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Ems homologs and area identity in the mammalian neocortex

The contribution of extrinsic and genetic mechanisms in determining areas of the mammalian neocortex has been a contested issue. This study analyzes the roles of the regulatory genes Emx2 and Pax6, which are expressed in opposing gradients in the neocortical ventricular zone. Emx2 is expressed in low rostral to high caudal and low lateral to high medial gradients, whereas Pax6 is expressed in low caudal to high rostral and low medial to high lateral gradients. Changes in the patterning of molecular markers and area-specific connections between the cortex and thalamus suggest that arealization of the neocortex is disproportionately altered in Emx2 and Pax6 mutant mice in opposing manners predicted from their countergradients of expression: rostral areas expand and caudal areas contract in Emx2 mutants, whereas the opposite effect is seen in Pax6 mutants. These findings suggest that Emx2 and Pax6 cooperate to regulate arealization of the neocortex and to confer area identity to cortical cells (Bishop, 2000).

The type II classical cadherins, Cadherin6 (Cad6) and Cadherin8 (Cad8), are expressed in areal patterns in the late embryonic mouse neocortex: Cad8 has been reported to mark the rostrally located motor area, and Cad6 marks the somatosensory area, located immediately caudal to the motor area, and the auditory area, located in the caudolateral neocortex. Because the cortex is reduced in size in both Emx2 and Pax6 mutants, as compared to wild type, the proportion of the cortical surface covered by domains of cadherin expression is determined as well as absolute domain size. No change in proportional sizes of cadherin expression domains would indicate that arealization per se has not been affected, suggesting that the full range of putative area identities is present in the smaller cortex and that all areas are uniformly reduced in size. A change in proportional sizes would indicate that areas are disproportionately affected in the mutant neocortex and therefore that Emx2 and/or Pax6 has a role in regulating arealization of the neocortex. This finding and interpretation would be most strongly supported by a change in absolute sizes of cadherin expression domains (Bishop, 2000).

The areal pattern of cadherin expression in the Emx2 homozygous mutant cortex is substantially different from that in wild type. The domain of Cad8 expression is expanded caudally. This expansion appears to be greater along the medial edge of the cortex than laterally, farther on, suggesting that Cad8 expression may also be expanded medially. The domain of Cad6 expression is shifted caudally and medially, as seen on the dorsal and lateral surfaces of the cortex. No changes in the cortical patterns of cadherin expression are observed in Emx2 heterozygous mice. The domain of Cad8 expression is significantly larger in the Emx2 homozygous mutant neocortex, in both proportional and absolute area, as well as in its proportional and absolute linear extent across the cortical surface. In fact, the absolute area of the Cad8 expression domain in Emx2 homozygous mutants is almost double that in wild type, even though the surface area of the dorsal cortex is reduced by one-third. In Emx2 homozygous mutants, the domain of Cad6 expression on the dorsal cortical surface is significantly increased in proportional area and in both proportional and absolute width, compared to that in wild type. In contrast, the area and length of the Cad6 expression domain on the lateral cortical surface of Emx2 homozygous mutants each exhibit both proportional and absolute reductions compared to that in wild type. These results suggest that areas located in rostral and lateral parts of the neocortex are expanded and shifted caudally and medially in the Emx2 mutant neocortex (Bishop, 2000).

Because Pax6 is expressed in a countergradient to Emx2, it was predicted that changes in domains of cadherin expression in Pax6 mutant mice (Sey/Sey mutants) would be in the opposite direction of those observed in Emx2 mutants. The domain of Cad8 expression is contracted rostrally in the Sey/Sey cortex compared to that in the wild type. The Cad6 expression domain is contracted both laterally and rostrally in the Sey/Sey cortex. The domains of Cad8 and Cad6 expression on the dorsal surface of the Sey/Sey cortex, which is about three-quarters the size of that in the wild type, show both proportional and absolute reductions in area. Heterozygous mice (Sey/+) show no changes in cortical cadherin expression compared to wild-type mice. These results suggest that areas located in rostral and lateral parts of the neocortex contract rostrally and laterally in Sey/Sey mutants (Bishop, 2000).

Changes in the expression domains of Cad6 and Cad8 in Emx2 and Pax6 mutants suggest corresponding changes in neocortical arealization. The primary neocortical areas (motor, visual, somatosensory, and auditory) receive area-specific inputs from the principal motor and sensory thalamic nuclei [ventrolateral (VL), dorsal lateral geniculate (dLG), ventroposterior (VP), and medial geniculate (MG), respectively]. During normal development, thalamocortical axons target and invade their neocortical areas in a precise manner. Therefore, as an additional assay for changes in area identity in the Emx2 mutant neocortex, retrograde and anterograde axon tracing were used to map thalamocortical projections. This analysis was not done in Sey/Sey mutants because thalamic axons do not reach the cortex in these mice. Retrograde labeling from the cortex of Emx2 homozygous mutants indicates a caudal shift in the border between the somatosensory and visual areas compared to that of the wild type. Injections confined to the cortical plate of the occipital cortex, the location of the primary visual area, normally backlabel neurons in the dLG nucleus. However, in Emx2 mutants, similarly placed injections label cells in the VP nucleus, which normally projects to the primary somatosensory area located rostral to the visual area. Deeper injections made into the subplate of the occipital neocortex in Emx2 mutants backlabel neurons in both dLG and VP nuclei, indicating that dLG thalamic axons extend through the subplate below the occipital cortex but fail to invade the overlying cortical plate. Retrograde tracing from anterior and posterior portions of the occipital cortex reveals the expected topography in wild-type mice but again indicates a caudal shift in the border between the somatosensory and visual areas in Emx2 mutants. In wild-type mice, injections into the anterior occipital cortex backlabel cells in the posterior dLG nucleus, and injections into the posterior occipital cortex backlabel cells in the anterior dLG nucleus. In contrast, in Emx2 mutants, injections into the anterior occipital cortex do not label cells in the dLG nucleus but instead label cells in the VP nucleus; injections in the very posterior occipital cortex do label cells in the dLG nucleus, but their number is significantly reduced compared to the wild type. These findings suggest that the visual area in Emx2 mutants is contracted and restricted to the extreme caudal part of the occipital cortex (Bishop, 2000).

Anterograde tracing of thalamocortical projections is consistent with the retrograde tracing results. Injections into the dLG nucleus of Emx2 mutants label axons in the subplate beneath the caudal occipital cortex, but in comparison to the wild type, few invade the cortical plate. Injections into the VP nucleus of Emx2 mutants label axons that extend farther caudally than in the wild type and aberrantly invade the cortical plate of occipital cortex, whereas in the wild type, VP axons invade the cortical plate of the more rostrally located parietal cortex (the location of the primary somatosensory area). Thalamocortical projections in heterozygous Emx2 mutants resemble those in the wild type. Overall, anterograde and retrograde tracing of thalamocortical projections in Emx2 mutants provides evidence for a contraction of the visual area and a caudal shift in the border between the somatosensory and visual areas (Bishop, 2000).

Area-specific connections between thalamic nuclei and neocortical areas are reciprocal. Injections into thalamic nuclei backlabel cortical neurons in wild-type mice and Emx2 mutants. Injections into the dLG nucleus backlabel significantly fewer cells in the occipital cortex of Emx2 mutants compared to the wild type. The few labeled cells in Emx2 mutants are restricted to the very caudal cortex. Injections into the VP nucleus in wild-type mice backlabel cells in the parietal cortex but backlabel none in the occipital cortex. In contrast, VP injections in Emx2 mutants backlabel a substantial number of cells in the occipital cortex. These findings suggest that, in Emx2 mutants, corticothalamic neurons located in the occipital cortex have acquired a somatosensory area identity instead of their usual visual area identity. These changes in area-specific corticothalamic projections in Emx2 mutants are consistent with the changes observed in area-specific thalamocortical projections. Together, they suggest that the primary visual area is substantially reduced and restricted to the very caudal part of the neocortex, with a corresponding caudal shift in the border between visual and somatosensory areas (Bishop, 2000).

Emx2 is reported to be expressed in a small patch of neuroepithelium in the ventral-most part of the dorsal thalamus. Although this part does not generate cells of the principal sensory and motor thalamic nuclei, several markers were used to confirm the normal development and organization of the dorsal thalamus in Emx2 mutants. Patterns of acetylcholinesterase (AChE) staining and Gbx2 expression and general morphology revealed by nuclear 4',6-diamidino-2-phenylindole staining are all normal in the dorsal thalamus of Emx2 mutants. Thus, alterations in thalamocortical and corticothalamic projections in Emx2 mutants can be presumed to be due to changes intrinsic to the neocortex (Bishop, 2000).

These findings implicate Emx2 and Pax6 in the genetic control of neocortical arealization. They cannot be explained by a potential delay in neocortical development, nor are the expansions and contractions of the cadherin expression domains secondary to a loss of thalamocortical input. In addition, the observed changes in cadherin expression domains are not simply a by-product of the reduced overall size of the cerebral cortex in Emx2 and Pax6 mutants, because the changes are disproportionate in each mutant and opposing in the two mutants as predicted from the countergradients of Emx2 and Pax6 expression. Similarly, the observed changes in thalamocortical and corticothalamic projections in the Emx2 mutants are not due simply to a caudal truncation of the neocortex with an associated loss of the visual area. Arguing against this possibility are the changes in cadherin expression in Emx2 mutants, especially the expansion of the Cad8 expression domain in the frontal cortex (the motor area), in both proportional and absolute size. Instead, the findings presented here indicate a disproportional, but orderly, arealization of the Emx2 mutant neocortex reflected by an expansion of rostral areas and a contraction of caudal areas, and an opposite effect on arealization in the Pax6 mutant neocortex (Bishop, 2000).

Changes in the areal expression patterns of the cadherins and the area-specific distribution of corticothalamic neurons in the mutants suggest that Emx2 and Pax6 confer area identities to cortical cells, including projection neurons. The changes in cadherin expression and, presumably, receptors for axon guidance molecules that control corticothalamic axon targeting may be indicative of a direct role for Emx2 and Pax6 in their regulation, or they may be an indirect effect of the regulation of area identity. Similarly, the changes in area-specific thalamocortical projections suggest that Emx2 and Pax6 are involved either directly or indirectly in the regulation of axon guidance molecules within the cortex that control thalamocortical axon targeting. The restricted cortical expression of Eph receptor tyrosine kinases and their ligands, the ephrins, which act as axon guidance molecules in several systems, makes these candidates for controlling the development of area-specific projections between the thalamus and cortex (Bishop, 2000).

Emx2 and Pax6 may act independently or in a combinatorial manner (possibly with other transcription factor genes) to specify neocortical areas. Because areas in the neocortex have sharp borders, it is likely (but not required) that the graded expression patterns of Emx2 and Pax6 are translated to regulate some downstream genes in restricted patterns with abrupt borders that relate to areas. Although the downstream targets of Emx2 and Pax6 in the cortex have yet to be identified, transcription factors such as T-brain1 and Id2 are expressed in the neocortex in discrete patterns with abrupt borders that may be controlled by upstream regulatory genes expressed in gradients (Bishop, 2000).

Emx2 and Pax6 appear to be independently regulated. The opposing gradients of Emx2 and Pax6 may be induced by signals secreted from the poles of the cortex. Several secreted proteins are candidates for these inductive signals, including the BMP, WNT (2b, 3a, 5a, and 7a), and FGF8 proteins. In addition, cortical expression of the transcription factor Gli3 is required for Emx2 expression. Thus, combinations of inductive signals and upstream transcription factors may specify gradients of Emx2 and Pax6. A better understanding of the roles of Emx2 and Pax6 in regulating neocortical arealization will require identifying the patterning mechanisms that establish their differential expression, identifying downstream targets, and defining the mechanisms by which they, in combination with other factors, intrinsic and extrinsic, control the process of arealization of the neocortex (Bishop, 2000).

The specification of area identities in the cerebral cortex is a complex process, primed by intrinsic cortical cues and refined after the arrival of afferent fibers from the thalamus. Little is known about the genetic control of the early steps of this process, but the distinctive expression pattern of the homeogene Emx2 in the developing cortex has prompted suggestions that it is critical in this context. This hypothesis was tested using Emx2 -/- mice. The normal spectrum of cortical areal identities is encoded in these mutants, but areas with caudal-medial identities are reduced and those with anterior-lateral identities are relatively expanded in the cortex (Mallamaci, 2000).

Comparison among Cadherin6 (Cad6), limbic system-associated membrane protein gene (Lamp) and H-2Z1 transgene cortical expression domains in Emx2-/- versus wild-type brains provided clues about the role of Emx2 in the process of cortical arealization. Neither Cad6 nor Lamp main expression domains are dramatically altered in their absolute mediolateral extension, but they are medially shifted, and medial cortical sectors where they are not expressed are reduced in size. Similarly, Cad6 and H-2Z1 expression domains are not remarkably changed in their rostrocaudal extensions, but they are caudally shifted, and caudal cortical sectors not expressing them are dramatically reduced in size. Additional hints came from the analysis of thalamocortical wiring of Emx2-/- mutant brains. Whereas the development of a huge, aberrant axon bundle extending from dorsal thalamus to basal-marginal telencephalon cannot be explained, the preferential connection of mutant caudal cortex with ventrobasal complex (VBC), a structure that normally projects to cortical somatosensory areas could reflect an enlargement of pallial regions with more anterior areal identity and/or a reduction of those with caudal identity. Nevertheless, even if reduced, medial and caudal areas display qualitatively normal areal traits. The medialmost mutant cortical wall expressed the antineural HLH gene Id3, normally restricted to the ventricular zone of the medial limbic area, as well as the CKI-p21, normally confined to the marginal zone of the same medial limbic area. At later gestational ages, a few proliferating cells have been specifically detected in the marginal zone of the medialmost cortical wall, possibly reminiscent of the secondary proliferative layer, which is normally set up at corresponding tangential locations under the pia, and provides the early-forming dentate gyrus with large numbers of granule neurons. At the same stage, again in the presumptive limbic area but at a more lateral location, a spot of Cad6 expression could be easily distinguished, possibly corresponding to the normal expression domain in the subiculum. The caudalmost cortical wall, around the occipital pole, expressed the NGF low-affinity receptor p75, normally detectable in the entire caudal half of the telecephalic vesicle. The presumptive Cad6 subicular domain was retricted to the caudalmost cortex also, whereas it normally extends up to the level of the foramen of Monro. Finally, the failure to find cortical areas with caudal identity connected to the LGN does not imply that these areas do not exist. They could simply be very small and therefore difficult to detect by the DiI retrograde labeling approach used. Thus, areal identies are generally encoded similarly in mutants and normal mice; however, the tangential distribution of these identities in mutant cortices, even if topologically correct, is geometrically distorted. Rostral-lateral areas, where Emx2 is poorly expressed, are unaffected or slightly enlarged; caudal-medial areas, where Emx2 products are normally very abundant, are strongly reduced (Mallamaci, 2000).

Nested expression among Otx and Emx genes has implicated their roles in rostral brain regionalization, but single mutant phenotypes of these genes have not provided sufficient information. In order to genetically determine the interaction between Emx and Otx genes in forebrain development, Emx2-/-Otx2+/- double mutants and Emx2 knock-in mutants into the Otx2 locus (Otx2+/Emx2) were examined. Emx2-/-Otx2+/- double mutants do not develop diencephalic structures such as ventral thalamus, dorsal thalamus/epithalamus and anterior pretectum. The defects are attributed to the loss of the Emx2-positive region at the three- to four-somite stage, when its expression occurs in the laterocaudal forebrain primordia. Ventral structures such as the hypothalamus, mammillary region and tegmentum developed normally. Moreover, dorsally the posterior pretectum and posterior commissure are also present in the double mutants. In contrast, Otx2+/Emx2 knock-in mutants display the majority of these diencephalic structures; however, the posterior pretectum and posterior commissure are specifically absent. Consequently, development of the dorsal and ventral thalamus and anterior pretectum requires cooperation between Emx2 and Otx2, whereas Emx2 expression is incompatible with development of the commissural region of the pretectum (Suda, 2001)

Regional patterning in the developing mammalian brain is partially regulated by restricted gene expression patterns within the germinal zone, which is composed of stem cells and their progenitor cell progeny. Whether or not neural stem cells, which are considered at the top of the neural lineage hierarchy, are regionally specified remains unknown. The cardinal properties of neural stem cells (self-renewal and multipotentiality) are conserved among embryonic cortex, ganglionic eminence and midbrain/hindbrain, but these different stem cells express separate molecular markers of regional identity in vitro, even after passaging. Neural stem cell progeny derived from ganglionic eminence but not from other regions are specified to respond to local environmental cues to migrate ventrolaterally, when initially deposited on the germinal layer of ganglionic eminence in organotypic slice cultures. Cues exclusively from the ventral forebrain in a 5 day co-culture paradigm can induce both early onset and late onset marker gene expression of regional identity in neural stem cell colonies derived from both the dorsal and ventral forebrain as well as from the midbrain/hindbrain. Thus, neural stem cells and their progeny are regionally specified in the developing brain, but this regional identity can be altered by local inductive cues (Hitoshi, 2002).

Neural stem cells self-renew to generate new stem cell sphere colonies after mechanical dissociation in serum-free medium. It was hypothesized that stem cells in the sphere colonies that derived from forebrain or midbrain/rostral hindbrain (MB/rHB) tissue of GFP transgenic mice and were then placed on the ganglionic eminence (GE) of cutured slices would maintain their 'stemness' after 5 days of in vitro slice co-culture. To test this, the largest fluorescent excised portion of each sphere/GE slice co-culture was excised under the fluorescence microscope, dissociated mechanically, and then the cells were plated at 10-50 cells/µl in serum-free medium with FGF2 and EGF. New GFP-positive sphere colonies were observed after 7 days in vitro from the co-cultures of the cortical, GE or MB/rHB GFP neurospheres and coronal slices. Whether the GFP-positive neural stem cell colonies maintain their donor regional identities, or alternatively whether they acquire a ventral forebrain (the GE host in the slice culture) regional identity was tested by analyzing the expression of Dlx2 in the newly generated GFP-positive secondary colonies. New colonies derived from co-cultures of GFP-positive cortical stem cell colonies expressed only Dlx2 (8/9, 88.9%), but were negative for their original regional identity marker Emx1. One secondary GFP-positive sphere colony expressed only Emx1 (1/9, 11.1%). In contrast, new colonies derived from co-cultures of GFP-positive MB/rHB stem cell colonies expressed only Dlx2 (6/9, 66.7%), or both Dlx2 and En1 (3/9, 33.3%). New colonies derived from co-cultures of GFP-positive GE stem cell colonies retained expression of Dlx2 (9/9, 100%). These findings suggest that specific cues from the ventral forebrain induce neural stem cells in cortical or MB/rHB colonies to acquire ventral forebrain identities and to suppress their original regional identities. Thus, even the early regional identities of neural stem cells are not irreversible and can be altered by local inductive cues (Hitoshi, 2002).

The early expression of transcription factors in the anterior neural plate (E8.5) already defines a regionalization pattern that persists after the onset of neurogenesis. When precursor cells derived from the early neural tube (E9.5-E10.5) are isolated in vitro, they maintain their regional specification. The E14.5 embryonic forebrain neural stem cells (from dorsal or ventral compartments), isolated from their in vivo environment, generate clonal colonies that express forebrain-specific regional markers (Emx1 or Dlx2), whereas neural stem cells isolated from the MB/rHB instead express a midbrain/rostral hindbrain-specific regional marker (En1), and those isolated from cHB express a caudal hindbrain-specific marker (Hoxb1). In addition, the Otx1 expression that is normally restricted to the forebrain and midbrain at E14.5 in vivo, was observed in cortical, GE, and MB/rHB neural stem cell colonies but not in caudal hindbrain colonies. These data reveal that neural stem cells in the E14.5 mammalian brain manifest a regional identity along the anteroposterior axis during development. It has been demonstrated that mouse E14.5 cortical neural stem cell colonies express a telencephalic-restricted Sox2 transgene or the Otx1 gene, but that these genes are not expressed by spinal cord-derived neural stem cell colonies. Thus, neural stem cell regionalization may be regulated throughout the entire developing CNS. Moreover, neural stem cell regionalization during development is not restricted to the anteroposterior axis. Even within the forebrain, most of the neural stem cell colonies derived from the dorsal compartment (cortex) express Emx1, but not Dlx2, and most of the neural stem cell colonies derived from the ventral compartment (GE) express Dlx2, but not Emx1. Thus, neural stem cells maintain a distinct dorsoventral identity within the forebrain, suggesting that neural stem cell regionalization can be regulated within distinct histogenic compartments rather than between broad CNS domains only (Hitoshi, 2002).

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

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

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

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

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

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

Three basic aspects of cerebral cortex development have been recently investigated, specification of cortical versus ganglionic identity, regionalization of the early cortical primordium and arealization of the developing cortex. Emx2 and Pax6 promote development of caudal-medial and rostral-lateral cortex, respectively, by properly shaping the early cortical protomap and possibly modulating the tangential growth ratio between medial and lateral cortical anlagen. By analyzing the brains of embryos bearing mutations for Emx2 and Pax6 in different combinations, it was found that both genes are necessary and sufficient for a more basic developmental choice, i.e. the specification of neuroblasts in the dorsal telencephalon as cortical versus ganglionic neuroblasts. The possible roles of the Emx2 paralog, Emx1, in these processes was investigated. By looking at embryos mutant for Emx1, Emx2 and Pax6 in various combinations, no evidence was found of Emx1 involvement in the process of cortical specification; conversely, this gene appeared to be involved to some extent in the process of regionalization of the cortical primordium along the medial-lateral axis, as a promoter of medial fates (Muzio, 2003).

Genetic studies of neocortical area patterning are limited, because mice deficient for candidate regulatory genes die before areas emerge and have other complicating issues. To define roles for the homeodomain transcription factor EMX2, nestin-Emx2 transgenic mice that survive postnatally were engineered that overexpress Emx2 in cortical progenitors coincident with expression of endogenous Emx2. Cortical size, lamination, thalamus, and thalamocortical pathfinding are normal in homozygous nestin-Emx2 mice. However, primary sensory and motor areas are disproportionately altered in size and shift rostrolaterally. Heterozygous transgenics have similar but smaller changes. Opposite changes are found in heterozygous Emx2 knockout mice. Fgf8 expression in the commissural plate of nestin-Emx2 mice is indistinguishable from wild-type, but Pax6 expression is downregulated in rostral cortical progenitors, suggesting that EMX2 repression of PAX6 specification of rostral identities contributes to reduced rostral areas. It is concluded that EMX2 levels in cortical progenitors disproportionately specify sizes and positions of primary cortical areas (Hamasaki, 2004).

Emx1 and Emx2 are mouse cognates of the Drosophila head gap gene, ems. The dentate gyrus is affected in Emx2 single mutants, and defects are subtle in Emx1 single mutants. In most of the cortical region Emx1 and Emx2 functions would be redundant. To test this assumption the Emx1 and Emx2 double mutant phenotype was examined. In the double mutants the archipallium is transformed into the roof without establishing the signaling center at the cortical hem and without developing the choroid plexus. It is proposed that Emx1 and Emx2 cooperate in generation of the boundary between the roof and archipallium; these genes develop the archipallium against the roof. This process probably occurs immediately after the neural tube closure concomitant with the Emx1 expression (Shinozaki, 2004).

The difference between Emx2 single and Emx1/2 double mutant defects is morphologically apparent at E12.5. The extent of this distinction involves the degree of invagination of the medial pallium and expansion of the roof; in particular, an intermediary structure (IMR) exists between the pallium and the expanded roof-like structure in Emx2 single mutants; however, the IMR is absent in Emx1/2 double mutants. A question first arose as to whether the entire expanded medial structure is indeed the roof. Morphologically its cellular composition is identical to that of the normal roof. Cell death is prominent and BrdU uptake is low. The expanded medial structure is Lhx5-, Msx1- and Bmps-positive and TTR-, Wnts-, Ngn2-, Pax6-, Lhx2-, Gli3- and BF1-negative. Thus, it is concluded that this structure is indeed the roof. However, the roof was dysmorphic in some ways. The roof has lost the noggin expression and ectopically expresses Otx2 and Ephb1. Moreover, it is the Fgf8-negative domain that emerges broadly in the midline of the roof. The cause and significance of these dysmorphies in the roof remain for future studies (Shinozaki, 2004).

Distinct actions of Emx1, Emx2, and Pax6 in regulating the specification of areas in the developing neocortex

The mammalian neocortex is organized into subdivisions referred to as areas that are distinguished from one another by differences in architecture, axonal connections, and function. The transcription factors EMX1, EMX2, and PAX6 have been proposed to regulate arealization. Emx1 and Emx2 are expressed by progenitor cells in a low rostrolateral to high caudomedial gradient across the embryonic neocortex, and Pax6 is expressed in a high rostrolateral to low caudomedial gradient. Recent evidence has suggested that EMX2 and PAX6 have a role in the genetic regulation of arealization. A panel of seven genes (Cad6, Cad8, Id2, RZRß, p75, EphA7, and ephrin-A5), representative of a broad range of proteins has been used as complementary markers of positional identity to obtain a more thorough assessment of the suggested roles for EMX2 and PAX6 in arealization, and in addition, to assess the proposed but untested role for EMX1 in arealization. Orderly changes in the size and positioning of domains of marker expression in Emx2 and Pax6 mutants strongly imply that rostrolateral areas (motor and somatosensory) are expanded, whereas caudomedial areas (visual) are reduced in Emx2 mutants and that opposite effects occur in Pax6 mutants, consistent with their opposing gradients of expression. In contrast, patterns of marker expression, as well as the distribution of area-specific thalamocortical projections, appear normal in Emx1 mutants, indicating that these mutants do not exhibit changes in arealization. This lack of a defined role for EMX1 in arealization is supported by finding of similar shifts in patterns of marker expression in Emx1; Emx2 double mutants as in Emx2 mutants. Thus, these findings indicate that EMX2 and PAX6 regulate, in opposing manners, arealization of the neocortex and impart positional identity to cortical cells, whereas EMX1 appears not to have a role in this process (Bishop, 2002).

Emx1 and Emx2 cooperate to regulate cortical size, lamination, neuronal differentiation, development of cortical efferents, and thalamocortical pathfinding

The homeobox transcription factors Emx1 and Emx2 are expressed in overlapping patterns that include cortical progenitors in the dorsal telencephalic neuroepithelium. Cooperation of Emx1 and Emx2 in cortical development was addressed by comparing phenotypes in Emx1; Emx2 double mutant mice with wild-type and Emx1 and Emx2 single mutants. Emx double mutant cortex is greatly reduced compared with wild types and Emx single mutants; the hippocampus and dentate gyrus are absent, and growth and lamination of the olfactory bulbs are defective. Cell proliferation and death are relatively normal early in cortical neurogenesis, suggesting that hypoplasia of the double mutant cortex is primarily due to earlier patterning defects. Expression of cortical markers persists in the reduced double mutant neocortex, but the laminar patterns exhibited are less sharp than normal, consistent with deficient cytoarchitecture, probably due in part to reduced numbers of preplate and Reelin-positive Cajal-Retzius neurons. Subplate neurons also exhibit abnormal differentiation in double mutants. Cortical efferent axons fail to exit the double mutant cortex, and TCAs pass through the striatum and approach the cortex but do not enter it. This TCA pathfinding defect appears to be non-cell autonomous and supports the hypothesis that cortical efferents are required scaffolds to guide TCAs into cortex. In double mutants, some TCAs fail to turn into ventral telencephalon and take an aberrant ventral trajectory; this pathfinding defect correlates with an Emx2 expression domain in ventral telencephalon. The more severe phenotypes in Emx double mutants suggest that Emx1 and Emx2 cooperate to regulate multiple features of cortical development (Bishop, 2003).

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

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

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

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

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

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

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

A novel mechanism for the transcriptional regulation of Wnt signaling in development

Axial patterning of the embryonic brain requires a precise balance between canonical Wnt signaling, which dorsalizes the nervous system, and Sonic hedgehog (Shh), which ventralizes it. The ventral anterior homeobox (Vax) transcription factors are induced by Shh and ventralize the forebrain through a mechanism that is poorly understood. This study therefore sought to delineate direct Vax target genes. Among these, an extraordinarily conserved intronic region was identified within the gene encoding Tcf7l2, a key mediator of canonical Wnt signaling. This region functions as a Vax2-activated internal promoter that drives the expression of dnTcf7l2, a truncated Tcf7l2 isoform that cannot bind β-catenin and that therefore acts as a potent dominant-negative Wnt antagonist. Vax2 concomitantly activates the expression of additional Wnt antagonists that cooperate with dnTcf7l2. Specific elimination of dnTcf7l2 in Xenopus results in headless embryos, a phenotype consistent with a fundamental role for this regulator in forebrain development (Vacik, 2011).

Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain

The forebrain consists of multiple structures necessary to achieve elaborate functions. Proper patterning is, therefore, a prerequisite for the generation of optimal functional areas. Only a few factors have been shown to control the genetic networks that establish early forebrain patterning. Using conditional inactivation, this study shows that the transcription factor Sp8 has an essential role in the molecular and functional patterning of the developing telencephalon along the anteroposterior axis by modulating the expression gradients of Emx2 and Pax6. Moreover, Sp8 is essential for the maintenance of ventral cell identity in the septum and medial ganglionic eminence (MGE). This is probably mediated through a positive regulatory interaction with Fgf8 in the medial wall, and Nkx2.1 in the rostral MGE anlage, and independent of SHH and WNT signaling. Furthermore, Sp8 is required during corticogenesis to sustain a normal progenitor pool, and to control preplate splitting, as well as the specification of cellular diversity within distinct cortical layers (Zembrzycki, 2007).

EMX homeobox genes regulate microphthalmia and alter melanocyte biology

Melanocytes are specialized cells that produce melanin, the pigment responsible for skin, hair and retina color. They derive during embryogenesis from the precursor cells melanoblasts, which are neural crest cells committed to the pigment cell lineage. The differentiation of melanoblasts into melanocytes involves the expression of melanocyte-specific genes, particularly those responsible for melanin production, such as Tyr, Tyrp-1 and Dct, the expression of which depends on the melanocyte-specific transcription factor microphthalmia (Mitf). A functional screen was developed and executed on melanocytes, with the aim of identifying genes involved in pigment cell biology. It was found that Emx1 and Emx2, two highly related homeobox genes that when overexpressed in melanocytes can downregulate Mitf, Tyrp1, Dct and Tyr. Constitutive expression of Emx alters pigment cell morphology and growth properties: it confers TPA independence but not the ability to grow in soft agar. Spatial and temporal expression of Emx and Mitf during embryonic development suggests that Emx could be one factor that regulates correct expression of Mitf by inhibiting its activation in neuroepithelial derivatives other than melanocytes (Bordogna, 2005).

Reduced hippocampal neurogenesis and skill reaching performance in adult Emx1 mutant mice

Mammalian homeobox gene Emx family is involved in the development of the rostral brain. Loss-of-function studies suggest that, despite the agenesis of corpus callosum, the Emx1 mutants display relatively modest defects compared to the Emx2 mutants. However, the role of the Emx1 in neurogenesis and brain function has never been explored. Unbiased stereology was used to determine the number of proliferating progenitors and immature neurons in the adult neurogenic zones. Although previous studies have established that the formation of the dentate gyrus (DG) requires Emx2, the adult Emx1 mutants also exhibited a smaller DG, reduced number of proliferating progenitor cells and immature neurons in the DG, in contrast to the indistinguishable level of neurogenesis in the subventricular zone when compared to the wild type mice. In view of the involvement of callosal projection neurons in mediating interhemispheric crosstalk and spatial coupling between the limbs, and the importance of DG in hippocampus-dependent function in learning and memory, motor and cognitive functions were assessed. Emx1 deletion impaired performance on a forelimb skill reaching task and attenuated training induced hippocampal neurogenesis, but it did not affect motor activity or basic motor function as evaluated in the open field, wire hanging and rotor rod tests. Unexpectedly, the adult Emx1 mutant mice did not exhibit impairment in spatial learning and memory in the Barnes maze test. These data suggest that deletion of the Emx1 gene reduces hippocampal neurogenesis and affects higher motor function that requires extensive learning (Hong, 2007).

The transcription factors Emx1 and Emx2 suppress choroid plexus development and promote neuroepithelial cell fate

The transcription factors Emx1 and Emx2 exert important functions during development of the cerebral cortex, including its arealization. This study addressed their role in development of the derivatives of the midline region in the telencephalon. The center of the midline region differentiates into the choroid plexus, but little is known about its molecular specification. Since a lack of Emx1 or 2 expression was noted in the midline region early in development, Emx1 and/or Emx2 were misexpressed in this region of the chick telencephalon. Ectopic expression of either Emx1 or Emx2 prior to HH 13 instructed a neuroepithelial identity in the previous midline region instead of a choroidal fate. Thus, Gli3 and Lhx2 normally restricted to the neuroepithelium expanded into the Emx misexpressing region. This was accompanied by down-regulation of Otx2 and BMP7, which implicates that these factors are essential for choroid plexus specification and differentiation. Interestingly, the region next to the ectopic Emx-misexpression then acquired a hybrid identity with some choroidal features such as Bmp7, Otx2 and Ttr gene expression, as well as some neuroepithelial features. These observations indicate that the expression levels of Emx1 and/or Emx2 restrict the prospective choroid plexus territory, a novel role of these transcription factors (von Frowein, 2006).

Vax, Floating head and Xnot, distant relatives of Empty spiracles

This work concerns the expression of two transcription factors during the development of the sea urchin Strongylocentrotus purpuratus: SpNot, the ortholog of the vertebrate Not gene, and SpBra, the ortholog of the vertebrate Brachyury gene are both expressed in the chordate notochord. It is of interest to see whether they are both expressed in a common structure in echinoderms. SpNot is a Hox gene distantly related to Drosophila empty spiracles, but more closely related to another Drosophila Hox gene, 90Bre, also termed E103, for which the literature is incomplete. SpNot transcripts are detected by in situ hybridization in the vegetal plate at the mesenchyme-blastula stage. Later the gene is expressed in the secondary mesenchyme, but expression is no longer detectable after gastrulation. SpNot is upregulated during larval development, in the invaginating vestibule of the adult rudiment. Transcripts are also found in several larva-specific tissues, including the epaulets, blastocoelar cells, and pigment cells. SpBra also displays a discontinuous pattern of expression. Much like SpNot, this gene is expressed during embryogenesis in the embryonic vegetal plate and secondary mesenchyme founder cells, and expression is then extinguished. The gene is upregulated over a week later in the feeding larva, in the vestibule of the adult rudiment. In contrast to SpNot, SpBra is also expressed in the mesoderm of both left and right hydrocoels, and it is not expressed in any larva-specific tissues (Peterson, 1999).

The spatial expression profile determined in this study is compared with that of the orthologous Brachyury gene in an indirectly developing enteropneust hemichordate, a representative of the sister group to the echinoderms within the deuterostomes. Both SpNot and SpBra are utilized discontinuously, displaying unconnected embryonic and larval phases of expression. Moreover, the expression of these two genes demonstrated in the vertebrate dorsal mesoderm is not found in the development of the echinoderm adult body plan. Thus, SpNot is not expressed at all in the coelomic mesoderm, but only in the invaginating vestibule of the rudiment. It is also expressed in various mesenchymal cells and larva-specific ectodermal derivatives. Expression of SpBra in larval stages occurs only in the mesodermal hydrocoel and the vestibule, i.e., in the major anlagen from which the echinoid body plan develops. Because Brachyury and Not are both expressed in the chordate notochord, precursors of the notochord were sought in the sea urchin, but none were found and it was concluded they do not exist in this species. The notochord is best considered a structure unique to chordates, and it would then follow that in the early evolution of chordates, these two genes were recruited for the specification of the notochord. The only overlapping expression domains of SpBra and SpNot in the larva are in the vestibule, a nonmesodermal structure exclusive to the euechinoids (Peterson, 1999).

These observations illuminate the genetic basis underlying the process of maximal indirect development in basal deuterostomes. Maximal indirect development requires the construction of two very different organisms, a relatively simple embryo/larva and a complex adult, which are formed by quantitively different modes of development. The embryo/larva is formulated by specification processes that proceed immediately to the institution of differentiation programs and the generation of cell types, beginning even in cleavage. Brachyury appears to be an excellent marker for the progeny of the set-aside cells of the sea urchin embryo. Patches of embryonic cells are set aside from the immediate specification-to-differentiation process of the embryo. Postembryonic development of the adult body plan can be considered to be initiated by genes encoding such pattern-forming transcription factors at the regulatory level, activated within the larval progeny of the set-aside cells (Peterson, 1999).

Xnot from Xenopus, Floating head from zebrafish, as well as related genes of higher vertebrates like Cnot from chicken are related to Empty spiracles and expressed in notochord. Information on Xnot and Floating head is provided below, but because these genes show a mesodermal expression pattern, it is doubtful whether they can be considered Empty spiracle homologs in a strict sense. Both Xnot and Floating head also have sequence affinity to Muscle segment homeobox, expressed in ectoderm and mesoderm and to NK1, expressed in mesoderm.

Xnot-2 is a homeobox gene expressed in Spemann's organizer. Microinjection of synthetic Xnot-2 mRNA leads to the formation of notochord. Microinjection into the dorsal side of the Xenopus embryo results in greatly expanded notochords. Nearby somitic and prechordal mesoderm becomes recruited into these enlarged notochords, which also affect CNS patterning. Two early genes expressed in the developing notochord, chd and XFKH-1, are activated by Xnot-2. Chordin, a homolog of Drosophila short gastrulation, is a dorsalizing factor secreted by the notochord that can be activated by Xenopus goosecoid and Xnot-2 in wild-type Xenopus embryos. Xenopus XFKH-1/Pintallavis, a homolog of Drosophila Forkhead and mammalian HNG3ß is expressed in the notochord and floor plate. It is concluded that gain-of-function of Xnot-2 promotes notochord formation (Gont, 1996).

Using fate mapping techniques, development of cells of the dorsal marginal region in wild-type and mutant zebrafish has been analyzed. A domain in the early gastrula has been defined that is located just at the margin and centered on the dorsal midline, in which most cells generate clones that develop exclusively as notochord. The borders of the notochord domain are sharp at the level of single cells, and coincide almost exactly with the border of the expression domain of the homeobox gene floating head (zebrafish homolog of Xnot), a gene essential for notochord development. In flh mutants, cells in the notochord domain generate clones of muscle cells. In contrast, notochord domain cells form mesenchyme in embryos mutant for no tail ( the zebrafish homolog of Brachyury). A minority of cells in the notochord domain in wild-type embryos develop as unrestricted mesoderm, invariably located in the tail, suggesting that early gastrula expression of flh does not restrict cellular potential to the notochord fate. The unrestricted tail mesodermal fate is also expressed by the forerunner cells, a cluster of cells located outside the blastoderm, adjacent to the notochord domain. Cells can leave the dorsal blastoderm to join the forerunners, suggesting that relocation between fate map domains might respecify notochord domain cells to the tail mesodermal fate. An intermediate fate of the forerunners is to form the epithelial lining of Kupffer's vesicle, a transient structure of the teleost tailbud. The forerunners appear to generate the entire structure of Kupffer's vesicle, which also develops in most flh mutants. Although forerunner cells are present in ntl mutants, Kupffer's vesicle never appears; this correlates with the later severe disruption of tail development (Melby, 1996).

Zebrafish floating head mutant embryos lack notochord and develop somitic muscle in its place. This may result from incorrect specification of the notochord domain at gastrulation, or from respecification of notochord progenitors to form muscle. In genetic mosaics, floating head acts cell autonomously. Transplanted wild-type cells differentiate into notochord in mutant hosts; however, cells from floating head mutant donors produce muscle rather than notochord in wild-type hosts. Consistent with respecification, markers of axial mesoderm are initially expressed in floating head mutant gastrulas, but expression does not persist. Axial cells also inappropriately express markers of paraxial mesoderm. Thus, single cells in the mutant midline transiently co-express genes that are normally specific to either axial or paraxial mesoderm. Since floating head mutants produce some floor plate in the ventral neural tube, midline mesoderm may also retain early signaling capabilities. These results suggest that wild-type floating head provides an essential step in maintaining, rather than initiating, development of notochord-forming axial mesoderm (Halpern, 1995).

The notochord is a midline mesodermal structure with an essential patterning function in all vertebrate embryos. Zebrafish floating head (flh) mutants lack a notochord, but develop with prechordal plate and other mesodermal derivatives, indicating that flh functions specifically in notochord development. The finding that some floor-plate cells and motor neurons are present in flh mutants might indicate that axial mesodermal cells in these animals can induce floor-plate and motor neuron differentiation for a limited time during gastrulation, even though these mesodermal cells do not differentiate as notochord later in embryogenesis. floating head is the zebrafish homolog of Xnot, a homeobox gene expressed in the amphibian organizer and notochord. The homeodomain sequence places the gene in the empty spiracles family, which includes Xnot, the chicken gene Cnot, Drosophila empty spiracles, 90Bre, and the Emx-1 and Emx-2 genes of mouse and human. There is a 75% identity to Xnot and a 53% identity to ems. There is an interaction between flh and the brachyury homolog no tail. In flh mutants, ntl expression in the germ ring and tail bud does not appear to be altered. Expression in the notochord precursors, however, is disrupted. At the end of gastrulation, ntl expression is largely absent from the midline of flh mutants, so that ntl is expressed strongly in the tail bud and faintly in a small axial region immediately adjacent to it. Earlier in gastrulation, cells expressing ntl are scattered along the axis, indicating that at least some axial mesodermal cells in flh mutants express ntl after leaving the margin to migrate towards the animal pole. These cells apparently do not maintain ntl expression, however, as they are not detected at the end of gastrulation. The regulatory interaction between flh and ntl is complex, as axial cells express lower levels of flh mRNA than those in wild-type embryos. It is proposed that flh regulates notochord precursor cell fate. The presence of floor plate in flh mutants can be explained by the fact that flh mutants transiently express sonic hedgehog, suggesting that flh only partially blocks the ability of axial mesodermal cells to induce floor-plate differentiation (Talbot, 1995).

In the roof of the zebrafish forebrain the first site at which neurogenesis occurs is the epiphysial region of the dorsal diencephalon. There are two classes of neurons in the zebrafish epiphysis (or pineal organ): photoreceptors and projection neurons. Analysis of Pax6 (Drosophila homolog eyeless) distribution in the CNS reveals that it is expressed in projection neurons. In contrast to Pax6, the islet-1 gene (a Lim homeodomain homolog of Drosophila islet) is found to be expressed by photoreceptors and projection neurons. Zash-1a, a zebrafish homolog of Drosophila achaete-scute proneural genes is also expressed in the epiphysis. Islet-1 positive cells are produced within the Zash-1 expression domain. The homeobox gene floating head is required for neurogenesis to proceed in the epiphysis. In flh mutant embryos the first few epiphysial neurons are generated, but beyond the 18 somite stage, neuronal production ceases. Islet-1 positive cells and mature neurons are reduced in the epiphysis of floating head mutants. In contrast, in masterblind mutant embryos, epiphysial neurons are generated throughout the dorsal forebrain. Thus mbl acts negatively, preventing the expression of flh in dorsal forebrain cells rostral to the epiphysis. Furthermore, epiphysial neurons are not ectopically induced in mbl/flh double mutant embryos, demonstrating that the epiphysial phenotype of mbl mutants is mediated by ectopic Flh activity. It is thought that Zash-1 is downstream of flh, and that Pax6 acts even further downstream, differentiating projection neurons from photoreceptors. A role for Flh is proposed in linking the signaling pathways that regulate regional patterning to the signaling pathways that regulate neurogenesis (Masai, 1997)

The vertebrate forebrain is formed at the rostral end of the neural plate under the regulation of local and specific signals emanating from both the endomesoderm and neuroectoderm. In particular, the development of the rostral and ventral forebrain was difficult to study, mainly because no specific markers have been available, to date. Vax1 is a novel homeobox-containing gene identified in mouse, Xenopus and human. It is closely related to members of the Not and Emx gene families, all of which are required for the formation of structures where they are expressed. In mouse and Xenopus, Vax1 expression first occurs in the rostral neural plate, in the medial anterior neural ridge and adjacent ectoderm. Later, at midgestation in the mouse and tadpole stage in Xenopus, the expression remains confined in the derivatives of this territory, which differentiate into rostromedial olfactory placode, optic nerve and disc, and anterior ventral forebrain. Together, these observations suggest that Vax1 could have an early evolutionary origin and could participate in the specification and formation of the rostral and ventral forebrain in vertebrates. Comparison of the limits of the expression territory of Vax1 with that of Dlx1, Pax6 and Emx1 indicates that the corticostriatal ridge is a complex structure with distinct identifiable genetic compartments. The study of Vax1 expression in Pax6-deficient homozygous brains also indicates that its regulation is independent of Pax6, although the expression patterns of these two genes appear complementary in wild-type animals. Vax1 chromosomal location is mapped at the distal end of the mouse chromosome 19, linked with that of Emx2. These two genes may have arisen by tandem duplication (Hallonet, 1998).

Mutations of the Drosophila empty spiracles (ems) gene affect embryonic head development and lead to loss of head structures. Therefore, ems acts as both a gap gene, responsible for the formation of a group of head segments, and a homeotic selector gene, specifying segment identity. Similarly, inactivation of Emx1 or Emx2 murine genes by homologous recombination in the mouse demonstrates that these genes are necessary for the formation of specific regions of the brain where they are expressed and for migration of some cortical neurons. Mutations in the human EMX2 genes have also been identified in patients with holoprosencephaly. Likewise, Not1 inactivation in the zebrafish leads to an absence of notochord where the gene would normally be expressed. Because it shares high sequence homology with these genes, Vax1 could be necessary for ventral forebrain specification and patterning. Moreover, the stability of Vax1 pattern of expression during neurulation in mouse and Xenopus suggests an important function of Vax1 in the specification and maintenance of basal forebrain identity. Vax1 is also expressed in the anterior neural ridge (ANR). This structure has inducing properties on the anterior neural plate that are mimicked by the FGF8 expressed at this level. It is therefore possible that Vax1 could participate in early neural plate induction (Hallonet, 1998 and references).

During early brain morphogenesis in mouse and Xenopus, Vax1 transcripts are detected in regions that correspond precisely to the derivatives of the area where the gene is expressed at the neural plate stage; this indicates that the early Vax1-expressing region is an embryological unit. This region could correspond to the part of the brain induced in Xenopus by Cerberus and specified by signals from rostral endoderm. Vax1 expression is simultaneous with neuronal progenitor proliferation and neural cell specification in the septum, preoptic area, and lateral ganglionic eminence (LGE). In addition, the subventricular zone where Vax1 is expressed in the ganglionic eminences, is considered to be a transition zone that is crossed by cells that originate in the ventricular zone. The ventricular zone is a region where cells can further proliferate and acquire information important for their final fate. Furthermore, Vax1 transcription occurring in the medial olfactory placode from E8 to E11 is no longer detected in the olfactory epithelium or in the vomeronasal organ at E13.5. The medial olfactory placode is the site of origin of the vomeronasal organ and of GnRH neurons that leave the placode from E11 onward, migrate out and settle in the septal and preoptic areas. Accordingly, Vax1 could function in early developing progenitors present in the medial olfactory placode before they migrate to the basal forebrain. In general, Vax1 could thus function in the early steps of the neuronal differentiation. Moreover, the derivatives of the embryonic brain region expressing Vax1 are widely interconnected and may be considered as a functional unit that appeared early in the evolution of vertebrates. Vax1 is thus a marker of a phylogenetically ancient embryological unit, which differentiates in a functionally central part of the forebrain (Hallonet, 1998 and references therein).

Vax genes and eye development

The mechanisms that establish the dorsal-ventral (DV) axis of the eye are poorly understood. Two homeobox genes have been isolated from mouse and chicken, mVax2 and cVax respectively, whose expression during early eye development is restricted to the ventral retina. Within the homeodomain, the deduced amino acid sequence is identical to that of another recently identified Emx-related gene, Vax1, which is expressed in the optic stalk and ventral forebrain during mouse and Xenopus development. Unlike mVax1, mVax2 is expressed exclusively in the ventral retina, both during embryonic development and in the adult. mVax2 maps to chromosome 6, linked to Cbl-ps1, D6Xrf91, Wnt7a, and Pang, a region syntenic to the chromosomal region harboring the EMX1 locus in humans. It is interesting to note that mVAX1 is found near the EMX2 locus, perhaps indicating that this family arose from a tandem duplication event. In chick, ectopic expression of either Vax leads to ventralization of the early retina, as assayed by expression of the transcription factors Pax2 and Tbx5, and the Eph family members EphB2, EphB3, ephrinB1, and ephrinB2, all of which are normally dorsally or ventrally restricted. Moreover, the projections of dorsal but not ventral ganglion cell axons onto the optic tectum show profound targeting errors following cVax misexpression. mVax2/cVax thus specify positional identity along the DV axis of the retina and influence retinotectal mapping (Schulte, 1999).

Vax2, a homolog of Drosophila empty spiracles, is a homeobox gene whose expression is confined to the ventral region of the prospective neural retina. Overexpression of this gene at early stages of development in Xenopus and in chicken embryos determines a ventralization of the retina, thus suggesting its role in the molecular pathway that underlies eye development. The generation and characterization of a mouse with a targeted null mutation of the Vax2 gene is described. Vax2 homozygous mutant mice display incomplete closure of the optic fissure that leads to eye coloboma. This phenotype is not fully penetrant, suggesting that additional factors contribute to its generation. Vax2 inactivation determines dorsalization of the expression of mid-late (Ephb2 and Efnb2) but not early (Pax2 and Tbx5) markers of dorsal-ventral polarity in the developing retina. Finally, Vax2 mutant mice exhibit abnormal projections of ventral retinal ganglion cells. In particular, almost complete absence of ipsilaterally projecting retinal ganglion cell axons in the optic chiasm and alteration of the retinocollicular projections is observed. All these findings indicate that Vax2 is required for the proper closure of the optic fissure, for the establishment of a physiological asymmetry on the dorsal-ventral axis of the eye and for the formation of appropriate retinocollicular connections (Barbieri, 2002).

The vertebrate retina is highly ordered along both its dorsoventral (DV) and nasotemporal (NT) axes, and this order is topographically maintained in its axonal connections to the superior colliculus of the midbrain. Although the graded axon guidance cues that mediate the topographic mapping of retinocollicular connections are increasingly well understood, the transcriptional regulators that set the DV and NT gradients of these cues are not. Genetic evidence is provided that Vax2, a homeodomain protein expressed in the ventral retina, is one such regulator. In Vax2 mutant mice, retinocollicular projections from the ventral temporal retina are dorsalized relative to wild type. Remarkably, however, this dorsalization becomes systematically less severe in progressively more nasal regions of the ventral retina. Vax2 mutants also exhibit flattened DV and NT gradients of the EphA5, EphB2, EphB3, ephrin-B1 and ephrin-B2 axon guidance cues. Together, these data identify Vax2 as a fundamental regulator of axial polarization in the mammalian retina (Mui, 2002).

During early formation of the eye, the optic vesicle becomes partitioned into a proximal domain that forms the optic nerve and a distal domain that forms the retina. In this study, the activities have been investigated of Nodal, Hedgehog (Hh) and Fgf signals and Vax family homeodomain proteins in this patterning event. Zebrafish vax1 and vax2 homologs of Drosophila ems are expressed in overlapping domains encompassing the ventral retina, optic stalks and preoptic area. Abrogation of Vax1 and Vax2 activity leads to a failure to close the choroid fissure and progressive expansion of retinal tissue into the optic nerve, finally resulting in a fusion of retinal neurons and pigment epithelium with forebrain tissue. Hh signals acting through Smoothened act downstream of the Nodal pathway to promote Vax gene expression. However, in the absence of both Nodal and Hh signals, Vax genes are expressed, revealing that other signals, which include Fgfs, contribute to Vax gene regulation. Pax2.1 and Vax1/Vax2 are likely to act in parallel downstream of Hh activity and the bel locus (yet to be cloned) mediates the ability of Hh-, and perhaps Fgf-, signals to induce Vax expression in the preoptic area. Taking all these results together, a model of the partitioning of the optic vesicle along its proximo-distal axis is presented (Take-uchi, 2003).

Loss of function studies in mouse and now in zebrafish have revealed requirements for Vax proteins in several different aspects of eye and forebrain midline development. The most conserved phenotype following abrogation of Vax activity is a failure in fusion of the choroid fissure. This phenotype is found in both mouse and fish lacking, or with reduced, Vax1 or Vax2 activity (with genetic background-dependent penetrance in mouse Vax2 mutants). The severity of this coloboma phenotype is increased in fish embryos compromised in both Vax1 and Vax2 activity, strongly suggesting that both Vax1 and Vax2 co-operate to regulate fusion of choroid fissure. The proteins that mediate fusion of the retina at the choroid fissure are unknown, but possible candidates include Eph receptors and their Ephrin ligands. Members of this family of signalling proteins have been implicated in fusion events in other epithelia, and several family members are expressed in the ventral retina. Indeed, in mouse, changes in ephrinB1, ephrinB2 and EphB2 expression occur in the ventral retina of vax2 mutants, and although these changes have primarily been considered in terms of retinal axon pathfinding, they could potentially contribute to the choroid fissure defects (Take-uchi, 2003),

A second conserved phenotype in Vax mutants is disruption to commissural axon guidance and targeting of retinal axons. In Vax1 mouse mutants, there is a severe disruption to midline development and consequently, severe disruption in the pathfinding of axons as they approach the midline. In vax2 mutants there are also variable defects in the development of ipsilateral and contralateral retinal projections. These may arise from incorrect assignment of identity to retinal neurons in the Vax2 mutants, but the possibility that midline tissue is also disrupted has not been excluded. Indeed, in fish, commissural axon pathfinding defects are much more obvious in Vax1/Vax2 double morphants than in either single morphant. This suggests, that at least in fish, Vax2 does co-operate with Vax1 to pattern midline tissue (Take-uchi, 2003),

The third conserved phenotype in animals compromised in Vax protein activity is a failure to limit retinal development to the optic cup. The initial indications of this phenotype came from analysis of mouse Vax1 mutants in which expression of genes normally restricted to retinal tissue (rx and pax6) was observed to encroach into the optic nerve. This study provides dramatic confirmation of the requirement for Vax protein activity to limit retinal development. In Vax1/Vax2 double morphants, there is a progressive expansion of retinal tissue along the optic nerve until by four days, neural and pigmented layers of the retina are in direct continuity with diencephalic cells of the optic chiasm. Early morphogenesis of the optic stalk and optic cup occurs relatively normally and indeed, retinal axons navigate out of the eye and along the stalk or nerve to the midline in the Vax1/Vax2 morphants. It is relatively late in development that differentiating retinal tissue expands to the midline. This phenotype probably reflects a change in the fate of optic stalk or nerve cells to retinal tissue. The possibility that overproliferation and evagination of retinal cells from the back of the eye may also contribute to the phenotype has not been ruled out (Take-uchi, 2003).

Although there are clear similarities in the phenotypes following abrogation of Vax function in mice and in fish, there are also differences. For instance, midline defects are more severe in vax1 mutant mice than in vax1/vax2 morphant fish. This of course could reflect true differences in Vax protein function but there are other possibilities. Perhaps the simplest would be that the vax1 MO does not remove all Vax1 function in fish. Although this possibility cannot be discounted, the severity of the retinal expansion phenotype in the Vax1/Vax2 morphants (which is much more severe than either vax1 or vax2 mutant mice) and the penetrance of the coloboma phenotype suggest that the MOs probably severely abrogate Vax function. A further possibility is the presence of other Vax genes in the fish genome. Indeed, it appears that expression of the known Vax genes is initiated a little later in fish than in other vertebrates, raising the question of whether another Vax gene, perhaps with early expression and a stronger role in midline development, might exist. The ongoing sequencing of the fish genome should soon allow a resolution of this issue (Take-uchi, 2003),

The vertebrate retina and optic nerve are strikingly different in terms of their size, organization, and cellular diversity, yet these two structures develop from the same embryonic neuroepithelium. Precursor cells in the most ventral domain of this epithelium give rise only to the astrocytes of the optic nerve, whereas immediately adjacent, more dorsal precursors give rise to the myriad cell types of the retina. Genetic evidence is provided that two closely related, ventrally expressed homeodomain proteins -- Vax1 and Vax2 -- control this neuroepithelial segregation. In the absence of both proteins, the optic nerve is transformed in its entirety into fully differentiated retina. This transformation results from the loss of ventralizing actvity in the developing eye field, and ventralization is mediated, at least in part, via Vax repression of the Pax6 gene, a potent inducer of retinal development (Mui, 2005).

Regulation of retinal axon growth by secreted Vax1 homeodomain protein

Retinal ganglion cell (RGC) axons of binocular animals cross the midline at the optic chiasm (OC) to grow toward their synaptic targets in the contralateral brain. Ventral anterior homeobox 1 (Vax1) plays an essential role in the development of the OC by regulating RGC axon growth in a non-cell autonomous manner. This study has identified an unexpected function of Vax1 that is secreted from ventral hypothalamic cells and diffuses to RGC axons, where it promotes axonal growth independent of its transcription factor activity. Vax1 binds to extracellular sugar groups of the heparan sulfate proteoglycans (HSPGs) located in RGC axons. Both Vax1 binding to HSPGs and subsequent penetration into the axoplasm, where Vax1 activates local protein synthesis, are required for RGC axonal growth. Together, these findings demonstrate that Vax1 possesses a novel RGC axon growth factor activity that is critical for the development of the mammalian binocular visual system (Kim, 2014).

Emergence of dorsal-ventral polarity in embryonic stem cell-derived retinal tissue

Mouse embryonic stem cell-derived retinal epithelium self-forms an optic cup-like structure. In the developing retina, the dorsal and ventral sides differ in terms of local gene expression and morphological features. This aspect has not yet been shown in vitro. This study demonstrates that embryonic stem cell-derived retinal tissue spontaneously acquires polarity reminiscent of the dorsal-ventral (D-V) patterning of the embryonic retina. Tbx5 (see Drosophila Omb) and Vax2 (see Drosophila Emx) were expressed in a mutually exclusive manner, as seen in vivo. Three-dimensional morphometric analysis showed that the in vitro-formed optic cup often contains cleft structures resembling the embryonic optic fissure. To elucidate the mechanisms underlying the spontaneous D-V polarization of embryonic stem cell-derived retina, the effects of the patterning factors were examined, and endogenous BMP signaling was found to play a predominant role in the dorsal specification. Further analysis revealed that canonical Wnt signaling, which was spontaneously activated at the proximal region, acts upstream of BMP signaling for dorsal specification. These observations suggest that D-V polarity could be established within the self-formed retinal neuroepithelium by intrinsic mechanisms involving the spatiotemporal regulation of canonical Wnt and BMP signals.

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empty spiracles: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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