empty spiracles


The amino acid sequence of the Muscle segment homeobox (MSH) homeodomain is highly homologous to the homeodomains of the Drosophila s59/NK1 and empty spiracles genes and the Hox 7 and Hox 8 family of vertebrate homeobox genes. In addition, the 5' end of MSH has 52% sequence identity to the 5' end of the Empty spiracles protein, encoding several stretches of amino acids rich in serine, alanine, proline, glutamine, and acidic amino acids, and thus indicating potential domains of regulatory activity (Lord, 1995).

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)

Several homeobox genes, for example those of the ems class, play important roles in animal head development. Expression pattern and function of ceh-2, the Caenorhabditis elegans ems/Emx ortholog, is reported. CEH-2 protein is restricted to the nuclei of one type of small muscle cell, one type of epithelial cell, and three types of neurons in the anterior pharynx in the head. A deletion allele of ceh-2 has been generated that removes the homeobox. Animals homozygous for this deletion are viable and fertile, but grow slightly slower and lay fewer eggs than wild type. The function was assayed of two types of pharynx neurons that express ceh-2, the pairs M3 and NSM. M3 activity is substantially reduced in electropharyngeograms of ceh-2 deletion mutants; this defect can account for the observed retardation in larval development, since M3 activity is known to be necessary for effective feeding. NSM function and metabolism are normal based on the assays used. All cells that express ceh-2 in wild type are present in the ceh-2 mutant and have normal morphologies. Therefore, unlike other ems/Emx genes, ceh-2 seems to be important for a late differentiation step and not for neuron specification or regional patterning. Because the CEH-2 homeodomain is well conserved, an assay was performed to see whether ceh-2 can rescue ems- brain defects in Drosophila, despite the apparent differences in biological roles. The C. elegans ems ortholog is able to substitute for fly ems in brain development, indicating that sequence conservation rather than conservation of biological function is important (Aspöck, 2003).

Recent comparative studies on expression patterns of homeobox genes in the development between ascidians (phylum Urochordata) and vertebrates have come to suggest a possibility that a common basic mechanism may exist in the patterning of the central nervous system (CNS). The ems/emx genes have been demonstrated to be involved in the formation and patterning of the anterior CNS in Drosophila and vertebrate embryos. In the present study, expression of Hremx, the acidian homolog of ems/emx has been examined with particular attention to whether it is expressed in the larval ascidian CNS. Expression of Hremx was detected in the anterior trunk and lateral tail epidermis but not in the anterior CNS. The two expression domains of the epidermis respond in different ways upon treatment with retinoic acid: the anterior expression domain is unaltered, while the posterior expression domain extends to the anterior. The present result suggests that Hremx may have a function in anterior patterning but not in the patterning of the CNS in the ascidian embryo. The possibility is suggested that the function of ems/emx genes in the patterning of the anterior CNS in Drosophila and vertebrate embryos might have been acquired independently in the lineages to Drosophila and vertebrates (Oda, 2001).

The structural organization and the regulation of Empty spiracles and Orthodenticle are remarkably conserved in evolution. Expression of four mouse homeobox genes related to two Drosophila genes have been studied. Structurally, Emx1 and Emx2 are related to Empty spiracles and Otx1 and Otx2 are related to Orthodenticle. The four vertebrate genes have a role in establishing cell fates within the limits of various embryonic brain regions. They act with a discrete progressive process, centered in the dorsal telencephalon. Each is expressed in a specific manner in the developing rostral brain of E10 mouse embryos. Otx2 is expressed in every dorsal and most ventral regions of telencephalon, diencephalon and mesencephalon. The Otx1 expression domain is similar to that of Otx2, but contained within it. The Emx2 expression domain is comprised of dorsal telencephalon and small diencephalic regions, both dorsally and ventrally. Finally, Emx1 expression is exclusively confined to the dorsal telencephalon (Boncinelli, 1993).

The expression pattern of Xemx1 and Xemx2 genes in Xenopus laevis have been isolated and characterized. Xemx genes are the homologs of mouse Emx genes, related to Drosophila empty spiracles. They are expressed in selected regions of the developing brain, particularly in the telencephalon, and, outside the brain, in the otic vesicles, olfactory placodes, visceral arches and the developing excretory system. Experiments have been carried out concerning the tissue and molecular signals responsible for Xemx gene's activation in competent ectoderm. Xemx genes are activated in ectoderm conjugated with head organizer tissue, but not with tail organizer tissue. Nor are they activated in animal cap, neither by noggin nor by Xnr3, suggesting that a different inducer or the integration of several signals may be responsible for their activation (Pannese, 1998).

Targeted disruption of the mouse Sonic hedgehog gene shows that Shh plays a critical role in patterning of embryonic tussues, including the brain and spinal cord, the axial skeleton and limbs. The earliest detectable defect occurs in the future forbrain region at embryonic day 9.5. In Shh mutants, the midline is indistinct, the ventral lips of the cephalic folds are fused, and the normally separate optic vesicles appear instead as a continous single vesicle protruding at the ventral midline, with optic stalks deficient or absent. There is no invagination to form the characteristic double-layered optic cups, and the fused eye tissue at the midline forms a pigmented epithelium with no apparent differentiation of retinal tissue. The cephalic defects become even more apparent when the neural tube closes, with an overall reduction in size of the brain and spinal cord (Chiang, 1996).

The zebrafish emx1 and emx2 homeoproteins consist of 233 and 247 amino acids, respectively. The zebrafish emx1 and emx2 mRNAs are present at 12 h after fertilization, when the presumptive brain is in a simple tubular structure, and before first primary neurons appear. During brain development, the emx1 mRNA is localized in the dorsal telencephalon, whereas the emx2 mRNA is distributed in the dorsal telencephalon, parts of the diencephalon and the otocyst. The differential expression patterns of the two emx homeoprotein mRNAs may define the subdivisions of the zebrafish telencephalon (Morita, 1995).

Emx family homeobox genes, Emx1 and Emx2, play an essential role in rostral brain development in mammalian embryos. A zebrafish emx family gene, emx1, is more similar to the mouse Emx1 gene than the previously reported zebrafish emx1 gene; it is proposed to rename that gene emx3. The expression of emx1 is first detected around the 10-somite stage in the pineal gland (epiphysis) primodium in the developing anterior brain and in the pronephric primodium within the intermediate mesoderm. emx1 expression in the epiphysis has not been reported in other species. Expression in the epiphysis is suppressed at 23 h post-fertilization (hpf) in the floating head (flh) mutant, in which development of the epiphysis is impaired. Subsequently, emx1 is expressed in the telencephalon, as reported in mammals, and can be detected in the olfactory placode and in a small group of cells in the forebrain at 25 hpf. In the mesoderm, emx1 expression is gradually concentrated in the posterior pronephric duct during somitogenesis, and becomes expressed predominantly in the urogenital opening at 25 hpf. Thus, emx1 displays a unique expression pattern that is distinct from the patterns of emx2 and emx3 (Kawahara, 2002).

During early embryogenesis, Emx2 is expressed in the presumptive cerebral cortex and olfactory bulbs; later it is found in the hippocampus proper and dentate gyrus. The latter are involved in memory processes. Homozygous Emx2 knockout mice die postnatally because of severe urogenital alterations. These mice present cerebral hemispheres with a reduced size and exhibit specific morphological alterations in allocortical structures of the medial wall of the brain. The dentate gyrus is missing and the hippocampus proper is reduced. The medial limbic cortex is also severely shortened. The development of the dentate gyrus is affected at the onset of its formation with defects in the neuroepithelium, from which it originates. These findings demonstrate that Emx2 is required for the development of several forebrain structures (Pellegrini, 1996).

The homeobox gene Emx2 is a mouse homolog of a Drosophila head gap gene, empty spiracles. Mouse Emx2 is essential for the development of the dorsal telencephalon. At the same time, Emx2 is expressed in the epithelial components of the developing urogenital system and, in Emx2 mutant mice, the kidneys, ureters, gonads and genital tracts are completely missing. Pax-2 and c-ret expressions in the Wolffian duct and WT-1 and GDNF expressions in the metanephric blastema are initially normal in the mutant. The ureteric bud grows and invades the metanephric mesenchyme where Pax-2 expression is normally induced. Subsequently, however, Pax-2, c-ret and Lim1 expressions in the ureteric bud and GDNF expression in the mesenchyme are greatly reduced. Wnt-4 expression is never found in the mesenchyme. The tip of the ureteric bud never dilates and branching of the bud does not occur. Neither pretubular cell aggregates nor epithelialization are found in the mesenchyme. Instead the ureteric bud soon degenerates and apoptotic figures are prominent in mesenchymal cells. These results suggest that in metanephrogenesis Emx2 is essential for the ureteric bud functions subsequent to Pax-2 induction in the metanephric mesenchyme (Miyamoto, 1997).

D/V patterning of the anterior neural plate is controlled by several signaling centers. Signals from the anterior neural ridge (ANR) regulate expression of the Forkhead related factor Brain factor1 (Bf1), which is required for growth and patterning of the telencephalon. Fgf8 represents an important component of this signal as Fgf8 applied to the prosencephalic neural plate mimics the effects of the ANR. In addition, the anterior non-neural ectoderm, the ANR and later the roof of the forebrain produce several secreted factors of the bone morphogenetic protein (Bmp) family. Bmps have been shown to induce the expression of Msx1 in the dorsal midline of the forebrain and to repress the expression of Bf1. Also, noggin, encoding a secreted protein that binds to Bmps and prevents the latter from interacting with its receptor, is expressed in the telencephalic roof plate, suggesting that Bmp activity is under stringent control during dorsal forebrain development (Theil, 1999 and references).

The prechordal mesendoderm represents a key determinant in the specification of the ventral forebrain and produces Sonic hedgehog protein. Shh is expressed throughout the axial mesendoderm and has been implicated in ventral patterning throughout the neuraxis. Mice mutant for Shh are cyclopic and exhibit disruptions of ventral forebrain formation. Mutations of the human SHH gene have also been identified in patients with holoprosencephaly. These studies therefore implicate Shh as an essential mediator of the inductive effects of the prechordal mesendoderm (Theil, 1999 and references).

The dentate gyrus and hippocampus as centers for spatial learning, memory and emotional behaviour have been the focus of much interest in recent years. The molecular information on their development, however, has been relatively poor. To date, only Emx genes are known to be required for dorsal telencephalon development. Forebrain development in the extra toes (XtJ) mouse mutant, which carries a null mutation of the Gli3 gene, is described. Gli3 is a mediator of Shh signaling. The XtJ defect leads to a failure to establish the dorsal di-telencephalic junction and finally results in a severe size reduction of the neocortex. In addition, XtJ/XtJ mice show absence of the hippocampus (AmmonÂ’s horn plus dentate gyrus) and the choroid plexus in the lateral ventricle. The medial wall of the telencephalon, which gives rise to these structures, fails to invaginate during embryonic development. On a molecular level, disruption of dorsal telencephalon development in XtJ/XtJ embryos correlates with a loss of Emx1 and Emx2 expression. Furthermore, the expression of Fgf8 and Bmp4 in the dorsal midline of the telencephalon is altered. However, expression of Shh, which is negatively regulated by Gli3 in the spinal cord, is not affected in the XtJ/XtJ forebrain. This study therefore implicates Gli3 as a key regulator for the development of the dorsal telencephalon and implies that Gli3 is upstream of Emx genes in a genetic cascade controlling dorsal telencephalic development (Theil, 1999).

Based on their expression pattern, Emx genes are candidates for playing a role in subdividing the prosencephalon. Furthermore, their Drosophila homolog, empty spiracles, functions as a gap gene as well as a segment identity gene during head segmentation suggesting that Emx genes might also be involved in specifying dorsal telencephalon identity. However, the phenotypes of mice in which Emx genes have been inactivated have not provided clues on these potential roles. Analysis of the forebrain phenotype of XtJ/XtJ mice therefore provides the first evidence for a gene controlling formation of the di-telencephalic boundary and specification of dorsal telencephalon identity (Theil, 1999).

The Gli3 mutation also affects development of the telencephalic roof and the juxtaposed medium pallium. Moreover, formation of the choroid plexus is disrupted in the lateral ventricle while its development occurs normally in the 4th ventricle. The expression patterns of several regulatory genes were found to be altered in the telencephalic dorsal midline of XtJ/XtJ embryos. While Fgf8 is ectopically activated in the trp, Bmp signaling is negatively affected by the Gli3 mutation as judged by the loss of Bmp4 and Msx1 expression and by the maintenance of noggin expression. Interestingly, Fgf8 and Bmp2/Bmp4 have been shown to act antagonistically on cell proliferation and differentiation in the dorsal forebrain. Ectopic Fgf8 expression and loss of Bmp signaling in the roof plate as observed in XtJ/XtJ embryos might therefore disrupt the balance between these two processes (Theil, 1999).

The appropriate control of proliferation of neural precursors has fundamental implications for the development of the central nervous system and for cell homeostasis/replacement within specific brain regions throughout adulthood. The role of genetic determinants in this process is largely unknown. The homeobox transcription factor Emx2 is expressed within the periventricular region of the adult telencephalon. This neurogenetic area displays a large number of multipotent stem cells. Adult neural stem cells isolated from this region do express Emx2 and down-regulate it significantly upon differentiation into neurons and glia. Abolishing or, increasing Emx2 expression in adult neural stem cells greatly enhances or reduces their rate of proliferation, respectively. Altering the expression of Emx2 affects neither the cell cycle length of adult neural stem cells nor their ability to generate neurons and glia. Rather, when Emx2 expression is abolished, the frequency of symmetric divisions that generate two stem cells increases, whereas it decreases when Emx2 expression is enhanced (Galli, 2002).

Leptomeningeal glioneuronal heterotopias are a focal type of cortical dysplasia in which neural cells migrate aberrantly into superficial layers of the cerebral cortex and meninges. These heterotopias are frequently observed as microscopic abnormalities in the brains of individuals with central nervous system (CNS) malformations and epilepsy. The function of Emx2 is essential for development of the cortical preplate, which gives rise to the marginal zone and subplate. However, transcriptional targets of EMX2 during CNS development are unknown. Leptomeningeal glioneuronal heterotopias form in Emx2–/– mice that are equivalent to human lesions. Additionally, ectopic expression of Wnt1 is observed in the embryonic roofplate organizer region and dorsal telencephalon. To determine the phenotypic consequences of such Wnt1 misexpression, a putative EMX2 DNA-binding site was deleted from the Wnt1 enhancer and this was used to misexpress Wnt1 in the developing murine CNS. Heterotopias were detected in transgenic mice as early as 13.5 days postcoitum, consistent with a defect of preplate development during early phases of radial neuronal migration. Furthermore, diffuse abnormalities of reelin- and calretinin-positive cell populations were observed in the marginal zone and subplate similar to those observed in Emx2-null animals. Taken together, these findings indicate that EMX2 is a direct repressor of Wnt1 expression in the developing mammalian telencephalon. They further suggest that EMX2-Wnt1 interactions are essential for normal development of preplate derivatives in the mammalian cerebral cortex (Ligon, 2003).

The preplate forms as the initial wave of neural precursors leaves the ventricular zone and migrates radially to the margin of the developing telencephalon. The PP, marginal zone and subplate then serve as a framework for the subsequent influx of neurons from the ventricular zone, cortical hem and ventral forebrain, potentially along tangential and radial processes of cells that reside in these layers. Because ectopic Wnt1 expression was never detected in the preplate or marginal zone of Emx2-null or Wnt1-Tg mice, it is likely that ectopic Wnt1 acts principally on the early ventricular zone progenitors of the dorsomedial cortex. It is now known that cells from this region, especially the cortical hem, appear much more migratory than originally assumed. Several reports suggest that neurons from the hem migrate extensively into the adjacent cortical primordium. As a result, ectopic Wnt1 is expressed early on in the same progenitor cells that will later migrate into and throughout the neocortex thereby creating the potential for very long-range functional effects. Evidently, Wnt1 signaling inhibits development or initial migration of preplate cells and their processes (Ligon, 2003).

On the origin and evolution of the tripartite brain

The many different nervous systems found in bilaterally symmetric animals may indicate that the tripartite brain appeared several times during the course of bilaterian evolution (see Bilaterian evolutionary tree). However, comparative developmental genetic evidence in arthropods, annelids, urochordates, and vertebrates suggests that the development of a tripartite brain is orchestrated by conserved molecular mechanisms. Similarities in the underlying genetic programs do not necessarily reflect a common origin of structures. Nevertheless, 3 lines of evidence support a monophyletic origin of the tripartite brain and possibly also an elongated central nervous system (CNS): structural homology, character identity networks, and the functional equivalence of character identity genes. Monophyly of the brain also implies that the brain was secondarily reduced and lost multiple times during the course of evolution, leading to extant brainless bilaterians. The likelihood of secondary loss can be estimated by metazoan divergence times and through reconstructed cases such as limb loss in tetrapods or eye loss in fish. When scaled to molecular clock dates, monophyly of the tripartite brain indicates that existing brainless Bilateria had several hundred million yearsÂ’ time for the secondary modification and eventual loss of a primitive/ancestral brain and CNS. To corroborate this conjecture, ancestral character identity genes of living brainless Bilateria can be tested for their potential to substitute Drosophila or Mus homologs in tripartite brain development (Hirth, 2010).

Comparative developmental genetic analyses in arthropods, annelids, urochordates, and vertebrates provide experimental evidence suggesting that brain and CNS development in these taxa is orchestrated by conserved molecular genetic mechanisms. Similarities in the underlying developmental genetic programs do not necessarily reflect a common origin of structures. However, monophyly of the tripartite brain is supported by 3 lines of evidence, namely (1) structural homology, (2) character identity networks (ChINs), and (3) functional equivalence of character identity genes. (Hirth, 2010).

Homology signifies common descent and can be defined as a relationship between traits of organisms that are shared as a result of common ancestry. Structural homology refers to a morphological character that is (a) derived from a common ancestor possessing this character, (b) built on the same basic plan, and (c) consists of comparable elements. The latter was already exemplified by Darwin, who referred to 'the relative position or connection in homologous parts; they may differ to almost any extent in form and size, and yet remain connected together in the same invariable order'. Textbook examples are the different forelimbs of tetrapods where similar bones are connected in the 'same invariable order', irrespective of the different functions they serve. The same principle of 'relative position or connection in homologous parts' applies to 'midbrain' structures in Drosophila and Mus, although function seems to be conserved as well; in both cases, GABAergic, serotonergic, and dopaminergic neural circuit elements involved in the control of locomotor behavior are located in the same relative position to other neural processing centers, namely posteriorly to light-sensing organs and anteriorly to gustatory and 'facial' innervation (Hirth, 2010).

The principle 'connected together in the same invariable order' of comparable elements may also apply to ventral/spinal cord motor neurons in Drosophila, Platynereis (Annelida), and Mus; they are located ventrally to nonneural tissue and dorsally to the midline. As a stand-alone criterion, however, 'relative position or connection in homologous parts' would be insufficient to signify homology because similar structures with similar positions can have multiple causes, including parallel or convergent evolution. However, related to brain evolution, this criterion is supported by developmental genetic evidence suggesting that insect and mammalian brains are built on the same basic plan, which is executed by the spatiotemporal activities of pleiotropic genes that constitute character identity networks (ChINs) (Hirth, 2010).

ChINs refer to genetic regulatory networks that control the developmental program and, hence, the specification of character identities, such as forelimbs. ChINs for brain and CNS development include Otx-Pax-Hox modules acting along the anterior-posterior axis, BMP-Msx-Nkx modules acting along a DV axis, and bHLH-Par-Numb modules acting along an apico-basal axis. Together, these modules are necessary for the correct formation of an orthogonal, species-specific CNS. Character identity genes are central to ChINs; their knockdown/mutational inactivation does obstruct the formation of a character identity. The majority of character identity genes are transcription factors, with textbook examples such as Pax6 genes, which are essential for the formation of light-sensing organs, or otd/Otx genes, which are essential for anterior brain formation. As stated earlier, similarities in developmental genetic programs do not necessarily reflect a common origin of character identities, which is exemplified, for example, by the role of distal-less(Dll/Dlx) genes in the formation of non-homologous appendages. However, the situation is different if taxa-specific ChINs, which comprise homologous genes, specify taxa-specific character identities to which structural homology applies, namely their construction depends on a conserved genetic program (basic plan) and consists of comparable elements that are arranged 'in the same invariable order'. This principle applies to the above-mentioned midbrain-derived neural circuit elements that are required for the control of locomotor behavior: in both flies and mice, FGF8, Engrailed, and Pax2/5/8 genes are essential for the formation and specification of these structures (Hirth, 2010).

ChINs of different taxa can comprise homologous genes that control the developmental program and, hence, the specification of character identities sharing a common descent. It has therefore been predicted that the phenotype caused by knockdown of a character identity gene (i.e. transcription factor) 'can be reversed with a gene from the clade that shares this character, but not by genes from species that diverged before the origin of this character'. Experimental evidence conforming to this prediction is available. Human Otx2 can rescue defective 'forebrain' formation in Drosophila orthodenticle (otd) mutants, and otd can replace mouse Otx2 in fore- and midbrain formation, but only if otd is accompanied by the Otx2 regulatory sequences required for epiblast-specific translational control. Drosophila engrailed can substitute for mouse Engrailed1 function in mid-hindbrain formation, but not for limb development. Mouse Emx1 can rescue brain defects in Drosophila empty spiracles(ems) mutants, but Acropora [Anthozoa, Cnidaria (radially symmetric organisms including corals, anemones and jelly fish)] Emx is not able to replace ems in fly brain development (Hirth, 2010).

Based on these results, and in line with Wagner's prediction of the existence of equivalent functions of character identity genes (Wagner, 2007), it can be concluded that: (1) The character of a 'fore-, mid-, and hindbrain' develops under the control of ChINs comprising otd/Otx, ems/Emx, and en/EN1 genes that are shared in a clade including flies and mice. (2) The failure of Drosophilaengrailed to rescue limb formation in MusEN1 mutants indicates that the appendages of mice and flies are not homologous. (3) Most likely, the epiblast did not exist in the last common ancestor of Drosophila and Mus and is an evolutionary novelty of the lineage leading to Mus (Boyl, 2001). (4) Drosophila and Acroporaems/Emx genes diverged before the origin of the character 'tripartite brain'. The last conclusion has major implications for the evolutionary origin of the tripartite brain and depends on the position of anthozoans/cnidarians relative to bilaterally symmetric animals in a phylogenetic tree (Hirth, 2010).

A phylogenetic tree is based on evolutionary relationships and can be reconstructed using cladistics. The cladistic concept is relative; it scores characters for their presence and absence and, if present, for their state in each of the taxa of interest. Current cladistics scores morphological as well as molecular characters (i.e. genes, genomes, and developmental pathways) against each other, and the resulting phylogenetic tree is based on sister and out groups in order to be rooted. For example sister groups like arthropods and onychophorans share a common panarthropod ancestor, and together they are a sister group to cycloneuralians (including nematodes), which together belong to the ecdysozoans, which are a sister group to lophotrochozoans. Out groups provide necessary additional information about the origin of a character in sister groups; they are used for character polarity which enables the application of the parsimony criterion in order to infer whether the character is primitive/ancestral or derived (Hirth, 2010).

Monophyly of bilaterally symmetric animals and subsequent interpretations about the origin and evolution of the brain and CNS hinge on the identification of genuine sister and out groups and, thus, on how deep a phylogenetic tree is rooted. Current cladistics is work in progress which is exemplified by the allocation of Cnidaria (but also Acoela) as either a sister or out group to bilaterians. The ambiguity is caused by mounting evidence suggesting that cnidarians possess genetic toolkits similar to those active in bilaterian axis and cell type specification, including neurogenesis (Gaillot, 2009). Depending on additional characters and genuine out groups to Cnidaria, this can be interpreted to mean that: (i) cnidarians are de facto bilaterians and a genuine sister group to the rest of bilaterally symmetric animals, namely P+D, or (ii) cnidarians are a genuine out group to P+D. It follows that the positioning of Cnidaria has an impact on the positioning of Urbilateria and the origin of a brain and CNS. Cnidarians possess nerve nets and nerve rings but, so far, no evidence of a centralized nervous system has been found [Gaillot, 2009]. Several extant Protostomia and Deuterostomia (P+D) also possess a net-like nervous system. In the first scenario, Urbilateria would be the last common ancestor of both Cnidaria and P+D, suggesting that a nerve net is a primitive/ancestral character. In the second scenario, Urbilateria would be the last common ancestor of P+D, and unrelated to Cnidaria, suggesting that the cnidarian nerve net is unrelated to the nerve net of extant bilaterally symmetric animals (the position of Acoela would require further considerations) (Hirth, 2010 and references therein).

As mentioned, the presence of genetic toolkits does not necessarily reflect a common origin of characters. The existence of equivalent functions of character identity genes, however, allows inferences as to whether genes diverged before the origin of a character or not (Wagner, 2007). Murine, but not the cnidarian AcroporaEmx gene, can rescue brain defects in Drosophila ems mutants, suggesting that Drosophila and Acroporaems/Emx genes diverged before the origin of the character 'tripartite brain', which is a primitive/ancestral character to mice and Drosophila. These data suggest that Acropora and the last common ancestor of mice and Drosophila did not share the character 'tripartite brain'; it may also indicate that the last common ancestor of Cnidaria and P+D possessed a nerve net-like nervous system. The latter notion is supported by the functional equivalence of another character identity gene. The cnidarian Hydra achaete scute homolog has proneural activity in Drosophila, can heterodimerize with daughterless, and is able to form ectopic sensory organs in the peripheral nervous system of Drosophila; in addition, it is also able to partially rescue adult external sensory organ formation in viable achaete scutecomplex mutations. Unfortunately, whether Hydra achaete scute is able to rescue defects in Drosophila achaete scute mutant brain and CNS development was not tested (Hirth, 2010).

Together, these data suggest that the morphological character 'tripartite brain' evolved after the Cnidaria-P+D split. It depends on the placement of cnidarians in- or outside the clade Bilateria, whether it is reasonable to propose that Urbilateria already possessed a tripartite brain and probably also a complex CNS, or whether Urbilateria was likely to be 'brainless'. If Cnidaria and P+D are true sister groups of a clade Bilateria, it follows that Urbilateria was brainless. Independent of the position of Urbilateria, the above-mentioned data corroborate monophyly of the tripartite brain, as well as its evolutionary origin after the cnidarian-P+D split but before the Protostomia-Deuterostomia (P/D) split. This concept, though, is challenged by the fact that several proto- and deuterostomians do not possess a brain and complex CNS. The conundrum is most obvious when the complex CNS of arthropods is compared to that of cycloneuralians, which are supposed to be sister groups. Comparative data for cycloneuralians are scant, except for Caenorhabditis elegans. The nematode does not possess a complex brain and CNS, yet monophyly of the brain is corroborated by functional equivalence of a character identity gene: the ems homolog ceh-2 of C. elegans is able to rescue ems mutant brain defects in Drosophila. These data suggest that C. elegans secondarily lost the character 'tripartite brain' and most likely also a complex CNS, as should be postulated for other brainless proto- and deuterostomians that are monophyletic to Drosophila and mice. (Hirth, 2010).

Monophyly of the tripartite brain and its evolutionary origin after the cnidarian-P+D split and before the P/D split implies that the brain and CNS were secondarily reduced and eventually lost multiple times and independently during the course of protostomian and deuterostomian evolution. For the quality and consistency of this conjecture, it is necessary to consider (1) metazoan divergence times, (2) the likelihood of secondary loss, as illustrated by reconstructed cases, and (3) the proposal of an experimental paradigm that can test ancestral character identity genes of brainless bilaterians for their potential to control brain development in Drosophila or mice. (Hirth, 2010).

Molecular clock dates suggest that the cnidarian-P+D split occurred somewhere around 630 million years ago (Mya), the P/D split around 555 Mya, and the Arthropoda-Priapulida split around 540 Mya. These estimates suggest that a primitive/ancestral tripartite brain likely evolved within 75 million years between 630 and 555 Mya and then continued to evolve into taxon- and species-specific characters such as the extant Drosophila and mouse brain. Monophyly implies that ancestral priapulids shared with arthropods the character of a tripartite brain and possibly a segmented CNS. Fossil evidence supports ancestral segmentation in priapulids (e.g. Markuelia) even though extant priapulids are nonsegmented and their CNS is 'only' composed of a nerve ring and a single ventral cord running the length of the body. Scaled against molecular clock dates, monophyly of the tripartite brain suggests that the derived character of the priapulid brain and CNS would have had several hundred million years' time for its secondary modification. Such a scale of divergence time can be extrapolated to other taxa as well, and implies that also other extant brainless P+D would have had several hundred million years' time for the secondary, independent modification and eventual loss of a tripartite brain and CNS (Hirth, 2010).

Comparative developmental genetics and phylogenomics reveal that morphological evolution is most likely driven by gene duplication and gene loss, together with changes in differential gene regulation, including mutations in cis-regulatory elements of pleiotropic developmental regulatory genes. These genetic modifications can account not only for the acquisition of novel morphological characters but also for the modification and eventual loss of a morphological character. The latter is illustrated by limbless tetrapods, such as whales, snakes, and flightless birds. Limbless tetrapods are descended from limbed ancestors, and limblessness has been shown to be polygenic, involving pleiotropic regulatory genes that act as modifiers to suppress limb development. In snakes, for example, differential regulation of HoxC genes accounts for the failure to activate the signaling pathways required for proper limb development, eventually leading to limbless snakes. Independent reduction and limb loss in tetrapods occurred repeatedly over several millions of years for lizards, and over 10-12 or up to 20 million years for whales (Hirth, 2010).

The secondary loss of morphological characters is also exemplified in fish. In different natural populations of threespined stickleback fish, the secondary loss of the pelvis occurred through regulatory mutations deleting a tissue-specific enhancer of the Pituitary homeobox transcription factor 1 (Pitx1) gene. The selective pressures causing secondary loss can be manifold, including energy limitation and environmental constraints which are most obvious for nervous system structures that are characterized by high energy consumption. For example, populations of cave fish have undergone convergent eye loss at least 3 times within the last 1 million years, whereas populations that continuously lived on the surface retained their eyes. These examples illustrate that the secondary loss of a morphological character can occur repeatedly during the course of evolution within a time frame of million years. In comparison, monophyly of the tripartite brain calibrated by metazoan divergence times suggests that extant brainless P+D would have had several hundred million years’ time, possibly from 555 Mya onwards, for the secondary modification and eventual loss of an ancestral/primitive tripartite brain and CNS, the mechanisms of which remain unknown (Hirth, 2010).

The secondary, independent loss of the brain and CNS multiple times during the course of protostomian and deuterostomian evolution is a conjecture that can be tested experimentally. Wagner's prediction states that the phenotype caused by knockdown of a character identity gene can be rescued with a gene from the clade that shares a particular character, but not by genes from species that diverged before the origin of this character (Wagner, 2007). Monophyly of the tripartite brain implies that extant brainless P+D species were once able to develop a primitive/ancestral tripartite brain; therefore, these brainless species should possess ChINs for the development and specification of a tripartite brain, unless they have secondarily lost the necessary genes during the course of evolution. The potential functional equivalence of character identity genes in brain development can be tested in those cases where brainless P+D species have retained the ChINs or at least a character identity gene. Thus, genes from species that have secondarily lost the character tripartite brain but have retained, for example, otd/Otx genes with an archetypical 'brain function' might be able to rescue, at least in part, brain phenotypes in Drosophila otd or Mus Otx2 mutants. These experiments are feasible and can be tested as homologs of character identity genes controlling tripartite brain development have been identified in brainless P+D species. It will be interesting to see whether Otx or Engrailed genes from brainless brachiopods or echinoderms like sea cucumber are able to substitute their Drosophila or murine homologs in the development and specification of a tripartite brain (Hirth, 2010).

Agenesis of the scapula in Emx2 homozygous mutants

The shoulder and pelvic girdles represent the proximal bones of the appendicular skeleton that connect the anterior and posterior limbs to the body trunk. Although the limb is a well-known model in developmental biology, the genetic mechanisms controlling the development of the more proximal elements of the appendicular skeleton are still unknown. The knock-out of Pax1 has shown that this gene is involved in patterning the acromion, while the expression pattern of Hoxc6 makes this gene into a candidate for involvement in scapula development. Surprisingly, scapula and ilium do not develop in Emx2 knock-out mice. In the homozygous mutants, developmental abnormalities of the brain cortex, the most anterior structure of the primary axis of the body, are associated with important defects of the girdles, the more proximal elements of the secondary axis. These abnormalities suggest that the molecular mechanisms patterning the more proximal elements of the limb axis are different from those patterning the rest of appendicular skeleton. While Hox genes specify the different segments of the more distal part of the appendicular skeleton forming the limb, Emx2 is concerned with the more proximal elements constituting the girdles (Pellegrini, 2001).

Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1

The genetic pathways underlying shoulder blade development are largely unknown, as gene networks controlling limb morphogenesis have limited influence on scapula formation. Analysis of mouse mutants for Pbx and Emx2 genes has suggested their potential roles in girdle development. In this study, by generating compound mutant mice, the genetic control of scapula development by Pbx genes and their functional relationship with Emx2 were examined. Analyses of Pbx and Pbx1;Emx2 compound mutants revealed that Pbx genes share overlapping functions in shoulder development and that Pbx1 genetically interacts with Emx2 in this process. A biochemical basis for Pbx1;Emx2 genetic interaction is provided by showing that Pbx1 and Emx2 can bind specific DNA sequences as heterodimers. Moreover, the expression of genes crucial for scapula development is altered in these mutants, indicating that Pbx genes act upstream of essential pathways for scapula formation. In particular, expression of Alx1, an effector of scapula blade patterning, is absent in all compound mutants. Pbx1 and Emx2 bind in vivo to a conserved sequence upstream of Alx1 and cooperatively activate its transcription via this potential regulatory element. These results establish an essential role for Pbx1 in genetic interactions with its family members and with Emx2 and delineate novel regulatory networks in shoulder girdle development (Capellini, 2010).

Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function

In the brain, Emx-1 normally restricted to the dorsal telencephalon corresponding to the neocortex, is detected throughout a single vesicle present in the midline, suggesting that the normal bilateral lobes of the telencephalon are fused to form a single midline structure and that forebrain structures are lost. Consistent with this interpretation, expression of the Otx-2 gene (Drosophila homolog: Orthodenticle), is lost in the ventral domains of the telencephalon and in the diencephalon. Reduced expression of Otx-2 in the mesencephalon is also consistent with the reduction in size and abnormal morphology of the midbrain in Shh knockouts. Expression of the En-1 gene (Drosophila homolog: Engrailed) at the isthmus is essentially normal, consistent with the presence of the midbrain/hindbrain constriction and also with the normal appearance of Pax-2 at this constriction (Chiang, 1996).

Expression of the Emx-1 and Dlx-1 homeobox genes define three molecularly distinct domains in the telencephalon of mouse, chick, turtle and frog embryos: implications for the evolution of telencephalic subdivisions in amniotes

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).

Absence of Cajal-Retzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic eminence in Emx1/2 double mutant cerebral cortex

The highly layered structure of the cerebral cortex is established through the pattern of neuronal cell migrations. The first step is the creation of the primordial layer, the preplate, consisting of radial glial cells and the earliest generated neurons. Among these neurons are the Cajal-Retzius neurons. In the next step, the preplate splits into a superficial (marginal) zone, where the Cajal-Retzius neurons reside, and a deep subplate wherein the neurons form. Neurons migrating from the subplate form the cortical plate. This migration takes place on the radial glial fibers. Emx1 and Emx2, mouse orthologs of the Drosophila head gap gene, ems, are expressed during corticogenesis. Emx2 null mutants exhibit mild defects in cortical lamination. Segregation of differentiating neurons from proliferative cells is normal for the most part, however, reelin-positive Cajal-Retzius cells are lost by the late embryonic period. Additionally, late-born cortical plate neurons display abnormal position. These types of lamination defects are subtle in the Emx1 mutant cortex. Emx1 and Emx2 double mutant neocortex is much more severely affected. Thickness of the cerebral wall is diminished with the decrease in cell number. Bromodeoxyuridine uptake in the germinal zone is nearly normal; moreover, no apparent increase in cell death or tetraploid cell number was observed. However, tangential migration of cells from the ganglionic eminence into the neocortex is greatly inhibited. The wild-type ganglionic eminence cells transplanted into Emx1/2-double mutant telencephalon do not move to the cortex. MAP2-positive neuronal bodies and RC2-positive radial glial cells emerge normally, but the laminar structure subsequently formed is completely abnormal. Furthermore, both corticofugal and corticopetal fibers are predominantly absent in the cortex. Most importantly, neither Cajal-Retzius cells nor subplate neurons are found throughout E11.5-E18.5. Thus, this investigation suggests that laminar organization in the cortex or the production of Cajal-Retzius cells and subplate neurons is interrelated to the tangential movement of cells from the ganglionic eminence under the control of Emx1 and Emx2 (Shinozaki, 2002).

Compromised generation of GABAergic interneurons in the brains of Vax1-/- mice

The subcortical telencephalon is the major source of GABAergic interneurons that, during development, tangentially migrate to the cerebral cortex, where they modulate the glutamatergic excitatory action of pyramidal cells. The transcription factor Vax1, an intracellular mediator of both Shh and Fgf signaling, is expressed at high levels in the medial and lateral ganglionic eminences (MGE and LGE, respectively), in the septal area (SA), in the anterior entopeduncular area (AEP) and in the preoptic area (POA). Vax1 expression in the neuroepithelium is graded: low in the ventricular zone (VZ) and high in the subventricular zone (SVZ), in a pattern that closely reproduces that of several members of the Dlx and Gsh family of homeobox transcription factors. Evidence is provided that Vax1 plays an important role in proliferation and differentiation of MGE, POA/AEP and septum, and that the last structure is completely absent in Vax1-/- mice. The absence of Vax1 causes a severe depletion of GABAergic neurons in the neocortex, ranging from 30% to 44%, depending on the cortical areas considered. Taken together, these data indicate that a loss of function mutation in the Vax1 gene generates abnormalities in basal ganglia subventricular zone development and that it prevents the formation of the septum, impairing GABAergic interneuron generation (Taglialatela, 2004).

Potential target genes of EMX2 include Odz/Ten-M and other gene families with implications for cortical patterning

EMX2 and PAX6 are expressed by cortical progenitors and specify area patterning. Representational difference analysis (RDA) was used to compare expressed RNAs from wild type and Emx2−/− cortex and 41 unique clones were identified. Using secondary screening by in situ hybridization, five genes were selected for further analysis, Cdk4, Cofilin1, Crmp1, ME2, and Odz4, involved in neuronal proliferation, differentiation, migration, and axon guidance. Each exhibits differential expression in wild type cortex. Odz4 is one of four members of a vertebrate gene family homologous to the Drosophila pair-rule patterning gene, Odd Oz (Odz), a transmembrane receptor. Odz genes are expressed in complementary patterns in cortex, as well as in nuclei-specific patterns in thalamus that relates to their area-unique cortical expression. In addition, each of the genes analyzed shows different expression patterns in wild type cortex, Emx2, and Pax6 mutant cortex, consistent with potential roles in area patterning. These findings identify potential targets of EMX2 that might account for its function and the defects in Emx2−/− cortex, and suggest that the Odz family of transmembrane proteins influences cortical area patterning downstream to EMX2 and PAX6 (Li, 2006).

CDK4 stimulates growth rate and is expressed exclusively by proliferating cells within neocortex. Down-regulated Cdk4 expression in Emx2−/− and Sey/Sey is consistent with decreased cortical sizes of both mutant mice. At E18.5, Cdk4 expression within ventricular zone (vz)/subventricular zone (svz) becomes broader and more diffused in Emx2−/− cortex while thinner in Sey/Sey cortex. This might be caused secondarily by different structural abnormalities of the mutant cortices (Li, 2006).

Absence of functional EMX2 leads to defective cell migration in telencephalon. Transplantation studies support that EMX2 mutation results in abnormal cortical milieu that impairs tangential cell migration from ganglionic eminence (GE) into neocortex. Defective radial migration is attributed to defective Cajal–Retzius cells that produce Reelin. Radial migration is robustly affected in Emx2−/− neocortex, while the penetration of impaired Reelin expression varies from zero to severe. Disorganized cortical lamination in Emx2−/− seems to be the result of more than defective Cajal–Retzius cells (Li, 2006).

Sema3A-mediated signaling plays important roles in directing axon guidance and neuronal migration. CRMP1, a component of Sema3A-receptor complex, is expressed in rostral-high to caudal-low gradient in neocortex. Cortical Crmp1 expression is down-regulated with flattened-out expression gradient in Emx2−/−, which might compromise neuronal sensitivity to semaphorins. Cell migration and axon guidance of cortical neurons would subsequently be affected, which has impact on cortical arealization (Li, 2006).

Cofilin1 regulates actin polymerization/depolymerization, which is the major machinery of cell motility. Cofilin1 is expressed in rostral-high to caudal-low gradient in cortex, and the expression is up-regulated with a flattened gradient in Emx2−/− while down-regulated in Sey/Sey. EMX2 and PAX6 might be involved in regulating neuronal motility by determining the dynamics of cytoskeletal components such as actin (Li, 2006).

bHLH transcription factors play important roles in a variety of developmental processes, including corticogenesis. ME2 belongs to class A bHLH family (Daughterless homolog) and is expressed by both proliferating and differentiating neurons. Within neocortex, ME2 is initially expressed in rostral-low to caudal-high gradient that becomes reversed as cortex matures. Cortical expression of ME2 is decreased and shifts caudally in Emx2−/− while becoming enhanced and shifts anteriorly in Sey/Sey. ME2 stimulates expression of Id2 that is capable of promoting expression of neurotrophin receptor, p75. EMX2 and PAX6 might regulate neocortical arealization indirectly through the neurotrophin-signaling pathway that is involved in neuronal differentiation, regeneration, survival, apoptosis, and cell migration (Li, 2006).

Odd Oz is a pair rule gene crucial for patterning Drosophila body plan which mouse orthologue, Odz4, is identified by RDA as down-regulated in Emx2−/−. In Drosophila, ODZ expression coincides with the developmental transition from syncytial to cellular blastoderm, which represents the first step in shifting segmentation mechanism from internuclear to intercellular signaling (Li, 2006).

In mouse, expression gradients of Odz2, Odz3, and Odz4 are increasing from lateral-anterior to medial-posterior neocortex. Genomics screens have also identified Odz2 and Odz4 as caudal genes. Odz1 expression gradient is decreasing from lateral-anterior to medial-posterior similar to Pax6 expression. While Emx2 and Pax6 expressions are restricted to proliferating neural progenitor cells, Odz4 is expressed by both proliferating and differentiating neurons. Odz1, Odz2, and Odz3 expressions are detected only in differentiating cells within cortical plate. The expression gradients suggest possible involvement of Odz in neocortical arealization. Indeed, Odz expressions are decreased and shrunk in Emx2−/− while increased and expanded in Sey/Sey cortex. EMX2 and PAX6 are not required to initiate or maintain Odz expression, but rather responsible for controlling the cortical position and area range that are permissive for Odz expression or settling of Odz-expressing cells (Li, 2006).

The arealization of neocortex depends on neuronal differentiation, positioning, and wiring. Migratory cells get instructions from diffusible chemical factors, electrical currents, or extracellular matrix (EM). Similar guiding mechanisms have been used by the neuronal growth cone to lead axons from cell soma to target tissue. Axons must grow upon solid substrate and growth cone orients with particular receptors for factors deposited in EM. There are three major EM components: collagen, proteoglycans, and large glycoproteins. ODZ has eight EGF repeats highly homologous to tenascin that is a large glycoprotein resembling fibronectin. ODZ4 is isolated as insoluble complex with cell membrane/EM during immunoprecipitation analyses. Electron microscopy combined with analytical ultracentrifugation demonstrates that ODZ can form homodimers through EGF repeat domains (Li, 2006).

ODZ may play important roles in axon guidance and cell migration. In vitro transfection studies suggested that homophilic binding of ODZ2 may be involved in the formation of synapses and fasciculation. During rat olfactory bulb regeneration, ODZ2 may facilitate precise wiring between receptor neurons and glomeruli. Interestingly, these data show that Odz2, Odz3, and Odz4 are strongly expressed in both visual cortex and dLG that are connected. ODZ may be involved in correct path-finding of TCAs to subplate, which underlies the neocortical area shift in Emx2−/− (Li, 2006).

At E18.5, there is a dramatic decrease and caudal shift of Odz4 expression in Emx2−/− dcp while the opposite is observed in Sey/Sey. Expression of Odz4 in layer 5 is, however, enhanced and evenly distributed throughout Emx2−/− cortex while there is no obvious change in Sey/Sey. Neurons of layer 5 project axons to specific subcortical targets, such as superior colliculus and spinal cord. Conceivably, corticotectal and corticospinal projections have to adjust to the shifted areas where ODZ4 might be involved (Li, 2006).

Odz1 separated from the other Odz members early during evolution and Odz1 expression pattern shifts from initially increasing to later decreasing gradient along the anterior–posterior axis during corticogenesis, suggesting that Odz1 may be important for the development of anterior cortical areas. Odz1 is also expressed in gradient by subplate cells that establish the initial connections between thalamic nuclei and neocortex. The graded expression of Odz1 polarizes the subplate, which may serve as guidance cues for invading TCAs. Interestingly, Odz1 is mapped to human X-chromosome in area Xq25, a region to which X-linked lymphoproliferative disease (XLP), X-linked mental retardation syndromes (XLMR), and “non-specific” mental retardation (MRX) have been mapped (Li, 2006).

Investigation on Odz is made complicated by the unsettled debate about whether this receptor family represents type I or type II transmembrane proteins, an important issue to resolve before designing any reasonable function studies. Based on expression data, it would be expected that Odz2, Odz3, and Odz4 may play overlapping roles in defining posterior cortical areas, while all their functions may not be redundant. Odz4 is the only Odz gene expressed in vz/svz which indicates possible roles in determining the fate of cortical progenitor cells. Odz4 is mapped to a region on mouse chromosome 7 where lethal ENU mutations exed (extraembryonic ectoderm development) and pid (preimplantation development) are localized. The mutant phenotype correlates with Odz4 expression patterns, suggesting that Odz4 might be indispensable for survival. It would be important to study in detail the functions of each Odz gene for a better understanding of cortical arealization (Li, 2006).

Emx1 and Emx2 show different patterns of expression during proliferation and differentiation of the developing cerebral cortex in the mouse

Insights into the complex structure of the forebrain and its regulation have recently come from the analysis of the expression of genes that are likely to be involved in regionalization of this structure. Four new homeobox genes have been cloned: Emx1, Emx2, Otx1 and Otx2. The expression domains of these genes in day 10 mouse embryos are within continuous regions of the developing brain and are contained within one another in the sequence Emx1 < Emx2 < Otx1 < Otx2. Recently, Otx1 has been found to be specifically expressed during neurogenesis of layer 5 and 6 in the developing cerebral cortex. In order to better understand the role of Emx1 and Emx2 in the maturation of the cortex, their expression patterns were analyzed in the developing mouse cerebral cortex, from embryonic day 12.5 to adulthood. Emx2 is expressed exclusively in proliferating cells of the ventricular zone whereas Emx1 is expressed in both proliferating and differentiated neurons throughout the cortical layers and during all the developmental stages examined. Therefore, Emx2 gene products might control some biological parameters of the proliferation of cortical neuroblasts or of the subsequent cell migration of postmitotic neurons as they leave the cortical germinal zone. Conversely, Emx1 expression, which is confined exclusively to the dorsal telencephalon, characterizes most cortical neurons during proliferation, differentiation, migration and postnatal development and maturation (Gulisano, 1996).

Emx1 and Emx2 functions in development of dorsal telencephalon

The genes Emx1 and Emx2 are mouse cognates of the Drosophila head gap gene empty spiracles. Their expression patterns in the mouse have suggested their involvement in regional patterning of the forebrain. Newborn Emx2 mutants display defects in archipallium structures that are believed to play essential roles in learning, memory and behavior: the dentate gyrus is missing, and the hippocampus and medial limbic cortex are greatly reduced in size. In contrast, defects are subtle in adult Emx1 mutant brain. In the early developing Emx2 mutant forebrain, the evagination of cerebral hemispheres is reduced and the roof between the hemispheres is expanded, suggesting the lateral shift of its boundary. Defects are not apparent, however, in the region where Emx1 expression overlaps that of Emx2, nor is any defect found in the early embryonic forebrain caused by mutation of the Emx1 gene. Expression of Emx1 principally occurs within the Emx2-positive region. Emx2 most likely delineates the palliochoroidal boundary in the absence of Emx1 expression during early dorsal forebrain patterning. In the more lateral region of telencephalon, Emx2-deficiency may be compensated for by Emx1 and vice versa. Phenotypes of newborn brains also suggest that these genes function in neurogenesis in patterns corresponding to their late regions of expression (Yoshida, 1997).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The same enhancer regulates the earliest Emx2 expression in caudal forebrain primordium, subsequent expression in dorsal telencephalon and later expression in the cortical ventricular zone

This study analyzed Emx2 enhancers to determine how Emx2 functions during forebrain development are regulated. The FB (forebrain) enhancer identified immediately 3' downstream of the last coding exon is well conserved among tetrapods and unexpectedly directed all the Emx2 expression in forebrain: caudal forebrain primordium at E8.5, dorsal telencephalon at E9.5-E10.5 and the cortical ventricular zone after E12.5. Otx, Tcf, Smad and two unknown transcription factor binding sites were essential to all these activities. The mutant that lacked this enhancer demonstrated that Emx2 expression under the enhancer is solely responsible for diencephalon development. However, in telencephalon, the FB enhancer did not have activities in cortical hem or Cajal-Retzius cells, nor was its activity in the cortex graded. Emx2 expression was greatly reduced, but persisted in the telencephalon of the enhancer mutant, indicating that there exists another enhancer for Emx2 expression unique to mammalian telencephalon (Suda, 2010).

Identification of novel binding partners for transcription factor Emx2

The mammalian homolog of Drosophila empty spiracles 2 (Emx2) is a homeobox transcription factor that plays central roles in early development of the inner ear, pelvic and shoulder girdles, cerebral cortex, and urogenital organs. The role for Emx2 is best understood within the context of the development of the neocortical region of the cortex, where Emx2 is expressed in a high posterior-medial to low anterior-lateral gradient that regulates the partitioning of the neocortex into different functional fields that perform discrete computational tasks. Despite several lines of evidence demonstrating an Emx2 concentration-dependent mechanism for establishing functional areas within the developing neocortex, little is known about how Emx2 physically carries out this role. Although several binding partners for Emx2 have been identified (including Sp8, eIF4E, and Pbx1), no screens have been used to identify potential protein binding partners for this protein. This study utilized a yeast two-hybrid screen using a library constructed from embryonic mouse cDNA in an attempt to identify novel binding partners for Emx2. This initial screen isolated two potential Emx2-binding partner proteins, Cnot6l and QkI-7. These novel Emx2-binding proteins are involved in multiple levels of mRNA metabolism that including splicing, mRNA export, translation, and destruction, thus making them interesting targets for further study (Groves, 2019).

Ems homologs and area identity in the mammalian neocortex

Continued: Empty spiracles Evolutionary homologs part 2/2

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

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