs are missing. These data reveal a requirement for Nkx6.1 and Nkx6.2 in sMN fate specification that is unrelated to their role in promoting Olig2 gene expression, and further indicate that Olig2, in the absence of Nkx6 protein function, is not sufficient to specify sMN fate in the hindbrain. These findings seem to exclude the possibility that Nkx6 and Olig proteins operate in a strict linear pathway. Since both Nkx6 and Olig proteins mediate their inductive activities by acting as repressors, it appears more likely that these proteins act in parallel to exclude different sets of repressor proteins from the sMN progenitor domain. If expressed in sMN progenitors in either Nkx6 or in Olig mutant mice, such Olig2 of Nkx6 regulated repressor proteins would be predicted to act independently of each other to block sMN generation at a step downstream of Olig2. This idea gains support by the fact that forced expression of Irx3 within the sMN progenitor domain, is sufficient to inhibit sMN generation (Pattyn, 2003 and references therein).
Within the developing vertebrate nervous system, specific subclasses of neurons are produced in vastly different numbers at defined times and locations. This implies the concomitant activation of a program that controls pan-neuronal differentiation and of a program that specifies neuronal subtype identity, but how these programs are coordinated in time and space is not well understood. Loss- and gain-of-function studies have defined Phox2b as a homeodomain transcription factor that coordinately regulates generic and type-specific neuronal properties. It is necessary and sufficient to impose differentiation towards a branchio- and viscero-motoneuronal phenotype and at the same time promote generic neuronal differentiation. The underlying genetic interactions have been examined. Phox2b has a dual action on pan-neuronal differentiation. It upregulates the expression of proneural genes (Ngn2) when expressed alone and upregulates the expression of Mash1 when expressed in combination with Nkx2.2. By a separate pathway, Phox2b represses expression of the inhibitors of neurogenesis Hes5 and Id2. The role of Phox2b in the specification of neuronal subtype identity appears to depend in part on its capacity to act as a patterning gene in the progenitor domain. Phox2b misexpression represses the Pax6 and Olig2 genes, which should inhibit a branchiomotor fate, and induces Nkx6.1 and Nkx6.2, which are expressed in branchiomotor progenitors. Phox2b behaves like a transcriptional activator in the promotion of both, generic neuronal differentiation and expression of the motoneuronal marker Islet1. These results provide insights into the mechanisms by which a homeodomain transcription factor through interaction with other factors controls both generic and type-specific features of neuronal differentiation (Dubreuil, 2002).
Genes belonging to the Nkx, Gsh and Msx families are expressed in similar dorsovental spatial domains of the insect and vertebrate central nervous system (CNS), suggesting the bilaterian ancestor used this genetic program during CNS development. The significance of these similar expression patterns was investigated by testing whether Nkx6 proteins expressed in ventral CNS of zebrafish and flies have similar functions. In zebrafish, Nkx6.1 is expressed in early-born primary and later-born secondary motoneurons. In the absence of Nkx6.1, there are fewer secondary motoneurons and supernumerary ventral interneurons, suggesting Nkx6.1 promotes motoneuron and suppresses interneuron formation. Overexpression of fish or fly Nkx6 is sufficient to generate supernumerary motoneurons in both zebrafish and flies. These results suggest that one ancestral function of Nkx6 proteins was to promote motoneuron development (Cheesman, 2004).
Flies possess a single Nkx6 gene orthologous to vertebrate Nkx6.1 and Nkx6.2 genes. Comparing zebrafish and fly proteins, there is 93% amino acid sequence identity within the homeodomain and 80% identity within the NK decapeptide. Outside these two conserved motifs there are several other regions of high amino acid identity (Cheesman, 2004).
Zebrafish nkx6.1 transcripts are first detected at the onset of gastrulation in the embryonic shield epiblast. Near the end of gastrulation, nkx6.1 is expressed in medial neurectoderm in two wide, diffuse stripes. By the 3-4 somite stage, nkx6.1 is confined to a tight stripe in medial neural keel, extending from the midbrain through the posterior neural plate. This pattern is maintained throughout somitogenesis; Nkx6.1 expression persists in ventral spinal cord until at least 48 hpf. nkx6.1 is detected in ventral hindbrain caudal to the midbrain-hindbrain boundary and in the pancreas at later stages. At all stages examined protein and RNA patterns appear indistinguishable. Cross-sections of 24 hpf embryos reveal Nkx6.1 expression in about five longitudinal cell rows in ventral spinal cord, including both medial and lateral floorplate; thus Nkx6.1-positive cells constitute approximately the ventral third of the spinal cord. This domain is similar to the olig2 expression domain; olig2 RNA is expressed in both progenitor and postmitotic cells, thus, Nkx6.1 must also be expressed in both of these cell types. The neurectodermal stripe of nkx6.1 includes the domain in which motoneuron progenitors undergo their final division. To determine whether postmitotic motoneurons express Nkx6.1, antibody double-label experiments were performed. Islet and Nkx6.1 proteins are colocalized in PMNs during early somitogenesis (14 hpf); later Nkx6.1 protein is downregulated. By 18 hpf, Nkx6.1 and Islet proteins are largely mutually exclusive and Nkx6.1 is expressed only in a few PMNs. Cross-sections at 48 hpf reveal Nkx6.1-positive SMN nuclei surrounded by Neurolin-positive plasma membranes, indicating co-expression in these cells. Thus both PMNs and SMNs express Nkx6.1 at least transiently (Cheesman, 2004).
Recent fate-mapping has revealed that in addition to motoneurons, several types of interneurons are derived from the olig2-positive ventral spinal cord domain. Whether one of these types of interneurons express Nkx6.1 was tested by labeling individual Ventral Longitudinal Descending (VeLD) interneurons with fluorescent dextrans, immediately fixing the embryos and examining whether the labeled cells co-expressed Nkx6.1. At 20 hpf most VeLDs are Nkx6.1-positive, however by ~24 hpf VeLDs has downregulated Nkx6.1 expression. Thus, it is inferred that VeLDs express Nkx6.1 early in development but downregulate it following axon extension (Cheesman, 2004).
Studies in chick and mouse suggest Hh induces expression of Nkx6.1 in ventral neural tube (Briscoe, 2000), thus, whether Hh establishes or maintains nkx6.1 expression in zebrafish was investigated. Injection of synthetic shh mRNA causes a dramatic dorsal expansion of nkx6.1 expression. At 3-5 somites and 18 hpf, injected animals have many ectopic nkx6.1-positive cells within the neural tube. Thus, Hh is sufficient to induce nkx6.1 expression in zebrafish CNS. However, nkx6.1 expression never expanded into the dorsalmost region of the neural tube (Cheesman, 2004).
Whether Hh is necessary for nkx6.1 expression was tested. At 24 hpf, Nkx6.1 expression appeared fairly normal in syu (shh) mutants, but was greatly reduced in smu (slow muscle omitted which encodes zebrafish Smoothened) mutants. smu mutants are more severe than syu mutants, however they still retain some early Hh signaling. To further suppress Hh signaling echidna hedgehog (ehh) and tiggy winkle hedgehog (twhh) morpholino antisense oligonucleotides (MOs) were injected into syu mutants. At 24 hpf nearly 25% of injected embryos displayed severely reduced nkx6.1 neural tube expression. This probably represents the syu mutant class with the greatest loss of Hh signals. About 75% of injected embryos had weaker spinal cord nkx6.1 expression than wild types, and thus probably constituted knockdown of ehh and twhh in a wild-type or heterozygous syu background. Earlier, at 12 hpf, about 25% of a clutch of syu mutants injected with ehh and twhh MOs had dramatically reduced nkx6.1 expression. Some embryos had a more severe loss than others, but in no embryo was nkx6.1 completely absent. From these data it is inferred that Hh signals are required early to induce at least the vast majority of nkx6.1 expression and later for its maintenance. These experiments suggest that zebrafish nkx6.1 acts downstream of Hh signaling, as in chick and mouse (Cheesman, 2004).
Because in zebrafish nkx6.1 is expressed in motoneurons and their progenitors, whether nkx6.1 was sufficient to generate these cells was tested. All zebrafish primary motoneurons (PMNs) initially express islet1 (isl1); later, two specific PMNs (CaP and VaP) downregulate isl1 and initiate expression of a related gene, islet2 (isl2) whereas two other PMNs (MiP and RoP) continue to express isl1. The presence of isl1-positive or isl2-positive PMNs was assayed for by RNA in situ hybridization at 18 hpf. The isl1 probe revealed supernumerary MiPs and RoPs and the isl2 probe revealed supernumerary CaPs and VaPs. Large clusters of zn1-positive motoneurons projecting axons into the periphery were observed as compared to wild types. Many of these supernumerary PMNs were located more dorsally than native PMNs, suggesting that Nkx6.1 converts some dorsal cells to a ventral fate. Injection of a GATA-2:GFP transgenic line that expresses GFP predominantly in SMNs with synthetic nkx6.1 mRNA revealed supernumerary SMNs at 24 hpf. Because Nkx6.1 is expressed in VeLD interneurons, whether overexpression of Nkx6.1 affected this cell type was tested. VeLDs are recognized by cell body position and GABA expression. Embryos ectopically expressing nkx6.1 RNA have fewer GABA-positive VeLDs than wild types at 24 hpf, suggesting cells that would normally become VeLDs become motoneurons instead (Cheesman, 2004).
Hh can induce nkx6.1, and nkx6.1 is sufficient for formation of supernumerary PMNs and SMNs, suggesting that nkx6.1 acts downstream of Hh. This by injecting synthetic nkx6.1 mRNA into clutches of embryos derived from smu heterozygotes; smu mutants have fewer PMNs and no SMNs because of reduced Hh signaling. At 18 hpf, approximately 25% of injected embryos lack isl2 expression in caudal spinal cord, just like 25% of embryos from a smu heterozygote cross, indicating that nkx6.1 is insufficient to restore PMNs in the absence of Hh. Similarly at 30 hpf, nkx6.1 is insufficient to restore SMNs in smu mutants. It is concluded that nkx6.1 alone is sufficient to induce motoneurons only in the presence of Hh, suggesting that Nkx6.1 collaborates with additional factors downstream of Hh during motoneuron induction (Cheesman, 2004).
To test whether Nkx6.1 is required for motoneuron formation, embryos were injected with an nkx6.1-specific MO. Surprisingly, nkx6.1 MO-injected animals have normal numbers of PMNs, revealed by isl1 expression at 12 hpf and isl2 expression at 18 hpf. MO-injected embryos initiate the spontaneous tail reflex around 18 hpf, thus PMNs are functional. However, in contrast to embryos injected with a mispaired control nkx6.1 MO, which appeared wild type at 36 and 48 hpf, nkx6.1 MO-injected embryos do not swim when touched, suggesting an absence of SMNs. Consistent with this, at 48 hpf, nkx6.1 MO-injected animals consistently had fewer SMNs. At this stage there are more than 30 SMNs per spinal hemisegment; these cells are difficult to count because their somata are closely packed. Thus, MO-injected embryos were divided into two categories: those with nearly wild-type numbers of SMNs, and those with less than half of the wild-type number. Most MO-injected embryos had less than half of the wild-type number of SMNs; in a few cases SMNs were entirely absent. To test whether SMNs were dying in MO-injected embryos, TUNEL assays were performed at several stages between 18 and 48 hpf. There was no discernible difference in the number of TUNEL-positive nuclei in the ventral spinal cords of MO-injected and wild-type embryos at any stage. Whether decreased proliferation accounted for the decrease in SMNs in MO-injected embryos was also tested. BrdU incorporation showed no difference between MO-injected and wild-type embryos at 24, 30 and 36 hpf Cheesman, 2004). It is concluded that lack of SMNs is not due to a change in birth or death of these cells, suggesting that in the absence of Nkx6.1, they undergo a fate change. There are many more GABA-positive cells in the VeLD position in nkx6.1 MO-injected embryos than in wild-type embryos, suggesting that in the absence of Nkx6.1, SMNs develop as VeLD interneurons. Supporting this hypothesis is the fact that this phenotype is the opposite that of embryos overexpressing Nkx6.1 (Cheesman, 2004).
Whether Nkx6 genes have conserved functions was tested by overexpressing the fly gene in zebrafish and the zebrafish gene in flies. It was first asked whether ectopic expression of fly or zebrafish Nkx6 produces the same phenotype in zebrafish embryos. 18 hpf zebrafish embryos overexpressing fly Nkx6 mRNA had supernumerary PMNs as compared to wild types, similar to the phenotype of embryos overexpressing zebrafish nkx6.1; both fish and fly Nkx6 appear equally potent at generating ectopic PMNs in zebrafish (Cheesman, 2004).
The ability of animals to carry out their normal behavioral repertoires requires exquisitely precise matching between specific motoneuron subtypes and the muscles they innervate. However, the molecular mechanisms that regulate motoneuron subtype specification remain unclear. This study used individually identified zebrafish primary motoneurons to describe a novel role for Nkx6 and Islet1 proteins in the specification of vertebrate motoneuron subtypes. Zebrafish primary motoneurons express two related Nkx6 transcription factors. In the absence of both Nkx6 proteins, the CaP motoneuron subtype develops normally, whereas the MiP motoneuron subtype develops a more interneuron-like morphology. In the absence of Nkx6 function, MiPs exhibit normal early expression of islet1, which is required for motoneuron formation; however, they fail to maintain islet1 expression. Misexpression of islet1 RNA can compensate for loss of Nkx6 function, providing evidence that Islet1 acts downstream of Nkx6. It is suggested that Nkx6 proteins regulate MiP development at least in part by maintaining the islet1 expression that is required both to promote the MiP subtype and to suppress interneuron development (Hutchinson, 2007).
In the developing spinal cord, early progenitor cells of the oligodendrocyte lineage are induced in the motor neuron progenitor (pMN) domain of the ventral neuroepithelium by the ventral midline signal Sonic hedgehog (Shh). The ventral generation of oligodendrocytes requires Nkx6-regulated expression of the bHLH gene Olig2 in this domain. In the absence of Nkx6 genes or Shh signaling, the initial expression of Olig2 in the pMN domain is completely abolished. In vivo evidence is provided for a late phase of Olig gene expression independent of Nkx6 and Shh gene activities and reveal a brief second wave of oligodendrogenesis in the dorsal spinal cord. In addition, genetic evidence is provided that oligodendrogenesis can occur in the absence of hedgehog receptor Smoothened, which is essential for all hedgehog signaling (Cai, 2005).
Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells. Nkx2.2 is the member of the vertebrate homeodomain transcription factor gene family that is most homologous to the Drosophila NK2/ventral nervous system defective (vnd) gene. Nkx2.2 was originally identified as a gene that is expressed in ventral regions of the developing vertebrate CNS. In addition to Nkx2.2, five other family members have been identified in mice: Nkx2.1, Nkx2.2, Nkx2.3 and Nkx2.4 are closely related, while Nkx2.5 and Nkx2.6 represent more divergent members of the family. NK2 family members have now been shown to be key regulators of development and differentiation in several tissues: Nkx2.1 is necessary for lung, thyroid and ventral forebrain development and Nkx2.5 is required for proper heart formation. Therefore, it is possible that Nkx2.2 may play a similar role in the development of the pancreas. The endocrine pancreas is organized into clusters of cells called islets of Langerhans comprizing four well-defined cell types: alpha, beta, delta and PP cells. While recent genetic studies indicate that islet development depends on the function of an integrated network of transcription factors, the specific roles of these factors in early cell-type specification and differentiation remain elusive. Within the pancreas, Nkx2.2 is expressed in alpha, beta and PP cells, but not in delta cells. Mice homozygous for a null mutation of Nkx2.2 develop severe hyperglycemia and die shortly after birth. Immunohistochemical analysis reveals that the mutant embryos lack insulin-producing beta cells and have fewer glucagon-producing alpha cells and PP cells. Remarkably, in the mutants there remains a large population of islet cells that do not produce any of the four endocrine hormones. These cells express some beta cell markers, such as islet amyloid polypeptide and Pdx1 (a homeodomain transcription factor that is an important factor in the proliferation and differentiation of the pancreatic buds to form a mature pancreas), but lack other definitive beta cell markers including glucose transporter 2 and Nkx6.1. It is proposed that Nkx2.2 is required for the final differentiation of pancreatic beta cells, and in its absence, beta cells are trapped in an incompletely differentiated state (Sussel, 1998).
Genetic studies are beginning to outline the hierarchy of transcription factors involved in beta cell development. For example, in Nkx2.2 mutant embryos, early expression of the beta cell specific transcription factor Nkx6.1 is unaffected. However, between E12.5 and E18.5, when there are major changes taking place within the pancreas (differentiation of exocrine tissue; beta cell proliferation; delta and PP cell formation) Nkx6.1 expression disappears. The data are consistent with a model where Nkx2.2 is required for the maintenance of Nkx6.1 expression as beta cells differentiate, and that continued expression of Nkx6.1 is necessary for complete beta cell differentiation. The genetic relationship between Nkx2.2 and Pdx1 is more complicated. Early in development, Pdx1 is expressed in all cells of the pancreatic bud, and is required for expansion of the bud. However, later in embryonic development, Pdx1 becomes progressively restricted to beta cells (and some delta cells); and at approximately E14.5, Pdx1 becomes upregulated in beta cells suggesting it plays a role in beta cell differentiation. In the Nkx2.2 mutant, early expression of Pdx1 is not affected. The later beta cell restriction of Pdx1 expression also occurs, but is quantitatively reduced in comparison to wild-type beta cells. Therefore, Nkx2.2 may be required for inducing high level expression of Pdx1 in beta cells, and the up-regulation of Pdx1 may be a necessary step in the final differentiation of the beta cell. In contrast to Nkx6.1 or Pdx1 expression, the expression of Isl1, Pax6 and Brn4 during embryogenesis does not appear to require Nkx2.2. These genes may therefore either lie upstream or in different pathways relative to the Nkx genes. Since Isl1 is expressed in islet cells soon after they exit the cell cycle, normal expression of Isl1 in the Nkx2.2 mutant suggests that all the islet cells are able to normally exit a proliferating state and proceed with a program of differentiation. This result supports the hypothesis that the immature beta cells are able to initiate beta cell development and it is subsequent steps of terminal differentiation that are blocked (Sussel, 1998 and references).
beta-Cell differentiation factor Nkx6.1 is a homeodomain protein expressed in developing and mature beta-cells in the pancreatic islets of Langerhans. To understand how it contributes to beta-cell development and function, its DNA binding and transactivation properties were characterized. A single copy of the homeodomain of Nkx6. 1 binds to a strictly conserved 8-base pair DNA consensus sequence, TTAATTAC; even minor variations to this consensus reduce DNA binding affinity significantly. Full-length Nkx6.1, however, has markedly reduced DNA binding affinity due to an acidic domain at the carboxyl end of the molecule that functions as a mobile binding interference domain capable of interrupting the interaction between DNA and DNA binding domains of the helix-turn-helix type. When expressed in fibroblast cell lines, Nkx6.1 represses transcription through isolated Nkx6.1 binding sites; in beta-cell lines, Nkx6.1 specifically represses the intact insulin promoter through TAAT-containing sequences. In Gal4 one-hybrid fusion studies, transcriptional repression maps to a discreet region within the amino terminus. These findings suggest a model in which Nkx6.1, regulated by interactions through its carboxyl terminus, directs the repression of specific genes in developing and mature beta-cells (Mirmira, 2000).
Most insulin-producing beta-cells in the fetal mouse pancreas arise during the secondary transition, a wave of differentiation starting at embryonic day 13. Disruption of homeobox gene Nkx6.1 in mice leads to loss of beta-cell precursors and blocks beta-cell neogenesis specifically during the secondary transition. In contrast, islet development in Nkx6.1/Nkx2.2 double mutant embryos is identical to Nkx2.2 single mutant islet development: beta-cell precursors survive but fail to differentiate into beta-cells throughout development. Together, these experiments reveal two independently controlled pathways for beta-cell differentiation, and place Nkx6.1 downstream of Nkx2.2 in the major pathway of beta-cell differentiation (Sander, 2000b).
In the mature pancreas, the homeodomain transcription factor Nkx6.1 is uniquely restricted to beta-cells. Nkx6.1 also is expressed in developing beta-cells and plays an essential role in their differentiation. Among cell lines, both beta- and alpha-cell lines express nkx6.1 mRNA; but no protein can be detected in the alpha-cell lines, suggesting that post-transcriptional regulation contributes to the restriction of Nkx6.1 to beta-cells. To investigate the regulator of Nkx6.1 expression, the promoter structure of the mouse nkx6.1 gene has been analyzed, and regions that direct cell type-specific expression were identified. The nkx6.1 gene has a long 5'-untranslated region (5'-UTR) downstream of a cluster of transcription start sites. nkx6.1 gene sequences from -5.6 to +1.0 kilobase pairs have specific promoter activity in beta-cell lines but not in NIH3T3 cells. This activity is dependent on sequences located at about -800 base pairs and on the 5'-UTR. Electrophoretic mobility shift assays demonstrate that homeodomain transcription factors PDX1 and Nkx2.2 can bind to the sequence element located at -800 base pairs. In addition, dicistronic assays establish that the 5'-UTR region functions as a potent internal ribosomal entry site, providing cell type-specific regulation of translation. These data demonstrate that complex regulation of both Nkx6.1 transcription and translation provides the specificity of expression required during pancreas development (Watada, 2000).
In the pancreas, the NK homeodomain transcription factor Nkx6.1 is required for the development of beta-cells and is believed to function as a potent repressor of transcription upon binding to A/T-rich sequences within the promoter region of target genes. Because the nkx6.1 promoter itself contains several such sequences, the possibility is considered that the expression level and restricted pattern of the nkx6.1 gene might be precisely regulated by one or more homeodomain transcription factors, including Nkx6.1 itself. In this report, a novel beta-cell-specific enhancer element is identified in the nkx6.1 gene between -157 and -30 bp (relative to the transcriptional start site) that harbors a conserved A/T-containing sequence flanked by G/C-rich stretches. Although the islet homeodomain-containing activator Pdx-1 is unable to stimulate transcription of a reporter gene through this enhancer element in mammalian cell lines, strikingly, Nkx6.1 robustly activates transcription through direct interaction with the A/T-rich sequence in this element. This activation is indeed transcriptional in nature (and not secondary to translational effects) and is mediated by a modular acidic sequence within the COOH-terminal domain of Nkx6.1. It has been shown by EMSAs that Nkx6.1 binds to the beta-cell-specific enhancer in vitro; by chromatin immunoprecipitation assays it has been shown that Nkx6.1 natively occupies this region in vivo in betaTC3 cells. It is therefore concluded that Nkx6.1 is a bifunctional transcription factor that serves to maintain the specific expression of its own gene during beta-cell differentiation while simultaneously effecting broader gene repression events (Iype, 2004).
In diabetic individuals, the imbalance in glucose homeostasis is caused by loss or dysfunction of insulin-secreting ß-cells of the pancreatic islets. As successful generation of insulin-producing cells in vitro could constitute a cure for diabetes, recent studies have explored the molecular program that underlies ß-cell formation. From these studies, the homeodomain transcription factor NKX6.1 has proven to be a key player. In Nkx6.1 mutants, ß-cell numbers are selectively reduced, while other islet cell types develop normally. However, the molecular events downstream of NKX6.1, as well as the molecular pathways that ensure residual ß-cell formation in the absence of NKX6.1 have remained largely unknown. This study shows that the Nkx6.1 paralog, Nkx6.2, is expressed during pancreas development and partially compensates for NKX6.1 function. Surprisingly, analysis of Nkx6 compound mutant mice reveals a previously unrecognized requirement for NKX6 activity in alpha-cell formation. This finding suggests a more general role for NKX6 factors in endocrine cell differentiation than formerly suggested. Similar to NKX6 factors, the transcription factor MYT1 has recently been shown to regulate alpha- as well as ß-cell development. Expression of Myt1 depends on overall Nkx6 gene dose, and therefore identifies Myt1 as a possible downstream target of Nkx6 genes in the endocrine differentiation pathway (Henseleit, 2005).
Despite much progress in identifying transcriptional regulators that control the specification of the different pancreatic endocrine cell types, the spatiotemporal aspects of endocrine subtype specification have remained largely elusive. This study addressed the mechanism by which the transcription factors Nkx6.1 (Nkx6-1) and Nkx6.2 (Nkx6-2) orchestrate development of the endocrine alpha- and beta-cell lineages. Specifically, an assay was performed for the rescue of insulin-producing beta-cells in Nkx6.1 mutant mice upon restoring Nkx6 activity in select progenitor cell populations with different Nkx6-expressing transgenes. Beta-cell formation and maturation was restored when Nkx6.1 was expressed in multipotential Pdx1+ pancreatic progenitors, whereas no rescue was observed upon expression in committed Ngn3+ (Neurog3+) endocrine progenitors. Although not excluding additional roles downstream of Ngn3, this finding suggests a first requirement for Nkx6.1 in specifying beta-cell progenitors prior to Ngn3 activation. Surprisingly, although Nkx6.2 only compensates for Nkx6.1 in alpha-but not in beta-cell development in Nkx6.1-/- mice, a Pdx1-promoter-driven Nkx6.2 transgene had the same ability to rescue beta-cells as the Pdx1-Nkx6.1 transgene. This demonstrates that the distinct requirements for Nkx6.1 and Nkx6.2 in endocrine differentiation are a consequence of their divergent spatiotemporal expression domains rather than their biochemical activities and implies that both Nkx6.1 and Nkx6.2 possess alpha- and beta-cell-specifying activities (Nelson, 2007).
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