ventral nervous system defective



Cloning and expression patterns of NK-2 homeobox genes

There are VND/NK2 related homeodomain proteins found in species ranging from planaria to mouse (Jimenez, 1995).

Expression of an NK-2 homeobox gene of the freshwater cnidarian, Hydris vulgaris is restricted to endodermal epithelial cells and is primarily expressed in the peduncle, the lower end of the body column. CnNK-2 expression invariably precedes foot formation as part of the normal tissue dynamics of the adult as well as during asexual reproduction by budding, foot regeneration, or ectopic food formation. Since hydra has no mesoderm, it is suggested that when trophoblastic animals appeared during evolution, expression of the genes of the NK class spread to this new tissue. In planaria, which diverged a little later than cnidarians during metazoan evolution, one of the two NK-2 homologs is expressed in an endodermal derivative; the other is expressed in the mesoderm. In nematodes, the single described NK gene, ceh-22, is expressed in mesodermal tissue, specifically pharyngeal muscle. Groups that appeared later, the vertebrates and arthropods, have several members of the NK-2 class. In these organisms, the range of tissues in which NK-2 genes are expressed has expanded to include not only endodermal derivatives and muscle, but also the central nervous system and other tissues (Grens, 1996 and references).

An amphioxus (Cephalochordata within the phylum Chordata) NK-2 homeobox gene (AmphiNk2-1), a homolog of vertebrate Nkx2-1, has been characterized that is involved in the development of the central nervous system and thyroid gland. At the early neurula stage of amphioxus, AmphiNk2-1 expression is first detected medially in the neural plate. By the mid-neurula stage, expression is localized ventrally in the nerve cord and also begins in the endoderm. During the late neurula stage, the ventral neural expression becomes transiently segmented in the posterior and is then down-regulated except in the cerebral vesicle at the anterior end of the central nervous system. Within the cerebral vesicle AmphiNk2-1 is expressed in a broad ventral domain, probably comprising both the floor plate and basal plate regions; this pattern is comparable to Nkx2-1 expression in the mouse diencephalon. In the anterior part of the gut, expression becomes intense in the endostyle (the right wall of the pharynx), which is the presumed homolog of the vertebrate thyroid gland. More posteriorly, there is transitory expression in the midgut and hindgut. In sum, the present results help to support homologies (1) between the amphioxus endostyle and the vertebrate thyroid gland and (2) between the amphioxus cerebral vesicle and the vertebrate diencephalic forebrain (Venkatesh, 1999).

The genome of amphioxus includes AmphiNk2-2, the first gene of the NK2 homeobox class to be demonstrated in any invertebrate deuterostome. AmphiNk2-2 encodes a protein with a TN domain, homeodomain, and NK2-specific domain; on the basis of amino acid identities in these conserved regions, AmphiNk2-2 is a homolog of Drosophila vnd and vertebrate Nkx2-2. During amphioxus development, expression of AmphiNk2-2 is first detected ventrally in the endoderm of late gastrulae. In neurulae, endodermal expression divides into three domains (the pharynx, midgut, and hindgut), and neural expression commences in two longitudinal bands of cells in the anterior neural tube. These neural tube cells occupy a ventrolateral position on either side of the cerebral vesicle (the probable homolog of the vertebrate diencephalic forebrain). The dynamic expression patterns of AmphiNkx2-2 suggest successive roles, first in regionalizing the endoderm and nervous system and later during differentiation of specific cell types in the gut (possibly peptide endocrine cells) and brain (possibly including axon outgrowth and guidance) (Holland, 1998).

Zebrafish nk2.2, a member of the Nk-2 family of homeobox genes, has been isolated. nk2.2 is expressed in a continuous narrow band of cells along a boundary zone demarcating the location at which two of the earliest nuclei in the brain differentiate. This band of cells is located within a few cell diameters of cells expressing the signaling protein Sonic hedgehog. Injection of shh mRNA results in ectopic expression of nk2.2 and concomitant abnormalities in the forebrain and eyes. Moreover, cyclops mutant embryos, which initially lack neurectodermal expression of shh, show a concomitant lack of nk2.2 expression. Together, these results suggest a requirement of Shh protein for the spatial regulation of nk2.2 expression (Barth, 1995).

One of the distinguishing features of vertebrate development is the elaboration of the anterior neural plate into forebrain and midbrain, yet little is known about the early tissue interactions that regulate pattern formation in this region or the genes that mediate these interactions. As an initial step toward analyzing the process of regionalization in the anterior-most region of the brain, an anterior neural Xenopus cDNA library was screened for homeobox clones: one such isolated clone is called XeNK-2 (Xenopus NK-2), because of its homology to the NK-2 family of homeobox genes. From neurula stages, when XeNK-2 is first detectable, through hatching stages, XeNK-2 mRNA is expressed primarily in the anterior region of the brain. By the free-swimming tadpole stages, XeNK-2 expression resolves into a set of bands positioned at the forebrain-midbrain and the midbrain-hindbrain boundaries; subsequently, XeNK-2 transcripts are no longer detectable. In addition to localized expression along the anterior-posterior axis, XeNK-2 may also play a role in the process of regionalization along the dorsal-ventral axis of the developing brain. At all stages examined, XeNK-2 mRNA is restricted to a pair of stripes that are bilaterally symmetrical in the ventral-lateral region of the brain. To begin to identify the tissue interactions that are required for the proper spatial and temporal localization of XeNK-2, a series of explant experiments was performed. Consistent with earlier work showing that the A/P axis is not fixed at mid-gastrula stages, it was shown that XeNK-2 expression is activated when assayed in gastrula stage explants taken from any region along the entire A/P axis and that the tissue interactions necessary to localize XeNK-2 along the A/P axis are not completed until later neurula stages (Saha, 1993).

The neural plate and neural tube show a longitudinal organization that is conserved in all of vertebrates. Expression of noggin (a protein that binds to and inactivates BMP-4, the vertebrate homolog of decapentaplegic), sonic hedgehog (Vertebrate homolog: Hedgehog) and Nkx-2.2 define longitudinal columns of cells that are present along the entire CNS axis. Within the forebrain, the expression of these genes, as well as that of Nkx-2.1 and BF-1, are in distinct longitudinal regions in the neural plate and tube. The earliest longitudinal axon pathways of the forebrain are spatially correlated with the longitudinal domain defined by Nkx-2.2. Expression of each of these genes, and Otx-1 and Emx-2, suggests that the cephalic neural plate is organized into molecularly distinct domains delimited by longitudinal and transverse borders (Shimamura, 1995).

A novel mouse homeobox-containing gene, Nkx-2.2, has been isolated. Nkx-2.2 is a member of a family of genes whose homeodomains are homologous to that of the Drosophila NK-2 gene. Nkx-2.2 transcripts are found in localized domains of the brain during mouse embryogenesis. Nkx-2.2 expression in the brain abuts and partially overlaps with the expression domains of two other related homeobox-containing genes, TTF-1 and Dlx (Drosophila homolog: Distal-less). The expression domains of the three genes in the developing prosencephalon coincide with anatomical boundaries, particularly apparent in the diencephalon. This result raises the possibility that these genes may specify regional differentiation of the developing diencephalon into its anatomically and functionally defined subregions. Nkx-2.2 may be involved in specifying diencephalic neuromeric boundaries (Price, 1992).

Several members of the chicken and mouse Nkx gene family are described. 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 (Drosophila homolog: Vnd) is expressed along the entire axis, whereas Nkx-2.1 is restricted to the forebrain; Nkx-6.1 and Nkx-6.2 (both without closely related Drosophila homologs) 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 (Qui, 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. 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).

Members of the NK-2 homeobox gene family are expressed in distinct parts of the central nervous system and in other non-neural territories not only in the fruitfly Drosophila melanogaster, but also in vertebrates. The murine Nkx2.1 (TTF-1, T/ebp) gene is indispensable in the developing forebrain, hypophysis, thyroid and lung. The early transcript distribution of the chick NKX2.1 gene is reported here. By whole-mount in situ hybridization a novel transient expression domain has been found in the early epiblast. Further expression occurs in the ventral medial endoderm, which becomes restricted to the anlage fields of the thyroid and lung, in the ventral diencephalon and telencephalon. These findings suggest that NKX2.1 is part of an Nkx code that specifies ventral territories in the vertebrate embryo (Pera, 1998).

The cortex and basal ganglia are the major structures of the adult brain derived from the embryonic telencephalon. Two morphologically distinct regions of the basal ganglia are evident within the mature ventral telencephalon: the globus pallidus (medially) and the striatum, which is positioned between the globus pallidus and the cortex. The globus pallidus develops from the medial ganglionic eminence (MGE), while the striatum develops from the lateral ganglionic eminence (LGE). These structures arise sequentially during development, with the MGE appearing immediately after anterior neuropore closure, followed later by the appearance of the LGE. Deletion of the Sonic Hedgehog gene in mice indicates that this secreted signaling molecule is vital for the generation of both these ventral telencephalic regions. Sonic hedgehog induces differentiation of ventral neurons characteristic of the medial ganglionic eminence, the embryonic structure which gives rise to the globus pallidus. While both Shh and Nkx2.1 are expressed strongly within the MGE, neither is present within the LGE. Sonic hedgehog induces ventral neurons with patterns of gene expression characteristic of the lateral ganglionic eminence. Although Ikaros is an LGE-specific marker, its expresssion only becomes detectable near birth, by in situ hybridization. Therefore, the absence or presence of Shh or Nkx2.1 in combination with the more widespred ventral telencephalic markers (Dlx, Evf-1, Islet-1/2 and GAD-16 ES) were used to distinguish LGE- and MGE-derived ventral neurons. An in vitro assay was used to evaluate the role of Shh in LGE induction. During a narrow window of competence, between E10.5-E11.5 of rat development (10 to 23 somites), either ventral telecephalic midline tissue or Shh protein can induce telencephalic tissue to express genes characteristic of the LGE. Even at the highest Shh concentration, the MGE/pallidal marker Nkx2.1 is not induced at this stage of development. Furthermore, if Shh is blocked between the time the MGE and LGE are induced, Dlx expression is greatly reduced. E11.5 explants in isolation are found to undergo a progressive loss of competence to express Dlx in response to Shh after one or two days in culture. These results suggest that temporally regulated changes in Sonic Hedgehog responsiveness are integral in the sequential induction of basal telencephalic structures (Kohtz, 1998).

The Brx1 homeobox gene has been isolated and shown to be expressed in the zona limitans intrathalamica (ZLI) of the mouse embryo. Brx1 is a member of the Brx gene family and comprises the genes for Brx1a and Brx1b, which differ in sequence in the region located on the 5'-terminal side of the homeobox. The complete amino acid sequences of the open reading frame of Brx1a and Brx1b were determined and each is found to be similar to that of Rgs, the mouse homolog of the Rieger syndrome associated human RIEG gene (RGS), to the extent that the sequence of Rgs has been clarified. Brx1 is strongly expressed in the mammillary area as well as in the ZLI of the mouse embryonic brain. Homologs of Brx1a and Brx1b were isolated in chick in which the expression of Brx1 in the ventral diencephalon is well conserved. The expression of Brx1 along with that of Sonic hedgehog (Shh), Nkx2.2, Dlx1 and Arx was examined at the time of the formation of ZLI in mouse embryos. The expression of Shh is initially noted in the ventricular zone of the presumptive ZLI and is then replaced by that of Brx1 at the time of radial migration of the neuroepithelial cells. Nkx2.2 is widely expressed in the ventricular zone of presumptive ZLI and also as a narrow band in the mantle zone. The expression of Dlx1 and Arx in the presumptive ventral thalamus extends as far as ZLI and overlaps with that of Brx1. The Dlx1- and Arx-expressing cells in ZLI, which extends towards the lateral (pial) surface of the diencephalic wall, differ from those expressing Nkx2.2 and Brx1. The embryonic ventral lateral geniculate nucleus present in the visual pathway is eventually formed from these cells. Each homeobox gene is also expressed regionally in the nucleus, suggesting that the nucleus is comprised of subdivisions (Kitamura, 1997).

Chicken Nkx-2.8 (cNkx-2.8) has been found to be 68% homologous to Ventral nervous system defective and Msh-2 of Drosophila, and more closely related to vertebrate Nkx-2.5 proteins. All NK-2 family members have two highly conserved amino acid sequences: (1) the amino terminal TN domain and (2) the NK-2-specific domain lying between the homeobox and the C-terminal sequences. cNkx-2.8 transcripts are first detectable at HH stage 7 in the splanchnopleura. At stage 10(+), the cNkx-2.8 gene is expressed in the linear heart tube and the dorsal half of the vitelline vein. However, after looping at HH stage 13, cNkx-2.8 is no longer expressed in the looped heart tube, but is expressed in the ventral pharyngeal endoderm. At stage 15, in addition to the pharyngeal expression pattern, cNkx-2.8 is expressed in the ectoderm of the pharyngeal arches and the aortic sac. By HH Stage 17, cNkx-2.8 expression is detectable in the lateral endoderm of the second and third pharyngeal pouches, the posterior portion of the aortic sac, and the sinus venosus. cNkx-2.8 binds to previously characterized Nkx2-1 and Nkx2-5 DNA-binding sites. Overexpression of cNkx-2.8 transactivates a minimal promoter, which contains multimerized Nkx-2 DNA-binding sites. cNkx-2.8 and serum response factor can coactivate a minimal cardiac alpha-actin promoter. These data are consistent with a model in which cNkx-2.8 performs a unique temporally and spatially restricted function in the developing embryonic heart and pharyngeal region. The coexpression of cNkx-2.5 and -2.8 raises the possibility that cNkx-2.8 may have a redundant role with respect to cNkx-2.5 in the coalescing heart tube and may play an important role in the transcriptional program(s) that underlies thymus formation. It is possible that the position and identities of various organ rudiments are determined by the combinatorial expression of Nkx genes (Reecy, 1997)

A novel cDNA was partially isolated from a HepG2 cell expression library by screening with the promoter-linked coupling element (PCE), a site from the alpha-fetoprotein (AFP) gene promoter. The remainder of the cDNA was cloned from fetal liver RNA using random amplification of cDNA ends. The cDNA encodes a 239-amino acid peptide with domains closely related to the Drosophila factor ventral nervous system defective (nk2). The new factor is the eighth vertebrate factor related to vnd, hence nkx-2.8. Northern blot and reverse transcriptase polymerase chain reaction analysis demonstrate mRNA in HepG2, in two other AFP-expressing human cell lines, and in human fetal liver. Transcripts are not detected in adult liver. Cell-free translation produces DNA binding activity that gel shifts a PCE oligonucleotide. Cotransfection of nkx-2.8 expression and PCE reporter plasmids into HeLa cells demonstrate transcriptional activation; NH2-terminal deletion eliminates this activity. Cotransfection into AFP-producing hepatocytic cells represses AFP reporter expression, suggesting that endogenous activity is already present in these cells. In contrast, cotransfection into an AFP-negative hepatocytic line produces moderate activation of the AFP gene. The cardiac developmental factor nkx-2.5 can substitute for nkx-2.8 in all transfection assays, whereas another related factor, thyroid transcription factor 1, shows a more limited range of substitution. Although the studies have yet to establish definitively that nkx-2.8 is the AFP gene regulator PCF, the two factors share a common DNA binding site, gel shift behavior, migration on SDS-acrylamide gels, and cellular distribution. Moreover, the nk-2-related genes are developmental regulators, and nkx-2.8 is the first such factor associated with liver development (Apergis, 1998).

Three novel mouse homeobox genes have been identified that are related to the Drosophila NK gene family. Two genes without direct homologues in Drosophila are designated Nkx-5.1 and Nkx-5.2; the third gene Nkx-1.1 constitutes the mouse homologue to NK-1. Nkx-5.1 and Nkx-5.2 are closely linked on mouse chromosome 7, whereas Nkx-1.1 is located on a different chromosome. Nkx-5.1 gene activity begins at Embryonic Day 10.5 in the developing ear, the neural tube, and dorsal root ganglia. It continues to be active throughout prenatal life in discrete regions of the brain with an anterior border in the ventral diencephalon at the optic chiasma and expression domains in mesencephalon, metencephalon, and myelencephalon. At midgestation, Nkx-5.1 is also expressed in mesenchyme of the head and branchial arches, and in some cranial ganglia, as well as in derivatives of neural crest, such as the truncus sympathicus and myenteric ganglia. The time pattern of Nkx-5.1 expression and its confinement to primarily postmitotic cells of the central and peripheral nervous system suggest that Nkx-5.1 may play a role in the specification of neuronal cell types (Boder, 1994).

Nkx homeobox transcription factors are expressed in diverse embryonic cells and presumably control cell-type specification and morphogenetic events. Nkx2-9 is a novel family member of NK2 genes that lacks the conserved TN-domain found in all hitherto known murine Nkx2 genes. The prominent expression of Nkx2-9 in ventral brain and neural tube structures defines a subset of neuronal cells along the entire neuraxis. During embryonic development, Nkx2-9-expressing cells shift from the presumptive floor plate into a more dorsolateral position of the neuroectoderm and later become limited to the ventricular zone. Nkx2-9 expression overlaps with that of Nkx2-2 but is generally broader. Initially, Nkx2-9 is expressed in close proximity to Sonic hedgehog; at later developmental stages, its expression domain clearly segregates from Sonic hedgehog. The dynamic expression pattern of Nkx2-9 in ventral domains of the CNS is consistent with a possible role in the specification of a distinct subset of neurons (Pabst, 1998).

Nkx-5.2 transcripts are first detected in E13.5 embryos where they colocalize with Nkx-5.1 mRNA in the developing central nervous system and the inner ear. However, the onset of Nkx-5.1 transcription begins much earlier in 10 somite stage embryos (E8.5) in the otic placode and the branchial region. Nkx-5.1 expression in the ear persists until birth, whereas in branchial arches it is transient between E8.5 to E11.5. Transcript distribution appears regionalized in the otic vesicle concentrating at the anterior and posterior margin and later at the dorsal side of the otocyst. These domains are distinct from regions expressing Pax-2 and sek, two other early markers for otic development. From E11.5 to birth several Nkx-5.1 expression domains appear in the brain between the ventral diencephalon and the myelencephalon. The same expression domains also exist for Nkx-5.2 beginning at E13.5. The regionally restricted expression pattern of both Nkx-5 genes during mouse development suggests their involvement in cell type specification of neuronal cells (Rinkwitz-Brandt, 1995).

Evolution of Antp-class genes and differential expression of Hydra Hox/paraHox genes in anterior patterning

The conservation of developmental functions exerted by Antp-class homeoproteins in protostomes and deuterostomes has suggested that homologs with related functions are present in diploblastic animals, in particular, in Hydra. Phylogenetic analyses show that Antp-class homeodomains belong either to non-Hox or to Hox/paraHox families. See Phylogenetic relationships among 200 Antp-class genes. Among the 13 non-Hox families, 9 reported here have diploblastic homologs: Msx, Emx, Barx, Evx, Tlx, NK-2 (Drosophila homolog Vnd), and Prh/Hex, Not, and Dlx. Among the Hox/paraHox, poriferan sequences are not found, and the cnidarian sequences form at least five distinct cnox families. Cnox-1 shows some affinity to paralogous group (PG) 1; this group includes Drosophila Labial. Cnox-2 is related to Drosophila Intermediate neuroblast defective. Cnox-3 and 5 show some affinity to PG9-10; this group includes Drosophila AbominalB. Cnox-4 has no counterparts in Drosophila or vertebrates. Intermediate Hox/paraHox genes (PG 3 to 8 and lox) do not have clear cnidarian counterparts. In Hydra, cnox-1, cnox-2, and cnox-3 are not found chromosomally linked within a 150-kb range and display specific expression patterns in the adult head. During regeneration, cnox-1 is expressed as an early gene whatever the polarity, whereas cnox-2 is up-regulated later during head but not foot regeneration. Finally, cnox-3 expression is reestablished in the adult head once the head is fully formed. These results suggest that the Hydra genes related to anterior Hox/paraHox genes are involved at different stages of apical differentiation. However, the positional information defining the oral/aboral axis in Hydra cannot be correlated strictly to that characterizing the anterior-posterior axis in vertebrates or arthropods (Gauchat, 2000)

Control of foot differentiation in Hydra: in vitro evidence that the NK-2 homeobox factor CnNK-2 autoregulates its own expression and uses pedibin as target gene

The foot of the simple metazoan Hydra is a highly dynamic body region of constant tissue movement, cell proliferation, determination and differentiation. Two genes have been shown to participate in the development and differentiation of this body region: homeodomain factor CnNK-2 and signal peptide pedibin. CnNk-2 functions as transcriptional regulator and is responsive to changes in the positional value while the secreted peptide pedibin serves as 'extrinsic' positional signal. Exposure of polyps to pedibin increases the spatial domain of CnNK-2 expression towards the gastric region, indicating that positional signals are integrated at the cis-regulatory region of CnNK-2. To elucidate the molecular basis of the interaction of CnNK-2 and pedibin, the 5' regulatory regions of both genes were characterized. Within the CnNK-2 5' upstream region, electrophoretic mobility shift assays showed that putative NK-2 binding motifs are specifically bound by both nuclear protein from Hydra foot and by recombinant CnNK-2, suggesting that CnNK-2 might autoregulate its own expression. This is the first indication for an autoregulatory circuit in Hydra. In addition, NK-2 binding sites were identified in the cis-regulatory region of the pedibin gene, indicating that this gene is one of the targets of the transcription factor CnNK-2. On the basis of these results, a model is presented for the regulatory interactions required for patterning the basal end of the single axis in Hydra that postulates that CnNK-2 together with pedibin orchestrates foot specific differentiation (Thomsen, 2004).

Molecular architecture of annelid nerve cord supports common origin of nervous system centralization in bilateria

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).

Anteroposterior patterning in hemichordates and the origins of the chordate nervous system

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, the hemichordate Saccoglossus kowalevskii was examined and the expression of orthologs of genes that are involved in patterning the chordate central nervous system was examined. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. It is proposed that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage (Lowe, 2003).

The adult S. kowalevskii has tripartite, tricoelomic organization. At the anterior is the muscular proboscis or prosome, used for burrowing and collecting food particles. It contains the heart, kidney, a section of the dorsal nerve cord, and the protocoel. The middle region, which is the collar or mesosome, contains the mouth, a section of dorsal nerve cord formed by neurulation, the paired mesocoels, and the base of the stomochord, which projects forward into the prosome. The posterior region or metasome contains the gill slits, the remainder of the dorsal nerve cord, the entire ventral nerve cord, paired metacoels, gonads, a long through-gut, and terminal anus. At juvenile stages, a ventral post-anal extension (called a tail or sucker) is present (Lowe, 2003).

Gastrulation entails uniform and simultaneous inpocketing of the vegetal half of the hollow blastula. As the blastopore closes, a gumdrop-shaped gastrula is formed. As the embryo lengthens, two circumferential grooves indent and divide the length into prosome, mesosome, and metasome regions. Mesodermal coeloms outpouch from the gut anteriorly and laterally. The first gill slit pair appears externally by day 5, and the animal bends from the dorsal side. The hatched juvenile elongates and adds further pairs of gill slits successively. The animal is nearly bilaterally symmetric, except that the prosome excretory pore (the proboscis pore) from the kidney is reliably on the left, defining a left-right asymmetry (Lowe, 2003).

The hemichordate adult nervous system is not centralized but is a diffuse intraepidermal, basiepithelial nerve net. Nerve cells are interspersed with epidermal cells and account for 50% or more of the cells in the proboscis and collar ectoderm and a lower percentage in the metasome. Axons form a meshwork at the basal side of the epidermis. The two nerve cords are through-conduction tracts of bundled axons and are not enriched for neurogenesis. This general organizational feature of the nervous system has been largely underemphasized in recent literature that focuses on possible homologies between chordate and hemichordate nerve cords (Lowe, 2003).

Twenty-two full-length coding sequences of orthologs associated with neural patterning in chordates were isolated. These genes are probably present as single copies in S. kowalevskii because orthologs of most of them are present as single copies in lower chordates and echinoderms, and many of the genes were recovered multiple times in the EST analysis without finding any closely related sequences (Lowe, 2003).

Using full-length probes for in situ hybridization, all 22 genes were found to be expressed strongly in the ectoderm as single or multiple bands around the animal, in most cases without dorsal or ventral differences (rx, hox4, nkx2-1, en, barH, lim1/5, and otx are exceptions). Circumferential expression is consistent with diffuse neurogenesis in the ectoderm. The domains resemble the circumferential expression of orthologs in Drosophila embryos. In chordates, by contrast, most of these neural patterning genes are expressed in stripes or patches only within the dorsal neurectoderm and not in the epidermal ectoderm. Also, in chordates, the domains are often broader medially or laterally within the neurectoderm, and there are usually additional expression domains in the mesoderm and endoderm. In most of the 22 cases in S. kowalevskii, the ectodermal domain is the only expression domain (six3, otx, gbx, otp, nkx2-1, dbx, hox11/13, and irx are exceptions) (Lowe, 2003).

Although each of the 22 genes has a distinct expression domain along the anteroposterior dimension of the chordate body, attempts were made to divide them into three broad groups to facilitate the comparison with hemichordates: anterior, midlevel, and posterior genes. Anterior genes are those which in chordates are expressed either throughout or within a subdomain of the forebrain. Midlevel genes are those expressed at least in the chordate midbrain, having anterior boundaries of expression in the forebrain or midbrain, and posterior boundaries in the midbrain or anterior hindbrain. Posterior genes are those expressed entirely within the hindbrain and spinal cord of chordates. Many of the chordate genes have additional domains of expression elsewhere in the nervous system and in other germ layers, but comparisons were restricted to domains involved in specifying the neuraxis in the anteroposterior dimension. Taking these groups of genes one at a time, it was asked where the orthologous genes are expressed in S. kowalevskii. In all comparisons, no morphological homology is implied between the subregions of the chordate and hemichordate nervous systems (Lowe, 2003).

Six genes were examined -- sine oculis-like or optix-like (six3), retinal homeobox (rx), distal-less (dlx), ventral anterior homeobox (vax: Drosophila homolog: Empty spiracles), nkx2-1, and brain factor 1 (bf-1). These six chordate neural patterning genes are expressed within the forebrain, each with its own contour and location (Lowe, 2003).

In S. kowalevskii, the orthologs of these six genes are expressed strongly throughout the ectoderm of the prosome. Within the prosome ectoderm, the domain of each gene differs in its exact placement and contours. vax is expressed just at the anterior tip of the prosome near the apical organ. six3 and rx are expressed throughout most of the prosome. rx expression is exclusively ectodermal. rx expression is absent in the apical region of ectoderm where vax is expressed. Six3 is expressed ectodermally and at low levels mesodermally in the developing prosome, and the domain extends slightly into the mesosome ectoderm. Expression of six3 is strongest in the most anterior ectoderm and attenuates posteriorly. dlx and bf-1 are both expressed strongly in a punctate pattern of numerous individual cells or cell clusters throughout most of the prosome ectoderm and also in a diffuse pattern at a lower level throughout the prosome ectoderm. The bf-1 domain is interrupted by a band of nonexpression in the midprosome. dlx expression is seen through the proboscis and individual cells strongly positive for dlx and ectodermal cells exhibiting low-level expression are seen in both apical and basal positions. dlx is also expressed more posteriorly in a dorsal midline stripe. nkx2-1 is specifically expressed in a ventral sector of the prosome ectoderm. In chordates, nkx2-1 is expressed in the ventral (subpallial) portion of the forebrain. It is also expressed less strongly in a ring in the hemichordate pharyngeal endoderm, a domain of interest in relation to this gene's involvement in the chordate endostyle and thyroid, in the hemichordate Ptychodera flava) (Lowe, 2003).

In conclusion, these six orthologs, whose chordate cognates are expressed entirely within the forebrain, all have prominent expression domains in the prosome ectoderm of S. kowalevskii, the hemichordate's most anterior body part (Lowe, 2003).

The 22 expression domains of orthologs of chordate neural patterning genes of S. kowalevskii correspond strikingly to those in chordates. There are differences such as the extent of overlap of edges of domains of otx, en, and gbx and other midlevel genes that are critical for forming boundaries within the chordate brain, but the relative domain locations are nonetheless very similar. This similar topography of domains is most parsimoniously explained by conservation in both lineages of a domain arrangement (a map) already present in the common ancestor, the ancestor of deuterostomes (Lowe, 2003).

At least 14 of the 22 conserved domains have similar locations in one or more protostome groups. Such similarities are most parsimoniously explained as a conservation of domains from the ancestral bilaterian. In the case of the hox genes, otx, emx, pax6, six3, gbx, and tll, there is strong evidence for such conservation, but less so for the others (barH and rx). At least four of the chordate-hemichordate conserved domains may not be shared by protostomes. Namely, three of these genes (dbx, vax, and hox11/13) are absent from the Drosophila genome and have not been cloned from other protostome groups. Also, one gene, engrailed, has no clear corresponding domain of expression known in protostomes. In Drosophila, en is expressed in the posterior compartments of 14 body segments and at three or more sites in the head that probably derive from ancient preoral segments. This pattern for en appears very different from the single ectodermal band in deuterostomes (Lowe, 2003).

The nerve net of hemichordates could represent the basal condition of the deuterostome ancestor, or it could represent the secondary loss of a central nervous system from an ancestor. Was the complex map of the ancestor associated with a complex diffuse nerve net or a central nervous system in the ancestor? It is suggested that the deuterostome ancestor may have had a diffuse basiepithelial nervous system with a complex map of expression domains, though not necessarily a diffuse net exactly like that of extant hemichordates. Hemichordates would then have retained a diffuse system in their lineage and early in the chordate lineage, centralization would have taken place. In this proposal, the domain map predates centralization and is carried into the nervous system. In this respect, the core questions of nervous system evolution would concern the modes of centralization utilized by the ancestor's various descendents rather than a dorsoventral inversion, per se. Thus, it is proposed that in chordates, especially vertebrates, the major innovation may have been the formation of a large contiguous nonneural (epidermogenic) region (Lowe, 2003).

Protein interactions of NK-2 proteins

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 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 patterning—including other Pax and Nkx proteins, and the Gsh, Msx, Gbx, and Tlx proteins—also 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).

Transcriptional regulation of NK-2 homeobox genes

Sonic hedgehog (Shh) acts as a morphogen to mediate the specification of distinct cell identities in the ventral neural tube through a Gli-mediated (Gli1-3) transcriptional network. Identifying Gli targets in a systematic fashion is central to the understanding of the action of Shh. This issue was examined in differentiating neural progenitors in mouse. An epitope-tagged Gli-activator protein was used to directly isolate cis-regulatory sequences by chromatin immunoprecipitation (ChIP). ChIP products were then used to screen custom genomic tiling arrays of putative Hedgehog (Hh) targets predicted from transcriptional profiling studies, surveying 50-150 kb of non-transcribed sequence for each candidate. In addition to identifying expected Gli-target sites, the data predicted a number of unreported direct targets of Shh action. Transgenic analysis of binding regions in Nkx2.2, Nkx2.1 (Titf1) and Rab34 established these as direct Hh targets. These data also facilitated the generation of an algorithm that improved in silico predictions of Hh target genes. Together, these approaches provide significant new insights into both tissue-specific and general transcriptional targets in a crucial Shh-mediated patterning process (Volkes, 2007).

Dlx1&2-dependent expression of Zfhx1b (Sip1, Zeb2) regulates the fate switch between cortical and striatal interneurons

Mammalian pallial (cortical and hippocampal) and striatal interneurons are both generated in the embryonic subpallium, including the medial ganglionic eminence (MGE). This study demonstrates that the Zfhx1b (Sip1, Zeb2) zinc finger homeobox gene is required in the MGE, directly downstream of Dlx1&2, to generate cortical interneurons that express Cxcr7, MafB, and cMaf. In its absence, Nkx2-1 expression is not repressed, and cells that ordinarily would become cortical interneurons appear to transform toward a subtype of GABAergic striatal interneurons. These results show that Zfhx1b is required to generate cortical interneurons, and suggest a mechanism for the epilepsy observed in humans with Zfhx1b mutations (Mowat-Wilson syndrome) (McKinsey, 2013).

Tcf/Lef repressors differentially regulate Shh-Gli target gene activation thresholds to generate progenitor patterning in the developing CNS

During neural tube development, Shh signaling through Gli transcription factors is necessary to establish five distinct ventral progenitor domains that give rise to unique classes of neurons and glia that arise in specific positions along the dorsoventral axis. These cells are generated from progenitors that display distinct transcription factor gene expression profiles in specific domains in the ventricular zone. However, the molecular genetic mechanisms that control the differential spatiotemporal transcriptional responses of progenitor target genes to graded Shh-Gli signaling remain unclear. The current study demonstrates a role for Tcf/Lef repressor activity in this process. Tcf3 and Tcf7L2 (Tcf4) were shown to be required for proper ventral patterning and function by independently regulating two Shh-Gli target genes, Nkx2.2 and Olig2, which are initially induced in a common pool of progenitors that ultimately segregate into unique territories giving rise to distinct progeny. Genetic and functional studies in vivo show that Tcf transcriptional repressors selectively elevate the strength and duration of Gli activity necessary to induce Nkx2.2, but have no effect on Olig2, and thereby contribute to the establishment of their distinct expression domains in cooperation with graded Shh signaling. Together, these data reveal a Shh-Gli-independent transcriptional input that is required to shape the precise spatial and temporal response to extracellular morphogen signaling information during lineage segregation in the CNS (Wang, 2011).

NK-2 homeobox genes and the patterning of the neural tube

Distinct classes of motor neurons (MNs) and ventral interneurons are generated by the graded signaling activity of Sonic hedgehog (See Drosophila Hedgehog). Three classes of ventral neurons are induced by distinct concentration thresholds of Shh. One class of interneurons, V1, is defined by coexpression of En1, Lim1/2 and Pax2 and is generated in the dorsal-most region of the ventral spinal cord. A second class of interneurons, V2, is defined by coexpression of Chx10, Lim3, and Gsh4 and is generated in the intermediate region of the ventral spinal cord, in a aregion that is ventral to V1 neurons. The third class, MNs, is defined by expression of Isl1 and is generated ventral to V2 interneurons. Shh controls neuronal fate by establishing different progenitor cell populations in the ventral neural tube that are defined by the expression of Pax6 and Nkx2.2 (a homolog of Drosophila Vnd. Pax6 (a homolog of Drosophila Eyeless) establishes distinct ventral progenitor cell populations and controls the identity of motor neurons and ventral interneurons, mediating graded Shh signaling in the ventral spinal cord and hindbrain. From stages 10 to 12, Pax6 is expressed by cells at all dorsoventral positions of the neural tube, with the exception of the ventral midline. Nkx2.2 is also detected at low levels but is restricted to ventral midline cells. From stages 12 to 16, the level of Pax 6 in cells adjacent to the floor plate decreases below the limit of detection; Nkx2.2 expression is initiated within these cells. Both V1 and V2 neurons derive from Pax 6 precursor cells, while ventrally positioned MNs derive from Nkx2.2 progenitor cells. In Pax6 mutant mice there is a marked increase in the number of Nkx2.2 cells and a dorsal expansion of the Nkx2.2 domain. Loss of Pax6 results in a dorsal-to-ventral transformation of one type of MNs to another (Ericson, 1997).

During vertebrate development, the specification of distinct cell types is thought to be controlled by inductive signals acting at different concentration thresholds. The degree of receptor activation in response to these signals is a known determinant of cell fate, but the later steps at which graded signals are converted into all-or-none distinctions in cell identity remain poorly resolved. In the ventral neural tube, motor neuron and interneuron generation depends on the graded activity of the signaling protein Sonic hedgehog (Shh). These neuronal subtypes derive from distinct progenitor cell populations that express the homeodomain proteins Nkx2.2 or Pax6 in response to graded Shh signaling. In mice lacking Pax6, progenitor cells generate neurons characteristic of exposure to greater Shh activity. However, Nkx2.2 expression expands dosally in Pax6 mutants, raising the possibility that Pax6 controls neuronal pattern indirectly. Evidence that Nkx2.2 has a primary role in ventral neuronal patterning. In Nkx2.2 mutants, Pax6 expression is unchanged but cells undergo a ventral-to-dorsal transformation in fate and generate motor neurons rather than interneurons. Thus, Nkx2.2 has an essential role in interpreting graded Shh signals and selecting neuronal identity (Briscoe, 1999).

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).

The expression of certain class I (Pax7, Dbx1, Dbx2, and Pax6) and class II (Nkx2.2) proteins is controlled by Shh signaling in vitro. The expression of class I proteins is repressed by Shh signaling, and the more ventral the boundary of class I protein expression in vivo, the higher is the concentration of Shh required for repression of protein expression in vitro. Conversely, Shh signaling is required to induce expression of the class II protein Nkx2.2 in vitro. Repression of Irx3 requires ~3 nM Shh-N, a concentration greater than that required for repression of Pax7, Dbx1, and Dbx2 expression, but less than that required for complete repression of Pax6. Conversely, induction of Nkx6.1 requires ~0.25 nM Shh-N -- a concentration lower than that required for induction of Nkx2.2 (3-4 nM). Thus, the link between the domains of expression of class I and class II proteins in vivo and the Shh concentration that regulates their expression in vitro extends to Irx3 and Nkx6.1. These findings support the idea that the differential patterns of expression of all class I and class II proteins depend initially on graded Shh signaling (Briscoe, 2000).

The boundaries of progenitor domains are sharply delineated in vivo, raising questions about the steps that operate downstream of Shh signaling to establish the nongraded domains of expression of class I and class II proteins. It was asked whether the domain of expression of class I proteins might be constrained by the action of the class II protein that abuts the same domain boundary, or vice versa. To test this, individual homeodomain proteins were misexpressed in the chick neural tube in mosaic fashion, and the resulting pattern of class I and class II protein expression was examined. Examined was the interaction between the class I protein Pax6 and the class II protein Nkx2.2 -- proteins that exhibit complementary domains of expression at the pMN/p3 boundary. To assess the influence of Pax6 on Nkx2.2, Pax6 was misexpressed ventral to its normal limit and the resulting pattern of Nkx2.2 expression was examined. After electroporation of Pax6, small clusters of ectopic Pax6+ cells were detected within the p3 domain. These cells lack Nkx2.2 expression, whereas expression of Nkx2.2 is maintained by neighboring p3 domain cells that lack ectopic Pax6 expression, arguing for a cell-autonomous action of Pax6. The expression of other class I and class II proteins is not affected by the deregulated expression of Pax6. Thus, Pax6 acts selectively to repress Nkx2.2 expression in p3 domain cells. These results complement studies showing a requirement for Pax6 activity in defining the dorsal limit of the p3 domain in vivo. The loss of Nkx6.1 function results in a ventral expansion in the extent of the p1 domain, without any change in Shh signaling. It is noteworthy that the boundaries of each of the five progenitor domains are sharply defined, yet class II proteins have been identified only at the pMN/p3 and p1/p2 boundaries. Thus, additional class II proteins may exist, with patterns of expression that complement the three orphan class I proteins (Briscoe, 2000).

This study has relied on ectopic expression methods to address the roles of Nkx6.1, Nkx2.2, and Irx3 in specifying the fate of V2 neurons, MNs, and V3 neurons. The results show that Nkx2.2 activity is sufficient to induce V3 neurons, that Nkx6.1 activity in the absence of Irx3 induces MNs, whereas Nkx6.1 activity in the presence of Irx3 induces V2 neurons. The inferences derived from these gain-of-function studies are supported by the switches in neuronal fate that occur in mice in which individual class I and class II proteins have been inactivated by gene targeting. In mice lacking Pax6 activity, the dorsal expansion in the domain of Nkx2.2 expression is accompanied by an expansion in the domain of V3 neuron generation, and by the loss of MNs. Conversely, the loss of Nkx2.2 results in the loss of V3 neurons and in the ectopic generation of MNs within the p3 domain. In addition, the loss of Nkx6.1 activity depletes the ventral neural tube of many MNs and V2 neurons (Briscoe, 2000 and references therein).

How do class I and class II proteins control neuronal subtype identity? The final cell division of certain ventral progenitors is accompanied by the onset of expression of a distinct set of homeodomain proteins, notably MNR2 and Lim3. Ectopic expression of MNR2 is able to induce MN differentiation independent of dorsoventral position, and ectopic expression of Lim3 induces V2 neurons. The studies indicate that class I and class II proteins function upstream of MNR2 and Lim3. Thus, within the pMN and p2 domains, the actions of progenitor homeodomain proteins in specifying neuronal subtype identity are likely to be mediated through MNR2 and Lim3. Subtype determinant factors with equivalent functions may therefore be expressed by cells in the other ventral progenitor domains (Briscoe, 2000).

A set of seven homeodomain proteins defines five neural progenitor domains with a fundamental role in the organization of ventral neural pattern. The analysis of these homeodomain proteins suggests that ventral patterning proceeds in three stages: (1) the regulation of class I and class II proteins by graded Shh signals; (2) the refinement and maintenance of progenitor domain identity by cross-repressive interactions between homeodomain proteins, and (3) the translation of a homeodomain protein code into neuronal subtype identity. The central features of this model may apply to other vertebrate tissues in which cell pattern is regulated by local sources of extrinsic signals. Consistent with this idea, cross-regulatory interactions between transcription factors have been suggested to refine cell pattern in the embryonic mesoderm and in the pituitary gland. The principles of the model of ventral patterning outlined here resemble those involved in subdividing the Drosophila embryo. Graded Shh signaling subdivides the ventral neural tube into five domains, just as graded levels of the dorsal protein establish five distinct regions of the early Drosophila embryo, suggesting an upper limit to the number of distinct cell fates that can be generated in response to a single gradient signaling system. In addition, the graded anterioposterior distribution of maternally supplied factors in the Drosophila embryo is known to initiate the expression of a set of proteins encoded by the gap genes. Subsequent cross-regulatory interactions establish and maintain sharp boundaries in the expression of gap proteins, and their activities within individual domains control later aspects of cell pattern. Thus, in the neural tube and the Drosophila embryo, the cross-repression of genes whose initial expression is controlled by graded upstream signals provides an effective mechanism for establishing and maintaining progenitor domains and for imposing cell type identity (Briscoe, 2000 and references therein).

Distinct classes of serotonergic (5-HT) neurons develop along the ventral midline of the vertebrate hindbrain. A Sonic hedgehog (Shh)-regulated cascade of transcription factors has been identified that acts to generate a specific subset of 5-HT neurons. This transcriptional cascade is sufficient for the induction of rostral 5-HT neurons within rhombomere 1 (r1) that project to the forebrain, but not for the induction of caudal 5-HT neurons, which largely terminate in the spinal cord. Within the rostral hindbrain, the Shh-activated homeodomain proteins Nkx2.2 and Nkx6.1 cooperate to induce the closely related zinc-finger transcription factors Gata2 and Gata3. Gata2 in turn is necessary and sufficient to activate the transcription factors Lmx1b and Pet1, and to induce 5-HT neurons within r1. In contrast to Gata2, Gata3 is not required for the specification of rostral 5-HT neurons and appears unable to substitute for the loss of Gata2. These findings reveal that the identity of closely related 5-HT subclasses occurs through distinct responses of adjacent rostrocaudal progenitor domains to broad ventral inducers (Craven, 2004).

The underlying transcriptional mechanisms that establish the proper spatial and temporal pattern of gene expression required for specifying neuronal fate are poorly defined. This study characterizes how the Hb9 gene is expressed in developing motoneurons in order to understand how transcription is directed to specific cells within the developing CNS. Non-specific general-activator proteins such as E2F and Sp1 are capable of driving widespread low level transcription of Hb9 in many cell types throughout the neural tube; however, their activity is modulated by specific repressor and activator complexes. The general-activators of Hb9 are suppressed from triggering inappropriate transcription by repressor proteins Irx3 and Nkx2.2. High level motoneuron expression is achieved by assembling an enhancesome on a compact evolutionarily-conserved segment of Hb9 located from -7096 to -6896. The ensemble of LIM-HD and bHLH proteins that interact with this enhancer change as motoneuron development progresses, facilitating both the activation and maintenance of Hb9 expression in developing and mature motoneurons. These findings provide direct support for the derepression model of gene regulation and cell fate specification in the neural tube, as well as establishing a role for enhancers in targeting gene expression to a single neuronal subtype in the spinal cord (Lee, 2004).

Nkx2.2, Nkx6.1, Pax6 and Irx3 control progenitor cell fate by repressing transcription. Since the deletion analysis of Hb9 indicated that repressor proteins might interact with the 2.5 kb distal segment from -8129 to -5575, tests were performed to see whether constructs with this DNA segment were repressed by Nkx2.2, Nkx6.1, Pax6 and/or Irx3 using 293 cell transfections. The Hb9 promoter was repressed ~50-500 fold by Nkx2.2 and Irx3, whereas Pax6 and Nkx6.1 were significantly less active. These findings suggest that progenitor cell factors such as Nkx2.2 and Irx3 expressed by non-motoneuron cells suppress the expression of Hb9 (Lee, 2004).

NK-2 homeobox genes and patterning of the brain and spinal cord

The Nodal and Hedgehog signaling pathways influence dorsoventral patterning at all axial levels of the CNS, but it remains largely unclear how these pathways interact to mediate patterning. In zebrafish, Nodal signaling is required for induction of the homeobox genes nk2.1a in the ventral diencephalon and nk2.1b in the ventral telencephalon. Hedgehog signaling is also required for telencephalic expression but may not be essential to establish diencephalic nk2.1a expression. Furthermore, Shh does not restore ventral diencephalic development in embryos lacking Nodal activity. In contrast, Shh does restore telencephalic nk2.1b expression in the absence of Nodal activity; this suggests that Shh acts downstream of Nodal activity to pattern the ventral telencephalon. Thus, the Nodal pathway regulates ventral forebrain patterning through both Hedgehog signaling-dependent and -independent mechanisms (Rohr, 2001).

Comparative analysis of the mouse and fish nk2.1 genes indicates that the expression domains of the two zebrafish nk2.1 genes cumulatively equate to those of the single mouse gene. Mouse nkx2.1 is expressed in the medial ganglionic eminence of the ventral telencephalon, the hypothalamus, lung, and thyroid gland. Zebrafish nk2.1a is expressed in the hypothalamus and the developing thyroid gland but not the ventral telencephalon. In contrast, nk2.1b is expressed strongly in the telencephalon, weakly in the hypothalamus, and not at all in the thyroid gland. Thus, both nk2.1 genes in fish have retained some sites of ancestral gene expression and lost others. The duplication-degeneration-complementation model of gene evolution suggests that both copies of duplicated genes may be retained if each gene accumulates mutations in regulatory regions that disrupt specific temporal or spatial patterns of expression. In this way, the functions of the ancestral gene are partitioned between the two paralogs, and both are retained within the genome. The two nk2.1 genes described in this study provide one of the most striking known examples of the segregation of expression domains between duplicated and retained orthologs of a single mammalian gene. The discrete expression domains of each gene provide an explanation as to why both copies have been retained over the several hundred million years since the genome duplication event is assumed to have occurred and provide compelling evidence in support of the duplication-degeneration-complementation model (Rohr, 2001).

The genetic program that underlies the generation of visceral motoneurons in the developing hindbrain remains poorly defined. The roles of Nkx6 (Drosophila homolog: HGTX) 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).

The ventral neural tube of vertebrates consists of distinct neural progenitor domains positioned along the dorsoventral (DV) axis that develop different types of motorneurons and interneurons. Several signalling molecules, most notably Sonic Hedgehog (Shh), retinoic acid (RA) and fibroblast growth factor (FGF) have been implicated in the generation of these domains. Shh is secreted from the floor plate, the ventral most neural tube structure that consists of the medial (MFP) and the lateral floor plate (LFP). While the MFP is well characterized, organization and function of the LFP remains unclear. The homeobox gene nkx2.2b is strongly expressed in the trunk LFP of zebrafish and thus represents a unique marker for the characterization of LFP formation and the identification of LFP deficient mutants. nkx2.2b and its paralog nkx2.2a (formerly known as nk2.2 and nkx2.2) arose by gene duplication in zebrafish. Both duplicates show significant differences in their expression patterns. For example, while prominent nkx2.2a expression has been described in the ventral brain, hardly any expression can be found in the trunk LFP, which is in contrast to nkx2.2b. Overexpression, mutant and inhibitor analyses show that nkx2.2b expression in the LFP is up-regulated by Shh, but repressed by retinoids and ectopic islet-1 (isl1) expression. In contrast to previously described zebrafish trunk LFP markers, like e.g. tal2 or foxa2, nkx2.2b is exclusively expressed in the LFP. Thus, nkx2.2b represents a unique tool to analyse the mechanisms of ventral neural tube patterning in zebrafish (Schafer, 2005).

NK-2 homeobox genes and the pituitary gland

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog, FGF, and BMP families. To better understand when and where Hh signaling influences pituitary development, the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants), has been examined. While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, the data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. The Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. Zebrafish Hh pathway mutants were used to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. These results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland (Sbrogna, 2003).

NK-2 genes and eye development

The diversity of cell types found within the vertebrate CNS arises in part from action of complex transcriptional programs. In the retina, the programs driving diversification of various cell types have not been completely elucidated. To investigate gene regulatory networks that underlie formation and function of one retinal circuit component, the bipolar cell, transcriptional regulation of three bipolar cell-enriched genes was analyzed. Using in vivo retinal DNA transfection and reporter gene constructs, a 200 bp metabotropic glutamate receptor 6 (Grm6) enhancer sequence, a 445 bp calcium-binding protein 5 (Cabp5) promoter sequence, and a 164 bp Chx10 enhancer sequence, were defined, each driving reporter expression specifically in distinct but overlapping bipolar cell subtypes. Bioinformatic analysis of sequences revealed the presence of potential paired-type and POU homeodomain-containing transcription factor binding sites, which were shown to be critical for reporter expression through deletion studies. The paired-type homeodomain transcription factors (TFs) Crx and Otx2 and the POU homeodomain factor Brn2 are expressed in bipolar cells and interacted with the predicted binding sequences as assessed by electrophoretic mobility shift assay. Grm6, Cabp5, and Chx10 reporter activity was reduced in Otx2 loss-of-function retinas. Endogenous gene expression of bipolar cell molecular markers was also dependent on paired-type homeodomain-containing TFs, as assessed by RNA in situ hybridization and reverse transcription-PCR in mutant retinas. Cabp5 and Chx10 reporter expression was reduced in dominant-negative Brn2-transfected retinas. The paired-type and POU homeodomain-containing TFs Otx2 and Brn2 together appear to play a common role in regulating gene expression in retinal bipolar cells (Kim, 2008).

Mutation of NK-2 homeobox genes

Vnd Evolutionary homologs part 2/2

ventral nervous system defective: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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