intermediate neuroblasts defective

EVOLUTIONARY HOMOLOGS

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

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

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

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

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

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

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

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

Six posterior neural domain genes were examined: gastrulation brain homeobox (gbx), hox1, 3, 4, 7/8, and 11/13. All of these genes are expressed in chordate neurectoderm in major domains entirely within the hindbrain and spinal cord regions of the nervous system. gbx was chosen in chordates because it has a role in forming the midbrain-hindbrain boundary and in establishing the site of the isthmic organizer (in vertebrates) by way of a mutual antagonism of gbx and otx expression. Its domain in chordates extends from the midbrain-hindbrain boundary back into the spinal cord. In Drosophila, it may serve an analogous function, delineating a neural boundary and antagonizing otx expression. This does not necessarily imply a structural homology between central nervous systems but, merely, a homologous use in anteroposterior patterning. In S. kowalevskii, the gbx ortholog is initially expressed in the entire metasome except for the ciliated telotroch region. Later, the anterior metasome becomes the site of strong expression, and posterior expression diminishes. An additional domain of gbx expression is detected only in early stages in the endoderm, with its anterior limit extending into the mesosome, beyond the anterior limit of the ectodermal domain. The ectodermal domain of gbx overlaps anteriorly with both the en and otx domains, whereas in chordates gbx overlaps en partially, but not otx (which it antagonizes). irx expression overlaps gbx, en, and otx expression in both chordates and hemichordates. Thus, the contiguity of ectodermal domains of gbx, en, otx, and irx resembles that in chordates, though with some differences of overlap (Lowe, 2003).

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

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

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

Gsh-2 is a novel murine dispersed homeobox gene. Analysis of its cDNA sequence, including the full open reading frame, reveals an encoded homeodomain that is surprisingly similar to the homeodomains of the Antennapedia-type clustered Hox genes. In addition, the encoded protein includes polyhistidine and polyalanine tracts, as observed for several other genes of developmental significance. In situ hybridizations show Gsh-2 expression in the developing central nervous system, including the ganglionic eminences of the forebrain, the diencephalon, which gives rise to the thalamus and hypothalamus, and in the hindbrain. A random oligonucleotide selection and PCR amplification procedure was used to define a target DNA binding sequence, CNAATTAG, as a first step toward the identification of downstream target genes (Hsieh-Li, 1995).

Gsh-1, a novel murine homeobox gene, produces a transcript of approximately 2 kb present during development at embryonic day 10.5, 11.5, and 12.5. The cDNA sequence encodes a proline rich motif, a polyalanine tract, and a homeodomain with strong homology to those homeodomains encoded by the clustered Hox genes. The Gsh-1 expression pattern has been determined for days E8.5 to E13.5 by whole mount and serial section in situ hybridizations. Gsh-1 transcription is restricted to the central nervous system. Expression is present in the neural tube and hindbrain as two continuous, bilaterally symmetrical stripes within neural epithelial tissue. In the mesencephalon, expression is seen as a band across the most anterior portion. There is also diencephalon expression in the anlagen of the thalamus and the hypothalamus as well as in the optic stalk, optic recess, and the ganglionic eminence. Through the use of fusion proteins containing the Gsh-1 homeodomain, the consensus DNA binding site of the Gsh-1 homeoprotein has been determined to be GCT/CA/CATTAG/A (Valerius, 1995).

The anterior pituitary regulates the function of multiple organ systems as well as body growth; in turn, it is controlled by peptides released by the hypothalamus. Mutation of the Gsh-1 homeobox gene results in pleiotropic effects on pituitary development and function. Homozygous mutants exhibit extreme dwarfism, sexual infantilism and significant perinatal mortality. The mutant pituitary is small in size and hypocellular, with severely reduced numbers of growth hormone- and prolactin-producing cells. Moreover, the pituitary content of a subset of pituitary hormones, including growth hormone, prolactin and luteinizing hormone, is significantly decreased. The hypothalamus, although morphologically normal, is also perturbed in mutants. The gsh-1 gene is essential for growth hormone-releasing hormone (GHRH) gene expression in the arcuate nucleus of the hypothalamus. Further, sequence and electrophoretic mobility shift data suggest that the Gsh-1 and GHRH genes are potential targets regulated by the Gsh-1-encoded protein. The mutant phenotype indicates a critical role for Gsh-1 in the genetic hierarchy of the formation and function of the hypothalamic-pituitary axis (Li, 1996).

The Gsh-2 nonclustered homeobox gene is expressed within the developing forebrain, midbrain, and hindbrain. Gsh-2 transcripts are shown to be particularly abundant in the hindbrain and within the developing ganglionic eminences of the forebrain. Homozygous Gsh-2 mutant mice uniformly fail to survive more than 1 day following birth. At the physiologic level the mutants experienced apnea and reduced levels of hemoglobin oxygenation. Histologically, the mutant brains have striking alterations of discrete components. In the forebrain, the lateral ganglionic eminence is reduced in size. In the hindbrain, the area postrema, an important cardiorespiratory chemosensory center, is absent. The contiguous nucleus tractus solitarius, involved in integrating sensory input to maintain homeostasis, is also severely malformed in mutants. Immunohistochemistry was used to examine the mutant brains for alterations in the distribution of markers specific for serotonergic and cholinergic neurons. In addition, in situ hybridizations were used to define expression patterns of the Dlx 2 and Nkx 2.1 homeobox genes in Gsh-2 mutant mice. The mutant lateral ganglionic eminences show an abnormal absence of Dlx 2 expression. These results better define the genetic program of development of the mammalian brain, support neuromeric models of brain development, and further suggest similar patterning function for homeobox genes in phylogenetically diverse organisms (Szucsik, 1997).

Screening of a medaka (Oryzias latipes) adult brain cDNA library, with a degenerated probe corresponding to the most conserved region of helix III of the homeodomain, led to the isolation of a gene homologous to a murine orphan Hox gene, named Gsh-1. This gene has been termed Ol-Gsh 1 (Oryzias latipes-Gsh 1). Molecular analysis of the Ol-Gsh 1 putative protein points to potential functional domains that are highly conserved between fish and mouse genes. Whole-mount in situ hybridization shows that Ol-Gsh 1 is expressed in several waves during embryonic development. Transcripts are found in many regions of the central nervous system: the spinal cord, dorsal rhombencephalon, optic tectum, dorsal diencephalon, hypothalamus anlagen and rostral telencephalon. This multimodal expression pattern, strikingly conserved between fish and mammals, is reminiscent of both clustered and orphan homeobox genes. In addition, each expression wave is initiated in the fish embryo earlier than in the mammalian embryo, relative to the time scale defined by somitogenesis. It is proposed that Ol-Gsh 1 may be involved in conserved developmental pathways and in particular may be linked to proliferation events. Mouse Gsh-1 has been shown to participate in neuro-endocrine functions of the hypothalamus. From late developmental stages onward, Ol-Gsh 1 expression is also restricted to the hypothalamus. The expression pattern in this structure raises interesting questions concerning a fully or partially conserved function for these genes (Deschet, 1998).

Described here is the successful application of a strategy that potentially provides for an efficient and universal screen for downstream gene targets. The promoter of the Gsh-1 homeobox gene was used to drive expression of the SV40 T-antigen gene in transgenic mice. The Gsh-1 homeobox gene is expressed in discrete domains of the ganglionic eminences, diencephalon, and hindbrain during brain development. Gsh-1-SV40 T transgenic mice show cellular hyperplasia in regions of the brain coincident with Gsh-1 expression. The Gsh-1-SV40 T transgene was introduced, by breeding, into Gsh-1 homozygous mutant mice, and Gsh-1 -/- cell lines were made. Clonal cell lines were generated and analyzed by Northern blot hybridizations and Affymetrix GeneChip probe arrays to determine gene expression profiles. The results indicate that the cell lines remain representative of early developmental stages. Further, immunocytochemistry shows uniformly high levels of nestin expression, typical of central nervous system progenitor cells, and the absence of terminal differentiation markers of neuronal cells. One clonal cell line, No. 14, was then stably transfected with a tet-inducible Gsh-1 expression construct and subcloned. The starting clone 14, together with the uninduced and induced subclones, provided cell populations with varying levels of Gsh-1 expression. Differential display and Affymetrix GeneChip probe arrays were then used to identify transcript differences that represent candidate Gsh-1 target genes. Of particular interest, the drm and gas1 genes, which repress cell proliferation, were observed to be activated in Gsh-1-expressing cells. These observations support models predicting that homeobox genes function in the regulation of cell proliferation (Li, 1999).

Inactivation of Gbx2 produces Hox-like phenotypes in the axial skeleton

Expression of Hoxa10 in the presomitic mesoderm is sufficient to confer a Hox group 10 patterning program to the somite, producing vertebrae without ribs, an effect not achieved when Hoxa10 is expressed in the somites. In addition, Hox group 11-dependent vertebral sacralization requires Hoxa11 expression in the presomitic mesoderm, while their caudal differentiation requires that Hoxa11 is expressed in the somites. Therefore, Hox gene patterning activity is different in the somites and presomitic mesoderm, the latter being very prominent for Hox gene-mediated patterning of the axial skeleton. This is further supported by the finding that inactivation of Gbx2, a homeobox-containing gene expressed in the presomitic mesoderm but not in the somites, produces Hox-like phenotypes in the axial skeleton without affecting Hox gene expression (Carapuco, 2005).

Gsh2 is required for the repression of Ngn1 and specification of dorsal interneuron fate in the spinal cord

The molecular programs that specify progenitors in the dorsal spinal cord remain poorly defined. The homeodomain transcription factor Gsh2 is expressed in the progenitors of three dorsal interneuron subtypes, dI3, dI4 and dI5 neurons, whereas Gsh1 is expressed only in dI4 and dI5 progenitors. Mice lacking Gsh2 exhibit a selective loss of dI3 interneurons that is accompanied by an expansion of the dI2 progenitor domain. In Gsh2 mutant embryos, expression of the proneural bHLH protein Mash1 is downregulated in dI3 neural progenitors, with Mash1 mutants exhibiting a concordant reduction in dI3 neurons. Conversely, overexpression of Gsh2 and Mash1 leads to the ectopic production of dI3 neurons and a concomitant repression of Ngn1 expression. These results provide evidence that genetic interactions involving repression of Ngn1 by Gsh2 promote the differentiation of dI3 neurons from class A progenitors (Kirks, 2005).

Gbx2 and the development of the midbrain

The anterior neural ridge (ANR), and the isthmic organizer (IsO) represent two signaling centers possessing organizing properties necessary for forebrain (ANR) as well as midbrain and rostral hindbrain (IsO) development. An important mediator of ANR and IsO organizing property is the signaling molecule FGF8. Previous work has indicated that correct positioning of the IsO and Fgf8 expression in this domain is controlled by the transcription factors Otx2 and Gbx2. In order to provide novel insights into the roles of Otx2 and Gbx2, mutant embryos carrying different dosages of Otx2, Otx1 and Gbx2 were studied. Embryos deficient for both OTX2 and GBX2 proteins (hOtx1²/hOtx1²; Gbx2^-/-) show abnormal patterning of the anterior neural tissue, that is evident at the presomite-early somite stage prior to the onset of Fgf8 neuroectodermal expression. Indeed, hOtx1²/hOtx1²; Gbx2^-/- embryos exhibit broad co-expression of early forebrain, midbrain and rostral hindbrain markers such as hOtx1, Gbx2, Pax2, En1 and Wnt1 and subsequently fail to activate forebrain and midbrain-specific gene expression. In this genetic context, Fgf8 is expressed throughout the entire anterior neural plate, thus indicating that its activation is independent of both OTX2 and GBX2 function. Analysis of hOtx1²/hOtx1²; Gbx2^-/- and Otx1^+/-; Otx2^+/- mutant embryos also suggests that FGF8 cannot repress Otx2 without the participation of GBX2. Embryos carrying a single strong hypomorphic Otx2 allele (Otx2^lambda) in an Otx2 and Gbx2 null background (Otx2^lambda/-; Gbx2^-/-) recover both the headless phenotype exhibited by Otx2^lambda/- embryos and forebrain- and midbrain-specific gene expression that is not observed in hOtx1²/hOtx1²; Gbx2^-/- mutants. Together, these data provide novel genetic evidence indicating that OTX2 and GBX2 are required for proper segregation of early regional identities anterior and posterior to the mid-hindbrain boundary (MHB) and for conferring competence to the anterior neuroectoderm in responding to forebrain-, midbrain- and rostral hindbrain-inducing activities (Martinez-Barbera, 2001).

The isthmic organizer, which patterns the anterior hindbrain and midbrain, is one of the most studied secondary organizers. In recent years, new insights have been reported on the molecular nature of its morphogenetic activity. Studies in chick, mouse and zebrafish have converged to show that mutually repressive interactions between the homeoproteins encoded by Otx and Gbx genes position this organizer in the neural primordia. Evidence is presented that equivalent (in addition to novel) interactions between these and other genes operate in Xenopus embryos to position the isthmic organizer. Use was made of fusion proteins in which Otx2 or Gbx2 homeodomains were combined with the E1A activation domain or the EnR repressor element; these were then injected into embryos. Otx2 and Gbx2 are likely to be transcriptional repressors, and these two proteins repress each other's transcription. The interaction between these two proteins is required for the positioning of the isthmic organizer genes Fgf8, Pax2 and En2. A novel in vitro assay has been developed for the study of the formation of this organizer. Conjugating animal caps previously injected with Otx2 and Gbx2 mRNAs recreates the interactions required for the induction of the isthmic organizer. This assay was used to determine which cells produce and which cells receive the Fgf signal. A novel genetic element, Xiro1, which encode another homeoprotein, was added to this process. Xiro1 expression domain overlaps with territories expressing Otx2, Gbx2 and Fgf8. By expressing wild-type or dominant negative forms of Xiro1, this gene is shown to activate the expression of Gbx2 in the hindbrain. In addition, Xiro1 is required in the Otx2 territory to allow cells within this region to respond to the signals produced by adjacent Gbx2 cells. Moreover, Xiro1 is absolutely required for Fgf8 expression at the isthmic organizer. A model is discussed where Xiro1 plays different roles in regulating the genetic cascade of interactions between Otx2 and Gbx2 that are necessary for the specification of the isthmic organizer (Glavic, 2002).

Development and differentiation of the vertebrate caudal midbrain and anterior hindbrain are dependent on the isthmic organizer signals at the midbrain/hindbrain boundary (MHB). The future MHB forms at the boundary between the Otx2 and Gbx2 expression domains. Recent studies in mice and chick have suggested that the apposition of Otx2- and Gbx2-expressing cells is instrumental for the positioning and early induction of the MHB genetic cascade. Otx2 and Gbx2 perform different roles in this process. Ectopically expressed Otx2 on its own can induce a substantial part of the MHB genetic network, namely En2, Wnt1, Pax-2, Fgf8 and Gbx2, in a concentration-dependent manner. This induction does not require protein synthesis and ends during neurulation. In contrast, Gbx2 is a negative regulator of Otx2 and the MHB genes. Based on the temporal patterns of expression of the genes involved, it is proposed that Otx2 might be the early inducer of the isthmic organizer genetic network while Gbx2 restricts Otx2 expression along the anterior-posterior axis and establishes an Otx2 gradient (Tour, 2002a).

Anterior-posterior patterning of the embryo requires the activity of multiple homeobox genes, among them Hox, caudal (Cdx, Xcad) and Otx2. During early gastrulation, Otx2 and Xcad2 establish a cross-regulatory network, which is an early event in the anterior-posterior patterning of the embryo. As gastrulation proceeds and the embryo elongates, a new domain forms, which expresses neither Otx2 nor Xcad2 genes. Early transcription of the Xenopus Gbx2 homolog, Xgbx2a, is spatially restricted between Otx2 and Xcad2. When overexpressed, Otx2 and Xcad2 repress Xgbx2a transcription, suggesting their role in setting the early Xgbx2a expression domain. Homeobox genes have been shown to play crucial roles in the specification of the vertebrate brain. The border between the transcription domains of Otx2 and Gbx2 is the earliest known marker of the region where the midbrain/hindbrain boundary (MHB) organizer will develop. Xgbx2a is a negative regulator of Otx2 and a weak positive regulator of Xcad2. Using obligatory activator and repressor versions of Xgbx2a, it has been demonstrated that during early embryogenesis, Xgbx2a acts as a transcriptional repressor. In addition, taking advantage of hormone-inducible versions of Xgbx2a and its antimorph, it has been shown that the ability of Xgbx2a to induce head malformations is restricted to gastrula stages and correlates with its ability to repress Otx2 during the same developmental stages. It is therefore suggested that the earliest known step of the MHB formation, the establishment of Otx2/Gbx2 boundary, takes place via mutual inhibitory interactions between these two genes and this process begins as early as midgastrulation (Tour, 2002b).

The organizer at the midbrain-hindbrain boundary (MHB) forms at the interface between Otx2 and Gbx2 expressing cell populations, but how these gene expression domains are set up and integrated with the remaining machinery controlling MHB development is unclear. The isolation, mapping, chromosomal synteny and spatiotemporal expression of gbx1 and gbx2 in zebrafish is reported. Focus was placed on the expression of these genes during development of the midbrain-hindbrain territory. The results suggest that these genes function in this area in a complex fashion, as evidenced by their highly dynamic expression patterns and relation to Fgf signaling. Analysis of gbx1 and gbx2 expression during formation of the MHB in mutant embryos for pax2.1, fgf8 and pou2 (noi, ace, spg), as well as Fgf-inhibition experiments, show that gbx1 acts upstream of these genes in MHB development. In contrast, gbx2 activation requires ace (fgf8) function, and in the hindbrain primordium, also spg (pou2). It is proposed that in zebrafish, gbx genes act repeatedly in MHB development, with gbx1 acting during the positioning period of the MHB at gastrula stages, and gbx2 functioning after initial formation of the MHB, from late gastrulation stages onwards. Transplantation studies furthermore reveal that at the gastrula stage, Fgf8 signals from the hindbrain primordium into the underlying mesendoderm. Apart from the general involvement of gbx genes in MHB development reported also in other vertebrates, these results emphasize that early MHB development can be divided into multiple steps with different genetic requirements with respect to gbx gene function and Fgf signaling. Moreover, these results provide an example for switching of a specific gene function of gbx1 versus gbx2 between orthologous genes in zebrafish and mammals (Rhinn, 2003).

The cerebellum develops from the rhombic lip of the rostral hindbrain and is organized by fibroblast growth factor 8 (FGF8) expressed by the isthmus. Irx2, a member of the Iroquois (Iro) and Irx class of homeobox genes is expressed in the presumptive cerebellum. When Irx2 is misexpressed with Fgf8a in the chick midbrain, the midbrain develops into cerebellum in conjunction with repression of Otx2 and induction of Gbx2. During this event, signaling by the FGF8 and mitogen-activated protein (MAP) kinase cascade modulates the activity of Irx2 by phosphorylation. These data identify a link between the isthmic organizer and Irx2, thereby shedding light on the roles of Iro and Irx genes, which are conserved in both vertebrates and invertebrates (Matsumoto, 2004).

Gsh2 and the telencephalon

The genetic mechanisms that regulate dorsal-ventral identity in the embryonic mouse telencephalon and, in particular, the specification of progenitors in the cerebral cortex and striatum have been examined. The respective roles of Pax6 and Gsh2 in cortical and striatal development were studied in single and double loss-of-function mouse mutants. Gsh2 gene function is essential to maintain the molecular identity of early striatal progenitors and in its absence the ventral telencephalic regulatory genes Mash1 and Dlx are lost from most of the striatal germinal zone. In their place, the dorsal regulators Pax6, neurogenin 1 and neurogenin 2 are found ectopically. Conversely, Pax6 is required to maintain the correct molecular identity of cortical progenitors. In its absence, neurogenins are lost from the cortical germinal zone and Gsh2, Mash1 and Dlx genes are found ectopically. These reciprocal alterations in cortical and striatal progenitor specification lead to the abnormal development of the cortex and striatum observed in Pax6 (small eye) and Gsh2 mutants, respectively. In support of this, double homozygous mutants for Pax6 and Gsh2 exhibit significant improvements in both cortical and striatal development compared with their respective single mutants. Taken together, these results demonstrate that Pax6 and Gsh2 govern cortical and striatal development by regulating genetically opposing programs that control the expression of each other as well as the regionally expressed developmental regulators Mash1, the neurogenins and Dlx genes in telencephalic progenitors (Toresson, 2000).

The Gsh1/2 homolog, intermediate neuroblasts defective (ind), is expressed in selected areas of the fly CNS including the intermediate region of the ventral nerve cord. ind mutants show a reduction in the number of neuroblasts in this region of the ventral nerve cord. The remaining neuroblasts, by all markers tested, appear to have acquired a dorsal fate. This phenotype is reminiscent of the mouse Gsh2 mutant phenotype where the progenitors in the LGE (i.e. the intermediate region of the telencephalon) have lost the expression of certain ventral genes and instead express the dorsal genes Pax6, neurogenin 1 and neurogenin 2. These findings therefore indicate that at least some aspects of Gsh/ind function in dorsal-ventral specification of the developing nervous system have been conserved throughout evolution (Toresson, 2000).

The telencephalon and diencephalon, which comprise the vertebrate forebrain, arise from the anterior-most region of the neuraxis. The telencephalon is subdivided into the pallial and subpallial domains. The pallium gives rise to dorsal structures, including the cerebral cortex, while the subpallium gives rise to ventral structures, including the globus pallidus and the striatum, which in combination form the majority of the basal ganglia. The basal ganglia arise from two major protrusions in the wall of the ventral telencephalon known as the medial ganglionic eminence (MGE) and the lateral ganglionic eminence (LGE), which primarily gives rise to the globus pallidus and the striatum, respectively. The appearance of these eminences occurs sequentially during development, with the more-medial MGE arising subsequent to neural-tube closure and the more lateral LGE arising shortly thereafter. The MGE and LGE are additionally hypothesized to be the source of a majority of the interneurons found in the olfactory bulb and the cerebral cortex (Corbin, 2000 and references therein).

Homeobox genes are important for the proper patterning of the mammalian telencephalon. One of these genes is Gsh2, whose expression in the forebrain is restricted to the ventral domain. Expression of Gsh2 is restricted in the mouse forebrain to the ventral domain. Gsh2 is first detected between E9 and E10 in the forebrain and is expressed in the later developing ventral thalamus, hypothalamus, MGE, LGE and caudal ganglionic eminence (CGE). To better understand the localization of Gsh2, its expression was assayed at a variety of stages of telencephalic development. At E9.5, Gsh2 mRNA expression is found the ventrolateral telencephalon, a region from where the LGE will putatively emerge. Interestingly, at E9.5, although a few GSH2-expressing cells appear to intermingle with those expressing NKX2.1, GSH2 and NKX2.1 are predominantly expressed in separate domains. By E10.5, coincident with the emergence of the MGE, GSH2 expression extends into the MGE and overlaps with NKX2.1 expression. By E12.5, Gsh2 is expressed in the MGE, LGE and septum. At E12.5, Gsh2 is also expressed in the CGE and the ventral diencephalon. Expression of Gsh2 mRNA is restricted to the VZ and is not expressed in the SVZ or differentiated structures of the developing ventral telencephalon (Corbin, 2000).

Gsh2 is a downstream target of sonic hedgehog and lack of Gsh2 results in profound defects in telencephalic development. Gsh2 mutants have a significant decrease in the expression of numerous genes that mark early development of the lateral ganglionic eminence, the striatal anlage. Accompanying this early loss of patterning genes is an initial expansion of dorsal telencephalic markers across the cortical-striatal boundary into the lateral ganglionic eminence. The homeodomain transcription factors Dlx1 and Dlx2 are expressed in the VZ and SVZ of multiple ventral forebrain structures, including the MGE, LGE, CGE and ventral diencephalon. At E12.5, in embryos lacking Gsh2 there is an absence of both Dlx1 and Dlx2 expression in all but the most ventral aspect of the LGE. Expression of Mash1, a bHLH transcription factor essential for proper striatal development, shows a similar reduced expression pattern. Furthermore, expression of Ebf1, a gene essential for the transition of cells from the SVZ to the striatal mantle, is significantly reduced in the mutant LGE, as is Gad67, the precursor enzyme that catalyzes the formation of the neurotransmitter GABA. Interestingly, as development proceeds, there is compensation for this early loss of markers that is coincident with a molecular re-establishment of the cortical-striatal boundary. Despite this compensation, there is a defect in the development of distinct subpopulations of striatal neurons. Moreover, while this analysis suggests that the migration of the ventrally derived interneurons to the developing cerebral cortex is not significantly affected in Gsh2 mutants, there is a distinct delay in the appearance of GABAergic interneurons in the olfactory bulb (Corbin, 2000).

At E18.5, a reduction of Enkephalin expression in the most ventral aspect of the nucleus accumbens and ventrolateral (perirhinal) region of the ventral telencephalon is observed in Gsh2 mutant embryos. Expression of Dopamine receptor 2 is also missing in both the nucleus accumbens and perirhinal regions at this age. There is also a partially penetrant loss of striatal marker DARPP32-positive cells in the striatal SVZ further suggesting a loss of subpopulations of early born striatal neurons. In contrast, development of later born neurons that comprise the striatal matrix appears unaffected. Ebf1 expression, although reduced at E15.5 and at E18.5, is present in the presumptive striatal matrix in Gsh2 mutants. Normal expression of calbindin within the striatal matrix further suggests this aspect of striatal development is unaffected by the loss of Gsh2. These results suggest that, despite the recovery of early patterning defects by later stages of telencephalic development, the generation of specific subpopulations of early, but not late born, striatal neurons is permanently affected. Taken together, these data support a model in which Gsh2, in response to sonic hedgehog signaling, plays a crucial role in multiple aspects of telencephalic development (Corbin, 2000).

The role of the two closely related homeobox genes, Gsh1 and Gsh2, in the development of the striatum and the olfactory bulb was examined. The neurons that comprise the striatum have their origin in two prominent elevations, called the medial and lateral ganglionic eminences (MGE and LGE, respectively). The GABAergic medium-sized spiny projection neurons, which constitute the vast majority of striatal neurons, are generated from the LGE. Conversely, the majority of striatal interneurons appear to be generated from the MGE. Gsh1 and Gsh2 are expressed in a partially overlapping pattern by ventricular zone progenitors of the ventral telencephalon. Gsh2 is expressed in both of the ganglionic eminences while Gsh1 is largely confined to the medial ganglionic eminence. Previous studies have shown that Gsh2^-/- embryos suffer from an early misspecification of precursors in the lateral ganglionic eminence leading to disruptions in striatal and olfactory bulb development. This molecular misspecification is present only in early precursor cells while at later stages the molecular identity of these cells appears to be normalized. Concomitant with this normalization, Gsh1 expression is notably expanded in the Gsh2^-/- LGE. While no obvious defects in striatal or olfactory bulb development were detected in Gsh1^-/- embryos, Gsh1/2 double homozygous mutants displayed more severe disruptions than were observed in the Gsh2 mutant alone. Accordingly, the molecular identity of LGE precursors in the double mutant is considerably more perturbed than in Gsh2 single mutants. These findings, therefore, demonstrate an important role for Gsh1 in the development of the striatum and olfactory bulb of Gsh2 mutant mice. In addition, the data indicate a role for Gsh genes in controlling the size of the LGE precursor pools, since decreasing copies of Gsh2 and Gsh1 alleles results in a notable decrease in precursor cell number, particularly in the subventricular zone (Toresson, 2001).

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).

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

The role was examined of the homeobox gene Gsh2 in retinoid production and signaling within the ventral telencephalon of mouse embryos. Gsh2 mutants exhibit altered ventral telencephalic development, including a smaller striatum with fewer dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32)-immunoreactive neurons than wild types. The expression of the retinoic acid (RA) synthesis enzyme, retinaldehyde dehydrogenase 3 (Raldh3, also known as Aldh1a3), is reduced in the lateral ganglionic eminence (LGE) of Gsh2 mutants. Moreover, using a retinoid reporter cell assay, it was found that retinoid production in the Gsh2 mutants is markedly reduced. The striatal defects in Gsh2 mutants are thought to result from ectopic expression of Pax6 in the LGE. Removal of Pax6 from the Gsh2 mutant background improves the molecular identity of the LGE in these double mutants; however, Raldh3 expression is not improved. The Pax6;Gsh2 double mutants possess a larger striatum than the Gsh2 mutants, but the disproportionate reduction in DARPP-32 neurons is not improved. These findings suggest that reduced retinoid production in the Gsh2 mutant contributes to the striatal differentiation defects. Since RA promotes the expression of DARPP-32 in differentiating LGE cells in vitro, whether exogenous RA can improve striatal neuron differentiation in the Gsh2 mutants was examined. Indeed, RA supplementation of Gsh2 mutants, during the period of striatal neurogenesis, results in a significant increase in DARPP-32 expression. Thus, in addition to the previously described role for Gsh2 to maintain correct molecular identity in the LGE, these results demonstrate a novel requirement of this gene for retinoid production within the ventral telencephalon (Waclaw, 2004).

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