ventral nervous system defective

EVOLUTIONARY HOMOLOGS (part 2/2)

Mutation of NK-2 homeobox genes

The mouse inner ear develops from the otic vesicle, a one-cell-thick epithelium, which eventually transforms into highly complex structures including the sensory organs for balance (vestibulum) and hearing (cochlea). Several mouse inner ear mutations with hearing and balance defects have been described, but most the underlying genes have not been identified; for example, the genes controlling the development of the vestibular organs. Inactivation of the homeobox gene, Nkx5-1, was accomplished by homologous recombination in mice. This gene is expressed in vestibular structures throughout inner ear development. Mice carrying the Nkx5-1 null mutation exhibit behavioral abnormalities that resemble the typical hyperactivity and circling movements of the shaker/waltzer type mutants. The balance defect correlates with severe malformations of the vestibular organ in Nkx5-1(-/-) mutants, which fail to develop the semicircular canals. Nkx5-1 is the first ear-specific molecule identified to play a crucial role in the formation of the mammalian vestibular system (Hadrys, 1998).

The telencephalon is organized into distinct longitudinal domains: the cerebral cortex and the basal ganglia. The basal ganglia primarily consists of a dorsal region (striatum) and a ventral region (pallidum). Within the telencephalon, the anlage of the pallidum expresses the Nkx2.1 homeobox gene. Nkx2.1 expression is first detectable in the basal telencephalon of the mouse at approximately the 11 somite stage. As the basal telencephalon develops, Nkx2.1 is maintained in regions that form morphologically distinct structures such as the medial ganglionic eminence (MGE), as well as parts of the septum, anterior entopeduncular area and preoptic area (POA). Focus was placed on the role of Nkx2.1 in the development of the MGE, a proliferative zone that gives rise to pallidal components of the basal ganglia. At E10.5 in the mouse, the MGE is a neuroepithelial protrusion from the wall of the anterobasal telencephalon into the lateral ventricle (LV). Nkx2.1 RNA is expressed uniformly throughout the MGE neuroepithelium. Between E11.5 and E12.5, a second morphologically distinct basal ganglia anlage, the lateral ganglionic eminence (LGE), emerges between the MGE and the cortex. At these stages the LGE lacks Nkx2.1 expression. By E12.5, the MGE is composed of three molecularly distinct cell layers. The ventricular zone (VZ), which is adjacent to the telencephalic ventricle, is composed of undifferentiated, rapidly dividing cells. VZ cells contribute to the subventricular zone (SVZ), a secondary proliferating population of cells. Postmitotic cells migrate from the proliferative zones to generate the mantle zone. Nkx2.1 is expressed in all three layers of the MGE. At E14.5 and later stages, Nkx2.1 expression is prominent in the developing globus pallidus (GP). As development continues, Nkx2.1 expression can be detected in several other ventral telencephalic structures including the bed nucleus of the stria terminalis (BNST), parts of the septum, the ventral pallidum and parts of the amygdala. A mouse deficient in Nkx2.1 function does not form pallidal structures; it lacks basal forebrain TrkA-positive neurons (probable cholinergic neurons) and shows reduced numbers of cortical cells that express GABA, DLX2 and calbindin. These cells all normally migrate from the pallidum through the striatum and into the cortex. Evidence is presented that these phenotypes result from a ventral-to-dorsal transformation of the pallidal primordium into a striatal-like anlage (Sussel, 1999).

Sonic hedgehog (SHH) secretion from the axial mesendoderm is required for patterning of the anteromedial neural plate, including the hypothalamus and basal telencephalon. In addition, SHH can induce markers of the basal telencephalon, such as Nkx2.1. Shh begins to be expressed in the VZ of the ventral-most regions of the basal telencephalon (preoptic and anterior entopeduncular areas; POA, AEP) between the 10-12 somite stage, at approximately the same time as Nkx2.1 begins to be expressed in the basal telencephalon. Subsequently, Shh expression spreads into the SVZ and mantle of the MGE. Because Shh is expressed early in MGE development and has a potential role in telencephalic patterning, its expression was assessed in the developing MGE of the Nkx2.1 mutant. Surprisingly, at E10.5 and E11.5, Shh expression is undetectable in the mutant basal telencephalon and hypothalamus, aside from trace levels of Shh in the rostral midline at E10.5. Shh expression in the midbrain and more posterior regions of the central nervous system appeared normal at all ages examined. To determine whether Nkx2.1 expression is required for induction or maintenance of Shh expression in the forebrain, E8.75-E9.5 Nkx2.1 mutant embryos were examined by whole-mount in situ hybridization. Even at these stages, which ordinarily show the expression of Shh in the forebrain, Shh transcripts in the hypothalamus and basal telencephalon are not detectable, while Shh expression in the anterior mesendoderm is normal (Sussel, 1999).

Nkx2.1 is related to the Drosophila gene ventral nervous system defective (vnd). vnd encodes the NK2 homeodomain protein that is expressed in the ventral part of central nervous system. The CNS in fly embryos lacking vnd has a ventral-to-dorsal transformation, analogous to the phenotype in the Nkx2.1 mutants. There are several Nkx genes expressed in the ventral CNS of vertebrates, including Nkx2.2 and Nkx6.1 (Drosophila homolog: Nkx6). Mutations of Nkx2.2 and Nkx6.1 also have ventral-to-dorsal transformations. In both of these cases, Shh expression is unaffected, suggesting that Nkx2.2 and Nkx6.1, like vnd, have primary roles in ventral specification. It is uncertain that Nkx2.1 alone is necessary for fate specification of the MGE, because Shh expression, which is essential for ventral specification, is also reduced in the Nkx2.1 knockout mice. However, the described functions of vnd, Nkx2.2 and Nkx6.1 support the hypothesis that Nkx2.1 has a primary role in regional specification, like its homologs. Furthermore, in all regions of the CNS, the Nkx genes are expressed before Shh, supporting the model that Nkx2.1 is upstream of Shh in the basal telencephalon. Finally, in Gli2 mutant mice, Shh is not expressed in the floor plate, yet Nkx2.2 is expressed and motor neurons form. Thus, while Shh expression in the axial mesendoderm is essential for ventral specification of the CNS, Shh expression in neural tissue may not have a major role in regionalization (Sussel, 1999 and references).

In the absence of Nkx2.1, the transformed MGE does not produce normal ventral telencephalic cell types. Evidence is accumulating that dorsoventral patterning of the vertebrate CNS is regulated by common mechanisms along the entire anteroposterior axis. For instance, the Nkx genes, which are induced by SHH in all ventral CNS regions, are required for ventral specification in the forebrain hindbrain and spinal cord. While there are a variety of ventral CNS cell types, all axial levels produce cholinergic neurons (e.g. basal forebrain cholingeric neurons and motor neurons in the midbrain, hindbrain and spinal cord). Nkx2.1 mutants lack TrkA-expressing cells in their telencephalons and therefore presumably lack cholinergic neurons. In addition to the loss of TrkA/cholinergic cells, Nkx2.1 mutants lack cells that migrate from the MGE through the LGE and into the entire cerebral cortex. Thus, the Nkx2.1 mutants, like the Dlx1/Dlx2 double mutants, have a reduction of GABAergic neurons in the cerebral cortex. However, whereas the Dlx1/Dlx2 double mutants have abnormal differentiation/migration of late born cells from both the LGE and the MGE, the Nkx2.1 mutants lack an MGE, but have a LGE. This has allowed an initial dissection of the relative contributions of the MGE and the LGE to telencephalic tangentially migrating cells. For instance, whereas the Dlx1/Dlx2 mutants lack GABAergic interneurons in the olfactory bulb, the olfactory bulb in the Nkx2.1 mutants appears normal. This shows that the MGE is not required to make interneuron precursors that follow the rostral migratory stream from the basal ganglia to the olfactory bulb. Likewise there are differences between the Dlx1/Dlx2 and the Nkx2.1 mutants in the distribution of interneuron loss in the paleocortex and neocortex. These results suggest that there are several distinct basal telencephalon sources that produce cells that migrate to the cortex. It is hypothesized that the Nkx2.1-positive ventral telencephalon (including the MGE, POA and AEP) produces cells expressing acetylcholine, calbindin and GABA, all of which migrate dorsally into the LGE. At least some of the calbindin-positive and GABAergic cells continue to migrate and populate the paleocortex, neocortex and hippocampus. It is suggested that the LGE produces GABAergic cells that migrate rostrally into the olfactory bulb and perhaps the cortex, and there may be a separate dorsal migration of GABAergic cells from the LGE to the cortex (Sussel, 1999 and references).

NKX2.1, a vertebrate homolog of Drosophila NK2, more properly termed Ventral nervous system defective, is a homeodomain transcriptional factor expressed in thyroid, lung, and parts of the brain. Septation of the anterior foregut along the dorsoventral axis into distinct tracheal and esophageal structures is blocked in mouse embryos carrying a homozygous targeted disruption of the Nkx2.1 locus. This is consistent with the loss of Nkx2.1 expression, which defines the dorsoventral boundary within the anterior foregut in wild-type E9 embryos. Failure in septation between the trachea and the esophagus in Nkx2.1(-/-) mice leads to the formation of a common lumen that connects the pharynx to the stomach, serving both as trachea and as esophagus, similar in phenotype to a human pathologic condition termed tracheoesophageal fistula. The main-stem bronchi bifurcate from this common structure and connect to profoundly hypoplastic lungs. The mutant lungs fail to undergo normal branching embryogenesis: the lung defect consists of highly dilated sacs that are not capable of sustaining normal gas exchange functions, and leads to immediate postnatal death. In situ hybridization suggests reduced Bmp-4 expression in the mutant lung epithelium, providing a possible mechanistic clue for impaired branching. Functional deletion of Nkx2.1 blocks pulmonary-specific epithelial cell differentiation marked by the absence of pulmonary surfactant protein gene expression. Altered expression of temporally regulated genes (such as Vegf) demonstrates that the lung in Nkx2.1(-/-) mutant embryos is arrested at an early pseudoglandular (E11-E15) stage. These results demonstrate a critical role for Nkx2.1 in morphogenesis of the anterior foregut and the lung as well as in differentiation of pulmonary epithelial cells (Minoo, 1999).

NKX2.3 of mouse is related to both Drosophila homeobox protein Vnd and to the Drosophila protein Scarecrow. Targeted disruption of the transcription factor NKX2.3 gene in mice results in anatomical defects of intestine and secondary lymphoid organs. Spleen and Peyer's patches of NKX2.3-deficient mice are considerably reduced in size and lack the ordered tissue architecture. T and B cells are misplaced within the spleen and mesenteric lymph nodes and fail to segregate into the appropriate T and B cell areas. Furthermore, splenic marginal zones, characterized by specific B cells and various types of macrophage-derived cells around the marginal sinus, are absent in mutants. Homozygous NKX2.3 mutants lack the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) that is normally expressed in mucosa-associated lymphoid tissue (MALT) and spleen. NKX2.3 can activate MAdCAM-1 transcription directly, suggesting that MAdCAM-1 is at least partly responsible for the migration and homing defects of lymphocytes and macrophages in mutants. Therefore, expression of MAdCAM-1 seems to be required for building functional structures in spleen and MALT, a prerequisite for unimpaired migration and segregation of B and T cells to and within these organs (Pabst, 2000).

The homeodomain-containing transcription factor Nkx2.9 is expressed in the ventralmost neural progenitor domain of the neural tube together with the related protein Nkx2.2 during early mouse embryogenesis. Cells within this region give rise to V3 interneurons and visceral motoneurons in spinal cord and hindbrain, respectively. To investigate the role of the Nkx2.9 gene, Nkx2.9 deficiency was generated by targeted gene disruption. Homozygous mutant animals lacking Nkx2.9 are viable and fertile with no apparent morphological or behavioral phenotype. The distribution of neuronal progenitor cells and differentiated neurons in spinal cord appears unaffected in Nkx2.9-deficient animals. This finding is in contrast to Nkx2.2-null mutants, which have been shown to exhibit ventral to dorsal transformation of neuronal cell fates in spinal cord. These results suggest that specification of V3 interneurons in the posterior CNS does not require Nkx2.9, most probably because of functional redundancy with the co-expressed Nkx2.2 protein. In hindbrain, however, absence of Nkx2.9 results in a significantly altered morphology of the spinal accessory nerve (XIth), which appears considerably shorter and thinner than in wild-type animals. Consistent with this phenotype, immature branchial motoneurons of the spinal accessory nerve, which normally migrate from a ventromedial to a dorsolateral position within the neural tube, are markedly reduced in Nkx2.9-deficient embryos at E10.5, while ventromedial motor column cells were increased in numbers. In addition, the vagal and glossopharyngeal nerves appear abnormal in approximately 50% of mutant embryos, which may be related to the observed reduction of Phox2b expression in the nucleus ambiguus of adult mutant mice. From these observations, it is concluded that Nkx2.9 has a specific function in the hindbrain as a determinant of the branchial motoneuron precursor cells for the spinal accessory nerve and possibly other nerves of the branchial-motor column. Like other Nkx genes expressed in the CNS, Nkx2.9 seems to be involved in converting positional information into cell fate decisions (Pabst, 2003).

Thus, expression of Nkx2.9 in brain parallels that of Nkx2.2 and continues until at least E11 of embryogenesis, in contrast to its early repression in spinal cord. In line with the prolonged presence of Nkx2.9 in brain domains and supporting the idea of redundant activities, Nkx2.2-deficient mice exhibit no obvious phenotypic switch in motoneuron identity within the hindbrain. In the reverse situation presented here by the Nkx2.9 knockout mouse, e.g. lack of Nkx2.9 but continuing presence of Nkx2.2, the phenotypic rescue is at least incomplete. In hindbrain neuronal progenitors of the p3 domain that co-express Nkx2.2 and Nkx2.9 give rise to branchiovisceral motoneurons. These cells can be identified by the expression of the transcription factor Phox2b. In E10.5 mutant embryos, Phox2b expression appears essentially normal in hindbrain at different axial levels, indicating that the formation of branchiovisceral motoneurons is not generally dependent on Nkx2.9 function but rather affects a subset or only some of these cells. Significantly, the population of presumptive branchial motoneurons of the spinal accessory nucleus, which are characterized by expressing Isl1 alone and their dorsolateral position in neural tube, is markedly reduced in the mutant mouse. Moreover, the population of somatic motoneurons of the median motor column, which co-express Isl1, Isl2 and Lim3, is increased. This result somewhat resembles the cell fate switch observed in the spinal cord of Nkx2.2 mutants with respect to the increase of somatic motoneurons at the expense of another neuronal subpopulation originating in the Nkx2.2/Nkx2.9 domain. Whether this reflects aberrant specification of neuronal identity in response to graded Shh signaling, as is the case in Nkx2.2 mutants, cannot be decided easily here, because the precise local relationship of the premigratory spinal accessory nucleus progenitors and the median motor column precursors is not clear. Consistent with the reduction of branchial motoneurons in the dorsolateral position of the neural tube at the level of C4-C3 a severely abnormal spinal accessory nerve was found in all mutant mice. The partial phenotype of the glossopharyngeal and the vagal nerves in ~50% of mutant embryos may be related to the finding that, in the absence of Nkx2.9, Phox2b expression in cells of the nucleus ambiguus is drastically reduced, although the number of cells appears largely unaltered. Since Nkx2.9 is only expressed in the progenitor domain and Phox2b-positive progenitors are present in normal numbers in the neuroepithelium of mutant embryos (E10.5), but not in the mature nucleus ambiguus, Nkx2.9 seems to have a function in maintaining the phenotypic trait of branchial motoneurons rather than specifying them. Another unknown transcription factor is possible involved acting downstream of Nkx2.9. Whether the alterations in the nucleus ambiguus also contribute to the mutant phenotype of the spinal accessory nerve appears disputable, since the existence of projections from the nucleus ambiguus has been recently questioned, at least in humans. The three nerves affected in the mutant belong to the branchial motor column, suggesting that Nkx2.9 in the hindbrain has a unique role in formation of branchial motoneurons, a function that can not be fully substituted for by Nkx2.2. Clearly, visceral motoneurons are not affected by the Nkx2.9 mutation. It is also interesting to note that the more rostrally located branchial motor nerves, such as the facialis and the trigeminus nerve, appear quite normal in mutants, suggesting a differential requirement for Nkx2.9 along the rostrocaudal axis. Whether the fractional loss of branchial motoneurons in hindbrain of Nkx2.9 mutants is due to partial rescue by redundant Nkx2.2 function or, alternatively, reflects the total loss of a neuronal subpopulation whose fate is entirely dependent on Nkx2.9, cannot be decided unequivocally by the available data. The latter possibility, however, seems less likely given the reduced size but not the complete absence of the affected nerves. Taken together, these data provide evidence that Nkx2.9 is a crucial transcription factor for the determination and/or differentiation of at least a subset of branchial motoneurons during hindbrain development. Its early role in establishing the p3-domain in spinal cord remains to be determined in Nkx2.2/Nkx2.9 double mutants (Pabst, 2003).

Regional patterning of the mammalian telencephalon requires the function of three homeodomain-containing transcription factors, Pax6, Gsh2 and Nkx2.1. These factors are required for the development of the dorsal, lateral and medial domains of the telencephalon, respectively. Pax6 and Gsh2 have been shown to cross-repress one another in the formation of the border between dorsal and lateral region of the telencephalon. This study examines whether similar interactions are responsible for the establishment of other boundaries of telencephalic gene expression. Surprisingly, despite the fact that, at specific times in development, both Pax6 and Gsh2 maintain a complementary pattern of expression with Nkx2.1, in neither case are these boundaries maintained through a similar cross-repressive mechanism. Rather, as revealed by analysis of double-mutant mice, Nkx2.1 and Gsh2 act cooperatively in many aspects to pattern the ventral telencephalon. By contrast, as indicated by both loss- and gain-of-function analysis, Gsh2 expression in the medial ganglionic eminence after E10.5 may negatively regulate Nkx2.1 dependent specification of oligodendrocytes. Taken together with previous studies, a hierarchy of gene expression for producing interneurons and oligodendrocytes is becoming apparent. Initiating the generation of these cell types in ventral regions are extrinsic cues, including Shh. These cues result in the expression of homeodomain genes, including Nkx2.1 and Gsh2, that ensure the expression of pan-ventral transcription factors, such as Dlx1/2, Mash1 and Olig2, in the MGE and LGE. These genes, in turn, may act as key effectors in the generation of specific ventral cell types, such as interneurons, and distinct populations of oligodendrocytes (Corbin, 2003).

In vitro studies have suggested that members of the GATA and Nkx transcription factor families physically interact, and synergistically activate pulmonary epithelial- and cardiac-gene promoters. However, the relevance of this synergy has not been demonstrated in vivo. This study shows that Gata6-Titf1 (Gata6-Nkx2.1) double heterozygous (G6-Nkx DH) embryos and mice have severe defects in pulmonary epithelial differentiation and distal airway development, as well as reduced phospholipid production. The defects in G6-Nkx DH embryos and mice are similar to those observed in human neonates with respiratory distress syndromes, including bronchopulmonary dysplasia, and differential gene expression analysis reveals essential developmental pathways requiring synergistic regulation by both Gata6 and Titf1 (Nkx2.1). These studies indicate that Gata6 and Nkx2.1 act in a synergistic manner to direct pulmonary epithelial differentiation and development in vivo, providing direct evidence that interactions between these two transcription factor families are crucial for the development of the tissues in which they are co-expressed (Zhang, 2007).

NK-2 homeobox genes and oligodendrocyte development

The role of Sonic hedgehog (Shh) in promoting the generation of oligodendrocytes in the mouse telencephalon is addressed in this study. In the forebrain, expression of the early oligodendrocyte markers Olig2, plp/dm20 and PDGFRalpha corresponds to regions of Shh expression. To directly test if Shh can induce the development of oligodendrocytes within the telencephalon, retroviral vectors were used to ectopically express Shh within the mouse embryonic telencephalon. Infections with Shh-expressing retrovirus at embryonic day 9.5 result in ectopic Olig2 and PDGFRalpha expression by mid-embryogenesis. By postnatal day 21, cells expressing ectopic Shh overwhelmingly adopt an oligodendrocyte identity. To determine if the loss of telencephalic Shh correspondingly results in the loss of oligodendrocyte production, Nkx2.1 mutant mice were studied, in which telencephalic expression of Shh is selectively lost. In accordance with Shh playing a role in oligodendrogenesis, within the medial ganglionic eminence of Nkx2.1 mutants, the early expression of PDGFRalpha is absent and the level of Olig2 expression is diminished in this region. In addition, in these same mutants, expression of both Shh and plp/dm20 is lost in the hypothalamus. Notably, in the prospective amygdala region where Shh expression persists in the Nkx2.1 mutant, the presence of plp/dm20 is unperturbed. Further supporting the idea that Shh is required for the in vivo establishment of early oligodendrocyte populations, expression of PDGFRalpha can be partially rescued by virally mediated expression of Shh in the Nkx2.1 mutant telencephalon. Interestingly, despite the apparent requirement for Shh for oligodendrocyte specification in vivo, all regions of either wild-type or Nkx2.1 mutant telencephalon are competent to produce oligodendrocytes in vitro. Furthermore, analysis of CNS tissue from Shh null animals definitively shows that, in vitro, Shh is not required for the generation of oligodendrocytes. It is proposed that oligodendrocyte specification is negatively regulated in vivo and that Shh generates oligodendrocytes by overcoming this inhibition. Furthermore, it appears that a Shh-independent pathway for generating oligodendrocytes exists (Nery, 2001).

Olig2, a basic helix-loop-helix (bHLH) transcription factor, is expressed in a restricted domain of the spinal cord ventricular zone that sequentially generates motoneurons and oligodendrocytes. Just prior to oligo-dendrocyte precursor formation, the domains of Olig2 and Nkx2.2 expression switch from being mutually exclusive to overlapping, and Neurogenins1 and 2 are extinguished within this region. Coexpression of Olig2 with Nkx2.2 in the spinal cord promotes ectopic and precocious oligodendrocyte differentiation. Both proteins function as transcriptional repressors in this assay. This effect is blocked by forced expression of Neurogenin1. By contrast, misexpression of Olig2 alone derepresses Neurogenins and promotes motoneuron differentiation. Olig2 therefore functions sequentially in motoneuron and oligodendrocyte fate specification. This dual action is enabled by spatio-temporal changes in the expression domains of other transcription factors with which Olig2 functionally interacts (Zhou, 2001).

If the domain of Olig gene expression indeed determines the site of origin of oligodendrocytes, then the timing of Olig gene expression within the spinal cord might determine the time at which such precursors are specified. Paradoxically, however, Olig2 is also expressed in the pMN domain at earlier stages, when motoneurons are being generated. Moreover, misexpression of Olig2 promotes motoneuron fate specification. These observations suggest either that Olig genes function in motoneuron and not oligodendrocyte fate determination, or alternatively that they play a role in both processes. To address this question, a chicken homolog of Olig2 has been identified, and its expression and function in relation to that of other transcription factors involved in neuronal differentiation in the ventral spinal cord has been examined (Zhou, 2001).

At E3.0, the time that motoneurons are being generated, the ventral boundary of the Olig2 expression domain is adjacent to, and nonoverlapping with, that of the underlying Nkx2.2 domain. However, just before oligodendrocyte precursors start to be produced (E6-E7), these two boundaries become overlapping, and numerous Olig2+, Nkx2.2+ cells are seen migrating away from the ventricular zone in a pattern characteristic of oligodendrocyte precursors. Combined misexpression of both Olig2 and Nkx2.2, but not of either gene alone, causes ectopic and precocious oligodendrocyte differentiation in the spinal cord. The normal generation of oligodendrocytes is also preceded by an extinction of the proneural bHLH factors Ngn1 and Ngn2 from the Olig2+ domain. Forced expression of Ngn1 blocks both ectopic oligodendrocyte differentiation induced by coexpression of Olig2+Nkx2.2 as well as normal oligodendrocyte differentiation. Taken together, these data suggest that Olig2 plays sequential roles in both motoneuron and oligodendrocyte fate specification. This dual action is enabled by spatio-temporal changes in the expression domains of other transcription factors expressed in the ventral spinal cord, with which Olig2 functionally interacts (Zhou, 2001).

In the vertebrate spinal cord, oligodendrocytes (OL) arise from the ventral part of the neuroepithelium, a region also known to generate somatic motoneurons. The emergence of oligodendrocytes, like that of motoneurons, depends on an inductive signal mediated by Sonic hedgehog. The precise timing of oligodendrocyte progenitor specification in the cervico-brachial spinal cord of the chick embryo has been determined. Ventral neuroepithelial explants, isolated at various development stages, are unable to generate oligodendrocytes in culture until E5 but become able to do so in an autonomous way from E5.5. This indicates that the induction of oligodendrocyte precursors is a late event that occurs between E5 and E5.5, precisely at the time when the ventral neuroepithelium stops producing somatic motoneurons. Analysis of the spatial restriction of oligodendrocyte progenitors, evidenced by their expression of O4 or PDGFRalpha, indicate that they always lie within the most ventral Nkx2.2-expressing domain of the neuroepithelium, and not in the adjacent domain characterized by Pax6 expression from which somatic motoneurons emerge. Shh is necessary between E5 and E5.5 to specify oligodendrocyte precursors but is no longer required beyond this stage to maintain ongoing oligodendrocyte production. Furthermore, Shh is sufficient to induce oligodendrocyte formation from ventral neuroepithelial explants dissected at E5. Newly induced oligodendrocytes express Nkx2.2 but not Pax6, correlating with the in vivo observation. Altogether, these results show that, in the chick spinal cord, oligodendrocytes originate from Nkx2.2-expressing progenitors (Soula, 2001).

It is now well established that somatic MNs derive from the ventral-most part of the Pax6+ region of the neuroepithelium in the chick spinal cord and not from the Nkx2.2+ domain. Recent evidence indicates that Nkx2.2 represses somatic MN genesis and restricts their emergence to the ventral-most part of the Pax6-expressing domain. In addition, a mechanism of mutual repression between the two factors leads to the formation of a sharp boundary between their two domains of expression. Double-staining experiments confirm that at E5.5 the last MNR2+ somatic MNs to be produced migrate from a level located dorsal to the OL/Nkx2.2 domain. Therefore, in chick spinal cord, OL and somatic MN precursors do not share the same transcription factors and do not originate from the same site in the neuroepithelium. The production of OLPs from a region different from the domain of somatic MN origin does not exclude the existence of common progenitor for OLs and other populations of ventral neurons. Indeed, in the spinal cord, the Nkx2.2+ neuroepithelial domain generates at least one population of uncharacterized V3 neurons, which in the mouse and chick embryos express the bHLH transcription factor Sim1. Similarly, in the hindbrain, OLPs could share a common progenitor with visceral MNs that are also generated from Nkx2.2-expressing neuroepithelial cells (Soula, 2001).

The relationship of Olig2+ and Nkx2.2+ oligodendrocyte progenitors (OLPs) has been investigated by comparing the expression of Olig2 and Nkx2.2 in embryonic chicken and mouse spinal cords before and during the stages of oligodendrogenesis. At the stages of neurogenesis, Olig2 and Nkx2.2 are expressed in adjacent non-overlapping domains of ventral neuroepithelium. During oligodendrogenesis stages, these two domains generate distinct populations of OLPs. From the Olig2+ motoneuron precursor domain (pMN) arise the Olig2+/Pdgfra+ OLPs, whereas the Nkx2.2+ p3 domain gives rise to Nkx2.2+ OLPs. Despite their distinct origins, both populations of OLPs eventually appear to co-express Olig2 and Nkx2.2 in the same cells. However, there is a species difference in the timing of acquiring Nkx2.2 expression by the Olig2+/Pdgfra+ OLPs. The co-expression of Nkx2.2 and Olig2 in OLPs is tightly associated with myelin gene expression in the normal and PDGFA^-/- embryos, suggesting a cooperative role for these transcription factors in the control of oligodendrocyte differentiation. In support of this suggestion, inhibition of expression of these two transcription factors in culture by antisense oligonucleotides has an additive inhibitory effect on OLP differentiation and proteolipid protein (PLP) gene expression (Fu, 2002).

Development of oligodendrocytes, myelin-forming glia in the central nervous system (CNS), proceeds on a protracted schedule. Specification of oligodendrocyte progenitors (OLPs) begins early in development, whereas their terminal differentiation occurs at late embryonic and postnatal periods. How these distinct steps are controlled remains unclear. The helix-loop-helix (HLH) transcription factor Ascl1 plays an important role in early generation of OLPs in the developing spinal cord. Ascl1 is also involved in terminal differentiation of oligodendrocytes late in development. Ascl1^-/- mutant mice showed a deficiency in differentiation of myelin-expressing oligodendrocytes at birth. In vitro culture studies demonstrate that the induction and maintenance of co-expression of Olig2 and Nkx2-2 in OLPs, and thyroid hormone-responsive induction of myelin proteins are impaired in Ascl1^-/-. Gain-of-function studies further showed that Ascl1 collaborates with Olig2 and Nkx2-2 in promoting differentiation of OLPs into oligodendrocytes in vitro. Overexpression of Ascl1, Olig2 and Nkx2-2 alone stimulates the specification of OLPs, but the combinatorial action of Ascl1 and Olig2 or Nkx2-2 is required for further promoting their differentiation into oligodendrocytes. Thus, Ascl1 regulates multiple aspects of oligodendrocyte development in the spinal cord (Sugimori, 2008).

NK-2 homeobox genes - thyroid and pancreas expression and function

The cDNA for TTF-1 (the same as Nkx2.1), a thyroid nuclear factor that binds to the promoter of thyroid specific genes, has been cloned. The protein encoded by the cDNA shows binding properties indistinguishable from those of TTF-1 present in nuclear extracts of differentiated rat thyroid cells. The DNA binding domain of TTF-1 is a novel mammalian homeodomain that shows considerable sequence homology to the Drosophila NK-2 homeodomain. TTF-1 mRNA and corresponding binding activity are detected in thyroid and lung. The chromosomal localization of the TTF-1 gene has been determined in humans and mice and corresponds to chromosomes 14 and 12, respectively, demonstrating that the TTF-1 gene is not located within previously described clusters of homeobox-containing genes (Guazzi, 1990).

A cDNA clone encoding a thyroid-specific enhancer-binding protein (T/EBP also known as Nkx2.1) was isolated from a rat thyroid-derived FRTL-5 cell lambda gt 11 expression library. Nucleotide and deduced amino acid sequences of the cDNA reveals that T/EBP is identical to the previously reported thyroid-specific transcription factor 1 (TTF-1), which binds to the promoter of the rat thyroglobulin gene and controls its thyroid-specific expression. Expression of the T/EBP cDNA under control of the human cytomegalovirus major immediate-early gene promoter confers thyroid-specific enhancer activity as high as 26-fold to nonpermissive human hepatoma HepG2 cells when cotransfected with a vector containing 6.3 kbp of upstream sequence of the human thyroid peroxidase gene connected to a luciferase reporter gene. T/EBP was further expressed in HepG2 cells by using the vaccinia virus expression system. The expressed protein was partially purified by using sequence-specific affinity column chromatography and was further shown to specifically bind to the enhancer-derived double-stranded oligonucleotide. These results clearly indicate that the binding of T/EBP (TTF-1) to the specific cis-acting enhancer element is largely responsible for thyroid-specific enhancer activity (Mizuno, 1991).

The thyroid-specific enhancer-binding protein (T/ebp or Nkx2.1) gene has been disrupted by homologous recombination in embryonic stem cells to generate mice lacking T/EBP expression. Heterozygous animals develop normally, whereas mice homozygous for the disrupted gene are born dead and lack the lung parenchyma. Instead, they have a rudimentary bronchial tree associated with an abnormal epithelium in their pleural cavities. Furthermore, the homozygous mice have no thyroid gland but have a normal parathyroid. In addition, extensive defects are found in the brain of the homozygous mice, especially in the ventral region of the forebrain. The entire pituitary, including the anterior, intermediate, and posterior pituitary, is also missing. In situ hybridization shows that the T/ebp gene is expressed in the normal thyroid, lung bronchial epithelium, and specific areas of the forebrain during early embryogenesis. These results establish that the expression of T/EBP, a transcription factor known to control thyroid-specific gene transcription, is also essential for organogenesis of the thyroid, lung, ventral forebrain, and pituitary (Kimura, 1996).

Most insulin-producing beta-cells in the fetal mouse pancreas arise during the secondary transition, a wave of differentiation starting at embryonic day 13. Disruption of homeobox gene Nkx6.1 in mice leads to loss of beta-cell precursors and blocks beta-cell neogenesis specifically during the secondary transition. In contrast, islet development in Nkx6.1/Nkx2.2 double mutant embryos is identical to Nkx2.2 single mutant islet development: beta-cell precursors survive but fail to differentiate into beta-cells throughout development. Together, these experiments reveal two independently controlled pathways for beta-cell differentiation, and place Nkx6.1 downstream of Nkx2.2 in the major pathway of beta-cell differentiation (Sander, 2000).

Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells. Nkx2.2 is the member of the vertebrate homeodomain transcription factor gene family that is most homologous to the Drosophila NK2/ventral nervous system defective (vnd) gene. Nkx2.2 was originally identified as a gene that is expressed in ventral regions of the developing vertebrate CNS. In addition to Nkx2.2, five other family members have been identified in mice: Nkx2.1, Nkx2.2, Nkx2.3 and Nkx2.4 are closely related, while Nkx2.5 and Nkx2.6 represent more divergent members of the family. NK2 family members have now been shown to be key regulators of development and differentiation in several tissues: Nkx2.1 is necessary for lung, thyroid and ventral forebrain development and Nkx2.5 is required for proper heart formation. Therefore, it is possible that Nkx2.2 may play a similar role in the development of the pancreas. The endocrine pancreas is organized into clusters of cells called islets of Langerhans comprizing four well-defined cell types: alpha, beta, delta and PP cells. While recent genetic studies indicate that islet development depends on the function of an integrated network of transcription factors, the specific roles of these factors in early cell-type specification and differentiation remain elusive. Within the pancreas, Nkx2.2 is expressed in alpha, beta and PP cells, but not in delta cells. Mice homozygous for a null mutation of Nkx2.2 develop severe hyperglycemia and die shortly after birth. Immunohistochemical analysis reveals that the mutant embryos lack insulin-producing beta cells and have fewer glucagon-producing alpha cells and PP cells. Remarkably, in the mutants there remains a large population of islet cells that do not produce any of the four endocrine hormones. These cells express some beta cell markers, such as islet amyloid polypeptide and Pdx1 (a homeodomain transcription factor that is an important factor in the proliferation and differentiation of the pancreatic buds to form a mature pancreas), but lack other definitive beta cell markers including glucose transporter 2 and Nkx6.1. It is proposed that Nkx2.2 is required for the final differentiation of pancreatic beta cells, and in its absence, beta cells are trapped in an incompletely differentiated state (Sussel, 1998).

Genetic studies are beginning to outline the hierarchy of transcription factors involved in beta cell development. For example, in Nkx2.2 mutant embryos, early expression of the beta cell specific transcription factor Nkx6.1 is unaffected. However, between E12.5 and E18.5, when there are major changes taking place within the pancreas (differentiation of exocrine tissue; beta cell proliferation; delta and PP cell formation) Nkx6.1 expression disappears. The data are consistent with a model where Nkx2.2 is required for the maintenance of Nkx6.1 expression as beta cells differentiate, and that continued expression of Nkx6.1 is necessary for complete beta cell differentiation. The genetic relationship between Nkx2.2 and Pdx1 is more complicated. Early in development, Pdx1 is expressed in all cells of the pancreatic bud, and is required for expansion of the bud. However, later in embryonic development, Pdx1 becomes progressively restricted to beta cells (and some delta cells); and at approximately E14.5, Pdx1 becomes upregulated in beta cells suggesting it plays a role in beta cell differentiation. In the Nkx2.2 mutant, early expression of Pdx1 is not affected. The later beta cell restriction of Pdx1 expression also occurs, but is quantitatively reduced in comparison to wild-type beta cells. Therefore, Nkx2.2 may be required for inducing high level expression of Pdx1 in beta cells, and the up-regulation of Pdx1 may be a necessary step in the final differentiation of the beta cell. In contrast to Nkx6.1 or Pdx1 expression, the expression of Isl1, Pax6 and Brn4 during embryogenesis does not appear to require Nkx2.2. These genes may therefore either lie upstream or in different pathways relative to the Nkx genes. Since Isl1 is expressed in islet cells soon after they exit the cell cycle, normal expression of Isl1 in the Nkx2.2 mutant suggests that all the islet cells are able to normally exit a proliferating state and proceed with a program of differentiation. This result supports the hypothesis that the immature beta cells are able to initiate beta cell development and it is subsequent steps of terminal differentiation that are blocked (Sussel, 1998 and references).

The homeodomain protein Nkx2.2 (Nkx2-2) is a key regulator of pancreatic islet cell specification in mice; Nkx2.2 is essential for the differentiation of all insulin-producing β-cells and of the majority of glucagon-producing alpha-cells, and, in its absence, these cell types are converted to a ghrelin cell fate. To understand the molecular functions of Nkx2.2 that regulate these early cell-fate decisions during pancreatic islet development, Nkx2.2-dominant-derivative transgenic mice were created. In the absence of endogenous Nkx2.2, the Nkx2.2-Engrailed-repressor derivative is sufficient to fully rescue glucagon-producing alpha-cells and to partially rescue insulin-producing β-cells. Interestingly, the insulin-positive cells that do form in the rescued mice do not express the mature β-cell markers MafA or Glut2 (Slc2a2), suggesting that additional activator functions of Nkx2.2 are required for β-cell maturation. To explore the mechanism by which Nkx2.2 functions as a repressor in the islet, the pancreatic expression was assessed of the Groucho co-repressors, Grg1, Grg2, Grg3 and Grg4 (Tle1-Tle4), which have been shown to interact with and modulate Nkx2.2 function. Grg3 is highly expressed in the embryonic pancreas in a pattern similar to Nkx2.2. Furthermore, Grg3 physically interacts with Nkx2.2 through its TN domain. These studies suggest that Nkx2.2 functions predominantly as a transcriptional repressor during specification of endocrine cell types in the pancreas (Doyle, 2007).

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