Yun, K., et al. (2002). Modulation of the notch signaling by Mash1

Notch signaling has a central role in cell fate specification and differentiation. Evidence is provided that the Mash1 (bHLH) and Dlx1 and Dlx2 (homeobox) transcription factors have complementary roles in regulating Notch signaling, which in turn mediates the temporal control of subcortical telencephalic neurogenesis in mice. Progressively more mature subcortical progenitors (P1, P2 and P3) are defined through their combinatorial expression of MASH1 and DLX2, as well as the expression of proliferative and postmitotic cell markers at E10.5-E11.5. In the absence of Mash1, Notch signaling is greatly reduced and 'early' VZ progenitors (P1 and P2) precociously acquire SVZ progenitor (P3) properties. Comparing the molecular phenotypes of the delta-like 1 and Mash1 mutants, suggests that Mash1 regulates early neurogenesis through Notch-and Delta-dependent and -independent mechanisms. While Mash1 is required for early neurogenesis (E10.5), Dlx1 and Dlx2 are required to downregulate Notch signaling during specification and differentiation steps of 'late' progenitors (P3). Dlx1/2 function appears to be required to specify and differentiate P3 progenitors by repressing the genes that are normally expressed in VZ progenitor cells (e.g. Mash1, Gsh1/2, Lhx2, COUP-TF1) and by activating genes expressed in the SVZ (e.g. Dlx5, Dlx6 and SCIP/Oct6) and MZ (e.g. Drd2). It is suggested that alternate cell fate choices in the developing telencephalon are controlled by coordinated functions of bHLH and homeobox transcription factors through their differential affects on Notch signaling (Yun, 2002).

Dlx1/2 mutants exhibit increased levels of Hes5 expression, implying that differentiation may be blocked due to increased levels of Notch signaling. At E11.5 Dll1 (a Delta homolog) and Mash1 expression are elevated in the SVZ; these abnormalities become more severe at later stages. As MASH1 and DLX2 are co-expressed in some progenitors (P3), a potential mechanism underlying this phenotype would be that Dlx1 and Dlx2 repress Mash1 expression (directly or indirectly) as P3 cells mature. In Dlx1/2 mutants, failure to downregulate Mash1 expression would lead to elevated levels of Dll1 expression; this, in turn, would increase Notch signaling and Hes5 expression in adjacent cells (Yun, 2002).

The Pax6 gene plays a developmental role in various metazoans as the master regulatory gene for eye patterning. Pax6 is also spatially regulated in particular regions of the neural tube. Because the amphioxus has no neuromeres, an understanding of Pax6 expression in the agnathans is crucial for an insight into the origin of neuromerism in the vertebrates. A single cognate cDNA of the Pax6 gene, LjPax6, has been isolated from a Lampetra japonica cDNA library and the pattern of its expression has been observed using in situ hybridization. Phylogenetic analysis has revealed that LjPax6 occurs as an sister group of gnathostome Pax6. In lamprey embryos, LjPax6 is expressed in the eye, the nasohypophysial plate, the oral ectoderm and the brain. In the central nervous system, LjPax6 is expressed in clearly delineated domains in the hindbrain, midbrain and forebrain. The pattern of LjPax6 expression was compared with that of other brain-specific regulatory genes, including LjOtxA, LjPax2/5/8, LjDlx1/6, LjEmx and LjTTF1. Most of the gene expression domains show a conserved pattern, which reflects the situation in the gnathostomes, conforming partly to the neuromeric patterns proposed for the gnathostomes. It is concluded that most of the segmented domains of the vertebrate brain were already established in the ancestor common to all vertebrates. Major evolutionary changes in the vertebrate brain may have involved local restriction of cell lineages, leading to the establishment of neuromeres (Murakami, 2001).

The LjOtxA-expressing domain terminates rostrally at the presumptive P2/P3 boundary (zona limitans), which is assumed from the position of the epiphysis. Rostral to this, LjPax6 and LjDlx1/6 are co-expressed in the dorsal diencephalon, and no expression is seen in the ventral diencephalon (hypothalamus). The expression of LjTTF1 appears to be complementary to the latter (in the ventral diencephalon or hypothalamus), and no transverse segmental boundaries in the forebrain can be detected for the telencephalon. Furthermore, the boundary between the LjPax6- LjDlx1/6 co-expressing domain and the LjTTF1-expressing domain may correspond to the alar-basal plate boundary (Murakami, 2001).

The morphology of the telencephalon is also problematic. Based on the expression patterns of the regulatory genes, the gnathostome telencephalon is assumed to be composed of three major components: the pallium (medial, dorsal, and lateral pallium), the intermediate zone (ventral pallium) and the subpallium (striatum). Emx and Pax6 are expressed in the pallium, and Dlx in the subpallium. In the stage 26 lampery forebrain, a transverse (morphologically horizontal) furrow is found, designated as the sulcus intraencephalicus anterior. Characteristic gene expression is observed in the part of the brain that is rostral and dorsal to this sulcus. LjPax6 is expressed in the dorsal part and LjDlx1/6 in the ventral part, possibly corresponding to the pallium and striatum in the lamprey, respectively. Furthermore LjEmx is restricted to a small dorsal domain that expresses this gene plus LjPax6, and resembles the dorsal pallium of the gnathostomes. These patterns of gene expression in this part of the lamprey brain are extremely reminiscent of the gnathostome telencephalon. Although this may also imply the presence of the P3/P4 boundary (pallio-subpallial boundary), it could not be followed into the dorsal diencephalic and hypothalamic regions. Finally, there is a region in the gnathostome telencephalon that includes the pallidum, in which Dlx and TTF1 are both expressed. The loss of TTF1 expression in the ventral telencephalic region of the lamprey forebrain may be related to the apparent absence of a pallidum in this animal (Murakami, 2001).

In conclusion, the present study of the lamprey brain primordium suggests the presence of the P1 and P2 segments, a longitudinally extending sulcus limitans that terminates rostrally, close to the optic chiasm, a hypothalamus and a tripartite telencephalon-like domain. All these features are directly comparable with those in the model established in the mouse. These results have not further clarified the number of segments in the rostralmost part of the brain. The shared morphological patterns described above are assumed to be very old in origin, possibly dating to the divergence of the lampreys and the gnathostomes. Since recent analyses based on several molecules place hagfishes as the sister group of the lamprey, the segments listed above were already present in the common ancestor of all the vertebrates. The recent discovery of the earliest fossil vertebrates in the early Cambrian period (490-545 million years ago) suggests that the segmental plan underlying vertebrate brain development may have an even longer history. The absence of compartments and the presence of similar anteroposterior regulation by various regulatory genes in cephalochordates imply that the vertebrate-specific compartments listed above were acquired by rough regionalization of the neurectoderm already present in the cephalochordates. It may have been proliferation of neurectodermal cells, as well as the restriction of local cell lineages to form boundaries, that facilitated this most curious evolutionary transition (Murakami, 2001).

The ectoderm of the pre-gastrula Xenopus embryo is at least partially patterned along the dorsal-ventral axis. The early expression of the anti-neural homeodomain gene Dlx3 is localized to the ventral ectoderm by a mechanism that occurs prior to gastrulation and is independent of the Spemann organizer. The repression of Dlx3 is mediated by signaling through beta-catenin, but is probably not dependent on the induction of the Xnr3 or chordin genes by beta-catenin. It is proposed that the establishment of the dorso-ventral axis in Xenopus, which occurs during the first cell cycle and requires an enrichment of beta-catenin in prospective dorsal cells, leads to the repression of Dlx3 in the most dorsal ectoderm, prior to the formation of the Spemann organizer. This inhibition could be mediated by repression of BMP-4 expression, but could also be direct. Injection experiments in which beta-catenin and BMP-4 are co-injected indicate that the repression of Dlx3 by beta-catenin is at least partially dependent on inhibition of BMP-4 expression. Since Dlx3 is an inhibitor of neural gene expression, this repression could account for the propensity of dorsal ectoderm to respond to neural inducers, and the tendency of ventral ectoderm to express epidermal markers. This model is compatible with the observation that ventral ectoderm can be induced to become neural by organizer transplants, or equivalent procedures which would repress expression of Dlx3. This model also predicts that neural induction will take place more readily with dorsal versus ventral ectoderm, which is what has been observed (Beanan, 2000).

Homologies between vertebrate forebrain subdivisions are still uncertain. In particular the identification of homologs of the mammalian neocortex or the dorsal ventricular ridge (DVR) of birds and reptiles is still a matter of dispute. To get insight about the organization of the primordia of the main telencephalic subdivisions along the anteroposterior axis of the neural tube, a fate map of the dorsal prosencephalon was obtained in avian chimeras at the 8- to 9-somite stage. At this stage, the primordia of the pallium, DVR and striatum are located on the dorsal aspect of the prosencephalon and ordered caudorostrally along the longitudinal axis of the brain. Expression of homeobox-containing genes of the Emx, Dlx and Pax families were used as markers of anteroposterior developmental subdivisions of the forebrain in mouse, chick, turtle and frog. Their expression domains delineate three main telencephalic subdivisions in all species at the onset of neurogenesis: the dorsal pallium (expressing Emx-1), an intermediate zone and striatal neuroepithelial (expressing Dlx-1) domains. The pattern of Pax-6 expression in the ventricular zone is similar in both chick and mouse and provides a good marker of the intermediate telecephalic territory. The fate of the intermediate subdivisions diverge, however, between species at later stages of development. Homologies between forebrain subdivisions are proposed based on the conservation and divergence of these gene expression patterns (Fernandez, 1998).

Expression of the single amphioxus Distal-less homolog during development is consistent with successive roles of this gene in global regionalization of the ectoderm, establishment of the dorsoventral axis, specification of migratory epidermal cells early in neurulation and the specification of forebrain. In the amphioxus gastrula AmphiDll is expressed throughout the animal hemisphere (presumptive ectoderm), but is soon downregulated dorsally (in the presumptive neural plate). During early neurulation, AmphiDll-expressing epidermal cells flanking the neural plate extend lamellipodia, appear to migrate over it and meet mid-dorsally. Midway in neurulation, cells near the anterior end of the neural plate begin expressing AmphiDll and, as neurulation terminates, these cells are incorporated into the dorsal part of the neural tube, which forms by curling of the neural plate. This group of AmphiDll-expressing cells and a second group expressing the gene a little later is evidently homologous to the craniate forebrain (Holland, 1996)

Mice mutant for the gene Mash1 display severe neuronal losses in the olfactory epithelium and ganglia of the autonomic nervous system, demonstrating a role for Mash1 in development of neuronal lineages in the peripheral nervous system. Mash1 function in the central nervous system, has been analyzed, focusing on the ventral telencephalon where it is expressed at high levels during neurogenesis. Mash1 mutant mice present a severe loss of progenitors, particularly of neuronal precursors in the subventricular zone of the medial ganglionic eminence. Discrete neuronal populations of the basal ganglia and cerebral cortex are subsequently missing. An analysis of candidate effectors of Mash1 function reveals that the Notch ligands Dll1 and Dll3, and the target of Notch signaling Hes5, fail to be expressed in Mash1 mutant ventral telencephalon. In the lateral ganglionic eminence, loss of Notch signaling activity correlates with premature expression of a number of subventricular zone markers by ventricular zone (VZ) cells. In mutant embryos, GAD67 is ectopically expressed by Z cells of the ventral telencephalon at all developmental stages examined. Similarly, Dlx-5 is ectopically expressed in the VZ in mutant embryos. Dlx-1 transcripts are also found in virtually all cells of the mutant VZ, an expression normally observed only in the subventricular zone. Expression of Lhx2 expression is strongly reduced in the VZ of the mutant median ganglionic eminence, but remains at significant levels in the lateral ganglionic eminence. This result suggests that, although mutant VZ cells have prematurely acquired subventricular zone characteristics, they have only partially changed phenotype in the lateral ganglionic eminence, since they maintain expression of a ventricular zone marker. Therefore, Mash1 is an important regulator of neurogenesis in the ventral telencephalon, where it is required both to specify neuronal precursors and to control the timing of their production. Negative regulation of Dlx-1/2 by Mash1 is probably due to a process of lateral inhibition, rather than to a cell-intrinsic mechanism. Mice mutant for Dlx-1 and Dlx-2 present a striatal defect, interpreted as a block in differentiation specifically affecting late-born matrix neurons (Casarosa, 1999).

To determine the function of Dlx-2, a null mutation in mice was generated using gene targeting. In homozygous mutants, differentiation within the forebrain is abnormal and the fate of a subset of cranial neural crest cells is respecified. The latter causes abnormal morphogenesis of the skeletal elements derived from the proximal parts of the first and second branchial arches. The affected skull bones from the first arch seem to have undergone a transformation into structures similar to those found in reptiles. These results show that Dlx-2 controls development of the branchial arches and the forebrain and suggests its role in craniofacial evolution (Qui, 1995).

Although previous analyses indicate that neocortical neurons originate from the cortical proliferative zone, more recent evidence suggests that a subpopulation of neocortical interneurons originates within the subcortical telencephalon. For example, gamma-aminobutyric acid (GABA)-expressing cells migrate in vitro from the subcortical telencephalon into the neocortex. The number of GABA-expressing cells in neocortical slices is reduced by separating the neocortex from the subcortical telencephalon. Mice lacking the homeodomain proteins DLX-1 and DLX-2 show no detectable cell migration from the subcortical telencephalon to the neocortex and also have few GABA-expressing cells in the neocortex (Anderson, 1997b).

Considerable data suggest that sonic hedgehog (Shh) is both necessary and sufficient for the specification of ventral pattern throughout the nervous system, including the telencephalon. The regional markers induced by Shh in the E9.0 telencephalon are dependent on the dorsoventral and anteroposterior position of ectopic Shh expression. This suggests that by this point in development regional character in the telencephalon is established. To determine whether this prepattern is dependent on earlier Shh signaling, the telencephalon was examined in mice carrying either Shh- or Gli3-null mutant alleles. This analysis revealed that the expression of a subset of ventral telencephalic markers, including Dlx2 and Gsh2, although greatly diminished, persists in Shh^-/- mutants, and that these same markers are expanded in Gli3^-/- mutants. To understand further the genetic interaction between Shh and Gli3, Shh/Gli3 and Smoothened/Gli3 double homozygous mutants were examined. Notably, in animals carrying either of these genetic backgrounds, genes such as Gsh2 and Dlx2, which are expressed pan-ventrally, as well as Nkx2.1, which demarcates the ventral most aspect of the telencephalon, appear to be largely restored to their wild-type patterns of expression. These results suggest that normal patterning in the telencephalon depends on the ventral repression of Gli3 function by Shh and, conversely, on the dorsal repression of Shh signaling by Gli3. In addition, these results support the idea that, in addition to hedgehog signaling, a Shh-independent pathways must act during development to pattern the telencephalon (Rallu, 2002).

A novel homeobox gene (Arx) has been isolated that is expressed in the mouse central nervous system and which shows striking similarity to the Drosophila aristaless gene in the homeodomain (85% identity) and in a 17 amino acid-sequence near the carboxyl-terminus. The C-peptide domain is found in several homeoproteins belonging to the paired-like class. Arx is highly conserved between mouse and zebrafish. Neuromeric expression in the forebrain and longitudinal expression in the floor plate are observed in mouse and zebrafish. The expression of Arx in the ganglionic eminence and ventral thalamus overlaps regionally with that of Dlx1, but the cell layer where Arx is expressed differs from that of the Dlx1. This gene is also expressed in the dorsal telencephalon (presumptive cerebral cortex) of mouse embryos. The structure and expression pattern of Arx with respect to any possible relationship to al and Dlx1 is discussed as well as the function of Arx in the floor plate. It is unlikely that Arx regulates Sonic Hedgehog in the floor plate since Arx is expressed later than Shh. Undue emphasis should not be placed on colocalization of Arx and the Dlx gene family expression in the forebrain, since expression of Arx and Dlx1 is found to differ in other regions (Miura, 1997)

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

The expression patterns of four genes that are potential regulators of development were examined in the CNS of the embryonic day 12.5 mouse embryo. Three of the genes, Dlx-1, Dlx-2 (Tes-1), and Gbx-2, encode homeodomain-containing proteins, and one gene, Wnt-3, encodes a putative secreted differentiation factor. These genes are expressed in spatially restricted transverse and longitudinal domains in the embryonic neural tube, and are also differentially expressed within the wall of the neural tube. Dlx-1 and Dlx-2 are expressed in two separate regions of the forebrain in an identical pattern. The Gbx-2 gene is expressed in four domains, two of which share sharp boundaries with the domains of the Dlx genes. One boundary is in the basal telecephalon between deep and superficial strata of the medial ganglionic eminence; the other boundary is in the diencephalon at the zona limitans intrathalamica. The Wnt-3 gene is expressed in a dorsal longitudinal zone extending from the hindbrain into the diencephalon, where its expression terminates at the zona limitans intrathalamica. Reciprocal patterns of expression are found within the dorsal thalamus for the Gbx-2 and Wnt-3 genes. These findings are consistent with neuromeric theories of forebrain development (Bulfone, 1993).

Identification of the earliest forebrain-specific markers should facilitate the elucidation of molecular events underlying vertebrate forebrain determination and specification. The sequence and characterization of fez (forebrain embryonic zinc finger), a gene that is specifically expressed in the embryonic forebrain of zebrafish, is reported. Fez encodes a putative nuclear zinc finger protein that is highly conserved in Drosophila (CG7312), zebrafish, Xenopus, mouse, and human. In zebrafish, the expression of fez becomes detectable at the anterior edge of the presumptive neuroectoderm by 70% epiboly. During the segmentation period, its expression is completely restricted to the rostral region of the prospective forebrain. At approximately 24 h postfertilization, fez expression is mostly confined to the telencephalon and the anterior-ventral region of the diencephalon. Although fez expression is present in one-eyed pinhead (oep) and cyclops (cyc) zebrafish mutants, the pattern is altered. Forced expression of fez induces ectopic expression of dlx2 and dlx6, two genes involved in brain development. Knockdown of fez function using a morpholino-based antisense oligo inhibits dlx2 expression in the ventral forebrain. These studies indicate that fez is one of the earliest markers specific for the anterior neuroectoderm and it may play a role in forebrain development by regulating Dlx gene expression (Yang, 2001).

The striatum has a central role in many neurobiological processes, yet little is known about the molecular control of its development. Inroads to this subject have been made, due to the discovery of transcription factors, such as the Dlx genes, whose expression patterns suggest that they have a role in striatal development. Mice lacking both Dlx-1 and Dlx-2 have a time-dependent block in striatal differentiation. In these mutants, early born neurons migrate into a striatum-like region that is enriched for markers of the striosome (patch) compartment. However, later born neurons accumulate within the proliferative zone. Several lines of evidence suggest that mutations in Dlx-1 and Dlx-2 produce abnormalities in the development of the striatal subventricular zone and in the differentiation of striatal matrix neurons (Anderson, 1997a).

Patterning of the embryonic ectoderm is dependent upon the action of negative (antineural) and positive (neurogenic) transcriptional regulators. Msx1 and Dlx3 are two antineural genes for which the anterior epidermal-neural boundaries of expression differ, probably due to differential sensitivity to BMP signaling in the ectoderm. In the extreme anterior neural plate, Dlx3 is strongly expressed while Msx1 is silent. While both of these factors prevent the activation of genes specific to the nascent central nervous system, Msx1 inhibits anterior markers, including Otx2 and cement gland-specific genes. Dlx3 has little, if any, effect on these anterior neural plate genes, instead providing a permissive environment for their expression while repressing more panneural markers, including prepattern genes belonging to the Zic family and BF-1. Zic3 is activated by chordin and suppressed by BMP4; overexpression of this factor results in conversion of ectoderm to anterior neural tissue. The finding that Dlx3 is able to suppress the activation of Zic3 suggests that a Dlx3-mediated regulatory step might exist between the initial disruption of BMP signaling and activation of this gene. To test this hypothesis, truncated BMP-4 receptor, Dlx3 and Zic3 RNAs were injected in combinations, followed by animal cap excision, culture and RNA isolation for Northern blot analysis. Dlx3 blocks the activation of the panneural marker Nrp1 by truncated BMP-4 receptor. Addition of Zic3 RNA to the injection mixture restores Nrp1 expression to levels comparable to those of truncated BMP-4 induced caps. Based on these results, it is concluded that the inductive effects of Zic3 function downstream of the antineurogenic stem mediated by Dlx3. These properties define a molecular mechanism for translating the organizer-dependent morphogenic gradient of BMP activity into spatially restricted gene expression in the prospective anterior neural plate (Feledy, 1999).

The adult basal ganglia arise from the medial and lateral ganglionic eminences, morphologically distinct structures found in the embryonic telencephalon. Temporal changes in sonic hedgehog responsiveness determine the sequential induction of embryonic neurons that populate the medial and lateral ganglionic eminences. LGE neurons do not express Nkx2.1. Shh-mediated differentiation of neurons that populate the lateral ganglionic eminence express different combinations of the homeobox-containing transcription factors Dlx, Mash1 and Islet 1/2. Dlx-expressing neurons are found in both the LGE and MGE, in both proliferating and differentiating zones. However, the numbers of Dlx-expressing progenitors were consistently greater in the LGE than the MGE. In addition there are more neurons co-expressing Dlx and Mash1 in the ventricular zone of LGE than the MGE. Dlx and Islet 1/2 are co-expressed in more differentiated neurons in both the LGE and MGE. Mash1 marks progenitors, whereas Islet 1/2 marks more differentiated neurons. These genes are present in distinct neurons in both the LGE and MGE. Individual Mash1- and Dlx2-expressing neurons incorporate BrdU (Kohtz, 2001).

N-terminal fatty-acylation of Shh significantly enhances its ability to induce the differentiation of rat E11 telencephalic neurons expressing Dlx, Islet 1/2 or Mash1. In utero injection of the E9.5 mouse forebrain with retroviruses encoding wild-type Shh induces the ectopic expression of Dlx2 and severe deformities in the brain. Shh containing a mutation at the site of acylation prevents either of these phenotypes. These results suggest that N-terminal fatty-acylation of Shh may play an important role in Shh-dependent signaling during rodent ventral forebrain formation (Kohtz, 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).

The expression of the Dlx homeobox genes is closely associated with neurons that express gamma-aminobutyric acid (GABA) in the embryonic rostral forebrain. To test whether the Dlx genes are sufficient to induce some aspects of the phenotype of GABAergic neurons, the electroporation method was adapted to ectopically express DLX proteins in slice cultures of the mouse embryonic cerebral cortex. This approach shows that ectopic expression of Dlx2 and Dlx5 induces the expression of glutamic acid decarboxylases (GADs), the enzymes that synthesize GABA. This method was also used to show cross-regulation between different Dlx family members. Dlx2 can induce Dlx5 expression, and Dlx1, Dlx2 and Dlx5 can induce expression from a Dlx5/6-lacZ enhancer/reporter construct. The results of this study suggest both redundant and distinct functions for different members of the Dlx gene family. Dlx1 and Dlx2 are redundant for the control of late-born neurons of the basal telencephalon to efficiently migrate away from the subventricular zone and to express markers of more differentiated neurons. Neither single mutant shows this phenotype. However, Dlx1 and Dlx2 show different abilities to ectopically induce GAD expression, and to regulate the Dlx5/6 enhancer. Thus, perhaps both Type A (Dlx2 and Dlx5) and Type B (Dlx1 and Dlx6) Dlx genes have redundant functions in regulating aspects of differentiation related to migration, but Type A and Type B Dlx genes may have distinct functions with respect to cross-regulation of Dlx genes and GAD expression. The hypothesis that Type A and B Dlx genes have different functions in vivo, is consistent with the observation that Dlx1 and Dlx2 mutants have distinct maxillary dysmorphologies, despite their similar expression patterns in the first branchial arch (Stühmer, 2002)

The prevailing model to explain the formation of topographic projections in the nervous system stipulates that this process is governed by information located within the projecting and targeted structures. In mammals, different thalamic nuclei establish highly ordered projections with specific neocortical domains and the mechanisms controlling the initial topography of these projections remain to be characterized. To address this issue, Ebf1^-/- embryos were examined in which a subset of thalamic axons does not reach the neocortex. Ebf1 (also known as Olf-1, O/E-1, COE1) encodes a HLH transcription factor. The projections that do form between thalamic nuclei and neocortical domains have a shifted topography, in the absence of regionalization defects in the thalamus or neocortex. This shift is first detected inside the basal ganglia, a structure on the path of thalamic axons, and one that develops abnormally in Ebf1^-/- embryos. A similar shift in the topography of thalamocortical axons inside the basal ganglia and neocortex was observed in Dlx1/2^-/- embryos, which also have an abnormal basal ganglia development. Furthermore, Dlx1 and Dlx2 are not expressed in the dorsal thalamus or in cortical projection neurons. Thus, this study shows that: (1) different thalamic nuclei do not establish projections independently of each other; (2) a shift in thalamocortical topography can occur in the absence of major regionalization defects in the dorsal thalamus and neocortex, and (3) the basal ganglia may contain decision points for thalamic axons' pathfinding and topographic organization. These observations suggest that the topography of thalamocortical projections is not strictly determined by cues located within the neocortex and may be regulated by the relative positioning of thalamic axons inside the basal ganglia (Garel, 2002).

If defects in structures located on the path of thalamic axons can shift the topography of thalamocortical axons, a similar phenotype should be found in other mutant mice that have basal ganglia defects. Thus Dlx1/2 mutants, where differentiation of the basal ganglia and ventral thalamus is abnormal, were examined. Furthermore, Dlx1 and Dlx2 are not expressed in the dorsal thalamus or in cortical projection neurons. In Dlx1/2 mutants, the formation of the internal capsule is perturbed, probably because of a block in basal ganglia differentiation and, as in Ebf1 mutants, the topography of thalamocortical projections is shifted in the neocortex and internal capsule. Thus, the phenotype of Dlx1/2^-/- mice supports the possibility that affecting structures on the path of thalamic axons can shift the topography of thalamocortical projections. It could be argued that the reduction of cortical interneurons in Dlx1/2 mutants might contribute to this phenotype. However, this is unlikely because Nkx2.1 mutants, which also have a major deficit in neocortical interneurons, have normal thalamocortical projections. Thus, the combined study of Ebf1 and Dlx1/2 mutants shows that the topography of thalamocortical axons can be systematically shifted in the absence of apparent abnormalities in the neocortex and dorsal thalamus, and suggests that this shift is due to defects in structures located on the path of the axons (Garel, 2002).

The lateral border of the neural plate is a major source of signals that induce primary neurons, neural crest cells and cranial placodes as well as provide patterning cues to mesodermal structures such as somites and heart. Whereas secreted BMP, FGF and Wnt proteins influence the differentiation of neural and non-neural ectoderm, members of the Dlx family of transcription factors position the border between neural and non-neural ectoderm and are required for the specification of adjacent cell fates. Inhibition of endogenous Dlx activity in Xenopus embryos with an Engrailed repressor (EnR) fusion protein with Dlx homeodomain (EnR-Dlx) expands the neural plate into non-neural ectoderm tissue whereas ectopic activation of Dlx target genes inhibits neural plate differentiation. Importantly, the stereotypic pattern of border cell fates in the adjacent ectoderm is re-established only under conditions where the expanded neural plate abuts Dlx-positive non-neural ectoderm. Experiments in which presumptive neural plate was grafted to ventral ectoderm reiterate induction of neural crest and placodal lineages and also demonstrate that Dlx activity is required in non-neural ectoderm for the production of signals needed for induction of these cells. It is proposed that Dlx proteins regulate intercellular signaling across the interface between neural and non-neural ectoderm that is critical for inducing and patterning adjacent cell fates (Woda, 2003).

The neural crest and sensory placodes arise from a region of the embryonic ectoderm that lies between the neural plate and future epidermis. While some of the signalling pathways that are involved in cell fate determination at the border of the neural plate have been characterized, it is still unclear how different signals are integrated. Transcription factors of the DLX gene family that may mediate such cell fate decisions are expressed at the border of the neural plate. DLX5 is demonstrated to be involved in positioning this border by repressing neural properties and simultaneously by promoting the formation of border-like cells that express the neural fold markers MSX1 and BMP4, and the preplacodal region marker SIX4. However, DLX5 is not sufficient to impart epidermal character or to specify cell fates that arise at the border of the neural plate, like neural crest or fully formed sensory placodes, in a cell-autonomous manner. Additional signals are generated when mature neural plate and epidermis interact and these are required for neural crest formation. It is proposed that patterning of the embryonic ectoderm is a multistep process that sequentially subdivides the ectoderm into regions with defined cell fates (McLarren, 2003).

Cranial placodes, which give rise to sensory organs in the vertebrate head, are important embryonic structures whose development has not been well studied because of their transient nature and paucity of molecular markers. Markers of pre-placodal ectoderm (PPE) (six1, eya1) have been used to determine that gradients of both neural inducers and anteroposterior signals are necessary to induce and appropriately position the PPE. Overexpression of six1 expands the PPE at the expense of neural crest and epidermis, whereas knock-down of Six1 results in reduction of the PPE domain and expansion of the neural plate, neural crest and epidermis. Using expression of activator and repressor constructs of six1 or co-expression of wild-type six1 with activating or repressing co-factors (eya1 and groucho, respectively), it has been demonstrated that Six1 inhibits neural crest and epidermal genes via transcriptional repression and enhances PPE genes via transcriptional activation. Ectopic expression of neural plate, neural crest and epidermal genes in the PPE demonstrates that these factors mutually influence each other to establish the appropriate boundaries between these ectodermal domains (Brugmann, 2004).

Members of the Dlx gene family represent some of the earliest genes expressed at the border between the neural plate and epidermis. In chick, dlx5 is expressed at the neural/non-neural border, overlapping with eya2 and six4 in the pre-placodal thickening where it is proposed to create a border zone in which lateral neurogenic fates can be expressed. In Xenopus there is a low level of dlx5/6 expression along the border of the neural plate, but the most intense stripe is adjacent to six1 expression along the anterior neural ridge, and overlapping with the lateral edge of the crescent of six1 PPE expression. In chick, dlx5 overexpression results in a weak upregulation of six4 expression, whereas in Xenopus wild-type dlx5/dlx6 both strongly reduce six1 expression, and activator Dlx constructs cause a loss of six1 expression. It is not clear whether these differences are due to species differences in the precise patterning of the embryonic ectoderm, as has been proposed for neural induction, or due to the fact that Six1 and Six4 belong to different subclasses of the Six gene family (Brugmann, 2004).

Regardless, it is clear that frog six1 has two effects on dlx5/6 expression. Most prominently, overexpression of six1 in the LNE pushes the dlx5/6 stripe laterally away from the neural plate midline. This phenotype is probably due to six1 causing an expansion of the pre-placodal ectoderm (eya1, sox11) and reduction of epidermis (keratin), resulting in the formation of a new border between the expanded LNE and the epidermis. This interpretation is consistent with the effects of six1 overexpression on keratin, and further suggests that the effect is not due to movement of the neural plate border because the sox2/3 domains do not change. Likewise, dlx5/6 negatively regulate six1 expression. A mutual regulation takes place between Dlx genes and six1: inhibition of endogenous Dlx activity relocates the six1 expression domain more laterally, whereas activation relocated it more medially. The second effect of six1 is complete repression of dlx5/6 expression in those cells expressing six1. This may result from Six1 either repressing dlx5/6 gene expression or causing changes in gene expression in the affected cells that secondarily create an environment that is not compatible with dlx5/6 expression. Interestingly, foxD3 overexpression has similar effects on dlx5/6, which could be direct, or, unlike six1, could be due to the expansion of sox2/3. Paradoxically similar dlx5/6 phenotypes occur by activator and repressor six1 construct expression and six1-MO injections. It is predicted that these can be explained by six1 effects on foxD3. The injection of six1VP16, six1-WT+eya-WT and six1-MO may indirectly reduce dlx5/6 by expansion of foxD3, whereas six1-WT alone and six1EnR constructs may directly reduce dlx5/6. These results support the proposal that dlx5/6 contribute to forming the LNE border zone, and additionally demonstrate that they do so by participating in a complex interplay with several genes expressed in adjacent domains. It will be important to determine the precise molecular interactions between these various gene pathways to fully understand their roles in specifying LNE fates (Brugmann, 2004).

Rohon-Beard sensory neurons, neural crest cells, and sensory placodes can be distinguished at the boundary of the embryonic epidermis (skin) and the neural plate. The inductive signals at the neural plate border region are likely to involve a gradient of bone morphogenic protein (BMP) in conjunction with FGF and Wnts and other signals. However, how these signals are transduced to produce the final cell fate remains to be determined. Recent evidence from Xenopus and chick suggest that Dlx genes are required for the generation of cell fates at the neural plate border. In the present study, these findings are extended to zebrafish, where dlx3b and dlx4b function in a dose-dependent manner to specify cell fates such as Rohon-Beard sensory neurons and trigeminal sensory placodes. dlx function was examined by inhibiting both protein levels [with antisense morpholino oligonucleotides (MOs)], and activity, by repressing the ability of dlx-homeodomain to bind to downstream targets (EnR-dlx3bhd mRNA; dlx3b homeodomain fused to Engrailed transcriptional repressor domain). Inhibition of dlx3b and dlx4b protein and activity results in the reduction or complete loss of Rohon-Beard (RB) sensory neurons and trigeminal (TG) sensory placodes. These data suggest that dlx3b and dlx4b function in the specification of RB neurons and trigeminal sensory placodes in zebrafish. Further, dlx3b and dlx4b function in a non-cell-autonomous manner for RB neuron development; dlx3b and dlx4b act to regulate bmp2b expression at the non-neural ectodermal border. These data suggest that the contribution of dlx3b and dlx4b to neural plate border formation is partially non-cell-autonomous acting via BMP activity (Kaji, 2004).

In the mouse telencephalon, Dlx homeobox transcription factors are essential for the tangential migration of subpallial-derived GABAergic interneurons to neocortex. However, the mechanisms underlying this process are poorly understood. This study demonstrates that Dlx1/2 has a central role in restraining neurite growth of subpallial-derived immature interneurons at a stage when they migrate tangentially to cortex. In Dlx1^−/−;Dlx2^−/− mutants, neurite length is increased and cells fail to migrate. In Dlx1^−/−;Dlx2^+/− mutants, while the tangential migration of immature interneurons appears normal, they develop dendritic and axonal processes with increased length and decreased branching, and have deficits in their neocortical laminar positions. Thus, Dlx1/2 is required for coordinating programs of neurite maturation and migration. In this regard, genetic evidence is provided that in immature interneurons Dlx1/2 repression of the p21-activated serine/threonine kinase PAK3, a downstream effector of the Rho family of GTPases, is critical in restraining neurite growth and promoting tangential migration (Cobos, 2007).

The Dlx genes are expressed in a coordinate manner, establishing proximal-distal polarity within the pharyngeal arches. In zebrafish, dlx2a is expressed in the migrating cranial neural crest that contributes to the pharyngeal arches. Expression of dlx2a in the arches is subsequently followed by overlapping expression of the physically linked dlx1a gene, and of other paralogues that include dlx5a/dlx6a and dlx3b/dlx4b. To investigate the patterning and establishment of arch proximodistal polarity in zebrafish, the function of dlx2a and dlx1a, were haracterized using antisense morpholino oligonucleotides (MOs). Embryos injected with dlx1a and dlx2a MOs exhibit reduced and dysmorphic arch cartilage elements. The combined loss of dlx1a and dlx2a causes severe arch cartilage dysmorphology, revealing a role for these genes in maturation and patterning of arch chondrogenesis. Knockdown of dlx2a affects migrating neural crest cells as evidenced by reduced expression of crestin, and sox9a transcripts, in addition to increased levels of apoptosis. During pharyngogenesis, loss of dlx2a results in aberrant barx1 expression and the absence of goosecoid transcripts in the dorsal region of the ceratohyal arch. Defects in the differentiation of ectomesenchymal derivatives, including sensory ganglia and cartilage elements, indicate a role for dlx2a in specification and maintenance of cranial neural crest (Sperber, 2008).