Otx2 expression in anterior neuroectoderm and forebrain/midbrain is directed by more than six enhancers.

Otx2 plays essential roles in each site at each step of head development. We previously identified the AN1 enhancer at 91kb 5' upstream for the Otx2 expressions in anterior neuroectoderm (AN) at neural plate stage before E8.5, and the FM1 enhancer at 75kb 5' upstream and the FM2 enhancer at 122kb 3' downstream for the expression in forebrain/midbrain (FM) at brain vesicle stage after E8.5. The present study identified a second AN enhancer (AN2) at 88kb 5' upstream; the AN2 enhancer also recapitulates the endogenous Otx2 expression in choroid plexus, cortical hem and choroidal roof. However, the enhancer mutants indicated the presence of another AN enhancer. The study also identified a third FM enhancer (FM3) at 153kb 5' upstream. Thus, the Otx2 expressions in anterior neuroectoderm and forebrain/midbrain are regulated by more than six enhancers located far from the coding region. The enhancers identified are differentially conserved among vertebrates; none of the AN enhancers has activities in caudal forebrain and midbrain at brain vesicle stage after E8.5, nor do any of the FM enhancers in anterior neuroectoderm at neural plate stage before E8.5.

Otx2 and Otx1 protect diencephalon and mesencephalon from caudalization into metencephalon during early brain regionalization.

Otx2 is expressed in each step and site of head development. To dissect each Otx2 function we have identified a series of Otx2 enhancers. The Otx2 expression in the anterior neuroectoderm is regulated by the AN enhancer and the subsequent expression in forebrain and midbrain later than E8.5 by FM1 and FM2 enhancers; the Otx1 expression takes place at E8.0. In telencephalon later than E9.5 Otx1 continues to be expressed in the entire pallium, while the Otx2 expression is confined to the most medial pallium. To determine the Otx functions in forebrain and midbrain development we have generated mouse mutants that lack both FM1 and FM2 enhancers (DKO: Otx2(ΔFM1ΔFM2/ΔFM1ΔFM2)) and examined the TKO (Otx1(-/-)Otx2(ΔFM1ΔFM2/ΔFM1ΔFM2)) phenotype. The mutants develop normally until E8.0, but subsequently by E9.5 the diencephalon, including thalamic eminence and prethalamus, and the mesencephalon are caudalized into metencephalon consisting of isthmus and rhombomere 1; the caudalization does not extend to rhombomere 2 and more caudal rhombomeres. In rostral forebrain, neopallium, ganglionic eminences and hypothalamus in front of prethalamus develop; we propose that they become insensitive to the caudalization with the switch from the Otx2 expression under the AN enhancer to that under FM1 and FM2 enhancers. In contrast, the medial pallium requires Otx1 and Otx2 for its development later than E9.5, and the Otx2 expression in diencepalon and mesencephalon later than E9.5 is also directed by an enhancer other than FM1 and FM2 enhancers.

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.

We have analyzed Emx2 enhancers to determine how Emx2 functions during forebrain development are regulated. The FB (forebrain) enhancer we 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.

Evolutionary origin of the Otx2 enhancer for its expression in visceral endoderm.

In the mouse, the Otx2 gene has been shown to play essential roles in the visceral endoderm during anterior-posterior axis formation and head induction. While these are primary processes in vertebrate embryogenesis, the visceral endoderm is a tissue unique to mammals. Two enhancers (VE and CM) have been previously found to direct Otx2 expression during early embryogenesis. This study demonstrates that in anterior visceral endoderm the CM enhancer does not have an activity by itself, but enhances the activity of the VE enhancer. These two enhancers also cooperate for the activities in anterior mesendoderm and cephalic mesenchyme. Comparative studies suggest that VE enhancer function was most likely established before the divergence of sarcopterygians into Actinistia, Dipnoi and tetrapods, while the nucleotide sequence corresponding to the VE enhancer was already present in the last common ancestor of bony fishes. The CM enhancer sequence and function would have been also established in ancestral sarcopterygians. The VE/CM enhancers and their gene cascades in the ancestral sarcopterygian head organizer would then have been co-opted by amphibian deep endoderm cells and mammalian visceral endoderm cells for the head development.

Evolution of Otx paralogue usages in early patterning of the vertebrate head.

To assess evolutional changes in the expression pattern of Otx paralogues, expression analyses were undertaken in fugu, bichir, skate and lamprey. Together with those in model vertebrates, the comparison suggested that a gnathostome ancestor would have utilized all of Otx1, Otx2 and Otx5 paralogues in organizer and anterior mesendoderm for head development. In this animal, Otx1 and Otx2 would have also functioned in specification of the anterior neuroectoderm at presomite stage and subsequent development of forebrain/midbrain at somite stage, while Otx5 expression would have already been specialized in epiphysis and eyes. Otx1 and Otx2 functions in anterior neuroectoderm and brain of the gnathostome ancestor would have been differentially maintained by Otx1 in a basal actinopterygian and by Otx2 in a basal sarcopterygian. Otx5 expression in head organizer and anterior mesendoderm seems to have been lost in the teleost lineage after divergence of bichir, and also from the amniotes after divergence of amphibians as independent events. Otx1 expression was lost from the organizer in the tetrapod lineage. In contrast, in a teleost ancestor prior to whole genome duplication, Otx1 and Otx2 would have both been expressed in the dorsal margin of blastoderm, embryonic shield, anterior mesendoderm, anterior neuroectoderm and forebrain/midbrain, at respective stages of head development. Subsequent whole genome duplication and the following genome changes would have caused different Otx paralogue usages in each teleost lineage. Lampreys also have three Otx paralogues; their sequences are highly diverged from gnathostome cognates, but their expression pattern is well related to those of skate Otx cognates.

Establishment of framework of the cortical area is influenced by Otx1.

Subcortical projection from layer 5 neurons is the major cortical output. A transcription factor, Otx1, which is expressed in the layer 5 subcortical projection neurons in the visual cortex, was reported to be responsible for the establishment of visual area-specific layer 5 subcortical projections by inducing the sensorimotor cortex to adopt a visual cortex identity. However, we here demonstrate that the area of corticospinal neurons shifted caudo-medially in the Otx1-null mice of which cortex is 9 tenths in size compared with that of the wild-type littermates, while the whole visual cortex did not convert to the sensorimotor cortex in the absence of Otx1. This suggests that Otx1 is not crucial for the development of visual cortex identity but for the determination of the proportion of cortical areas to the whole neocortex.

A stream of cells migrating from the caudal telencephalon reveals a link between the amygdala and neocortex.

The amygdaloid complex consists of diverse nuclei that belong to distinct functional systems, yet many issues about its development are poorly understood. Here, we identify a stream of migrating cells that form specific amygdaloid nuclei in mice. In utero electroporation showed that this caudal amygdaloid stream (CAS) originated in a unique domain at the caudal telencephalic pole that is contiguous with the dorsal pallium, which was previously thought to generate only neocortical cells. The CAS and the neocortex share mechanisms for specification (transcription factors Tbr1, Lhx2 and Emx1/2) and migration (reelin and Cdk5). Reelin, a critical cue for migration in the neocortex, and Cdk5, which is specifically required for migration along radial glia in the neocortex, were both selectively required for the normal migration of the CAS, but not for that of other amygdaloid nuclei. This is first evidence of a dorsal pallial contribution to the amygdala, demonstrating a developmental and mechanistic link between the amygdala and the neocortex.

Evolutionary constraint on Otx2 neuroectoderm enhancers-deep conservation from skate to mouse and unique divergence in teleost.

Otx2 is a paired type homeobox gene that plays essential roles in each step and site of head development in vertebrates. In the mouse, Otx2 expression in the anterior neuroectoderm is regulated primarily by two distinct enhancers: anterior neuroectoderm (AN) and forebrain/midbrain (FM) enhancers at 92 kb and 75 kb 5'of the Otx2 locus, respectively. The AN enhancer has activity in the entire anterior neuroectoderm at headfold and early somite stages, whereas the FM enhancer is subsequently active in the future caudal forebrain and midbrain ectoderm. In tetrapods, both AN and FM enhancers are conserved, whereas the AN region is missing in teleosts, despite overt Otx2 expression in the anterior neuroectoderm. Here, we show that zebrafish and fugu FM regions drive expression not only in the forebrain and midbrain but also in the anterior neuroectoderm at headfold stage. The analysis of coelacanth and skate genomic Otx2 orthologues suggests that the utilization of the two enhancers, AN and FM, is an ancestral condition. In contrast, the AN enhancer has been specifically lost in the teleost lineage with a compensatory establishment of AN activity within the FM enhancer. Furthermore, the AN activity in the fish FM enhancer was established by recruiting upstream factors different from those that direct the tetrapod AN enhancer, yet zebrafish FM enhancer is active in both mouse and zebrafish anterior neuroectoderm at the headfold stage.

The c-Jun N-terminal kinase activator dual leucine zipper kinase regulates axon growth and neuronal migration in the developing cerebral cortex.

Mammalian corticogenesis substantially depends on migration and axon projection of newborn neurons that are coordinated by a yet unidentified molecular mechanism. Dual leucine zipper kinase (DLK) induces activation of c-Jun N-terminal kinase (JNK), a molecule that regulates morphogenesis in various organisms. We show here, using gene targeting in mice, that DLK is indispensable for establishing axon tracts, especially those originating from neocortical pyramidal neurons of the cerebrum. Direct and quantitative analysis of radial migration of pyramidal neurons using slice culture and a time-lapse imaging system revealed that acceleration around the subplate was affected by DLK gene disruption and by administration of a JNK inhibitor. Phosphorylation of JNK substrates, including c-Jun and doublecortin, and of JNK itself at the activation loop were partially affected in brains of DLK-deficient mouse embryos. These data suggest that DLK plays a significant role in the coordinated regulation of radial migration and axon projection by modulating JNK activity.

Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions.

Fez and Fez-like (Fezl) are zinc-finger genes that encode transcriptional repressors expressed in overlapping domains of the forebrain. By generating Fez;Fezl-deficient mice we found that a redundant function of Fez and Fezl is required for the formation of diencephalon subdivisions. The caudal forebrain can be divided into three transverse subdivisions: prethalamus (also called ventral thalamus), thalamus (dorsal thalamus) and pretectum. Fez;Fezl-deficient mice showed a complete loss of prethalamus and a strong reduction of the thalamus at late gestation periods. Genetic marker analyses revealed that during early diencephalon patterning in Fez;Fezl-deficient mice, the rostral diencephalon (prospective prethalamus) did not form and the caudal diencephalon (prospective thalamus and pretectum) expanded rostrally. Fez;Fezl-deficient mice also displayed defects in the formation of the zona limitans intrathalamica (ZLI), which is located on the boundary between the prethalamus and thalamus. Fez and Fezl are expressed in the region rostral to the rostral limit of Irx1 expression, which marks the prospective position of the ZLI. Transgene-mediated misexpression of Fezl or Fez caudal to the ZLI repressed the caudal diencephalon fate and affected the formation of the Shh-expressing ZLI. These data indicate that Fez and Fezl repress the caudal diencephalon fate in the rostral diencephalon, and ZLI formation probably depends on Fez/Fezl-mediated formation of diencephalon subdivisions.

A new serine/threonine protein kinase, Omphk1, essential to ventral body wall formation.

Here, we report a new serine/threonine protein kinase of the SNF1 subfamily Omphk1. Two Omphk homologues exist in each vertebrate species, and one homologue exists in Drosophila and Caenorhabditis elegans; the kinase domain is highly conserved among these homologues, and several domains are conserved among vertebrate Omphk. Omphk1 expression dynamically changes in the developing central nervous system, is found ubiquitously in epidermis, and is present uniquely in several other tissues. Its expression is also found in each tissue associated with the ventral body wall closure: the primary body wall composed of primitive ectoderm and each component of the secondary body wall. Concomitantly, its null mutant exhibits omphalocele with a failure in closure of the secondary body wall. There are no apparent gross morphological defects in brain, however, despite the unique Omphk1 expression in this tissue.

Generation of trans-mitochondrial mice carrying homoplasmic mtDNAs with a missense mutation in a structural gene using ES cells.

Generation of various kinds of trans-mitochondrial mice, mito-mice, each carrying mtDNAs with a different pathogenic mutation, is required for precise investigation of the pathogenesis of mitochondrial diseases. This study used two respiration-deficient mouse cell lines as donors of mtDNAs with possible pathogenic mutations. One cell line expressed 45-50% respiratory activity due to mouse mtDNAs with a T6589C missense mutation in the COI gene (T6589C mtDNA) and the other expressed 40% respiratory activity due to rat (Rattus norvegicus) mtDNAs in mouse cells. By cytoplasmic transfer of these mtDNAs to mouse ES cells, we isolated respiration-deficient ES cells. We obtained chimeric mice and generated their F(6) progeny carrying mouse T6589C mtDNAs by its female germ line transmission. They were respiration-deficient and thus could be used as models of mitochondrial diseases caused by point mutations in mtDNA structural genes. However, chimeric mice and mito-mice carrying rat mtDNAs were not obtained, suggesting that significant respiration defects or some deficits induced by rat mtDNAs in mouse ES cells prevented their differentiation to generate mice carrying rat mtDNAs.

Application of ES cells for generation of respiration-deficient mice carrying mtDNA with a large-scale deletion.

In a previous study, we used mouse zygotes as recipients of mtDNA with a large-scale deletion mutation (DeltamtDNA) and generated respiration-deficient mice (mito-mice) carrying DeltamtDNA. In this study, we used mouse ES cells as recipients of DeltamtDNA, and generated mito-mice with DeltamtDNA only when the ES cells carried 17% DeltamtDNA. No chimera mice or their F(1) progenies were obtained from ES cells carrying more than 61% DeltamtDNA. These observations suggest that respiratory defects of ES cells inhibit their normal differentiation into chimera mice and mito-mice, and that ES cells are more effective than zygotes for generation of mito-mice carrying mtDNAs without significant pathogenic mutations.

Sphingosine 1-phosphate acts as a signal molecule in ceramide signal transduction of TNF-alpha-induced activator protein-1 in osteoblastic cell line MC3T3-E1 cells.

We previously demonstrated that tumor necrosis factor (TNF)-alpha stimulated the production of activation protein (AP)-1, a transcriptional factor, in mouse osteoblastic MC3T3-E1 cells. Recent studies have shown the importance of ceramide and its metabolites as signal molecules for TNF-alpha-induced gene expression in several cell types. Therefore, our interest was to investigate whether sphingosine metabolites are involved in TNF-alpha-induced signaling in MC3T3-E1 cells. DL-threo-1-phenyl-2-hexadecanoyl-amino-3-pyrrolidino-1-propanol (PPPP), which causes accumulation of intracellular ceramide, stimulated the TNF-alpha-induced expression of the c-fos and c-jun genes. Gel shift assay clearly showed that PPPP increased the cytokine-induced specific binding of nuclear proteins to the 12-tetra-decanoyl phorbol 13-acetate-responsive element (TRE), a consensus sequence for AP-1. In addition, cell-permeable ceramide (N-acetylsphingosine, N-hexanoylsphingosine or N-octanoylsphingosine) stimulated expression of the c-fos and c-jun genes and nuclear protein binding to TRE. Interestingly, DL-threo-dihydrosphingosine (DHS), an inhibitor of sphingosine kinase, clearly blocked the ceramide analogue-induced stimulation. Sphingosine 1-phosphate (SPP) actually induced expression of these oncogenes and activated AP-1. Although TNF-alpha stimulated the AP-1-mediated expression of the monocyte chemoattractant JE/MCP-1, this stimulation was inhibited by DHS. SPP also stimulated JE/MCP-1 gene expression. The present study thus suggests that SPP acts as a signal molecule in ceramide-dependent signal transduction in TNF-alpha-induced AP-1 in osteoblastic MC3T3-E1 cells.

Emx2 and Pax6 function in cooperation with Otx2 and Otx1 to develop caudal forebrain primordium that includes future archipallium.

One of the central issues in developmental neurobiology is how the forebrain is organized ontogenetically. The traditional view is that the anterior neuroectoderm first develops into mesencephalic and prosencephalic vesicles; the latter vesicle subsequently develops into the diencephalon and secondary prosencephalon, of which dorsal parts protrude to generate the telencephalon. The diencephalon yields the pretectum, thalamus, and prethalamus, and the telencephalon produces the archipallium, neopallium, and ganglionic eminences. By identifying cell descendants that once expressed Emx2 with use of the Cre knock-in mutant into the Emx2 locus and analyzing phenotypes of double mutants between Emx2 and Otx2/Otx1 and between Emx2 and Pax6, we propose that at the 3-6 somite stage, the anterior neuroectoderm develops into three primordia: midbrain, caudal forebrain, and rostral forebrain. The caudal forebrain primordium generates not only the pretectum, thalamus, and prethalamus but also the archipallium, cortical hem, choroid plexus, choroidal roof, and eminentia thalami. The primordium corresponds to the Emx2- or Pax6-positive region at the 3-6 somite stage that most probably does not include the future neopallium or commissural plate. Otx2 and Otx1 that are expressed in the entire future forebrain and midbrain cooperate with this Emx2 and Pax6 expression in the development of the caudal forebrain primordium; Emx2 and Pax6 functions are redundant. In the embryonic day 9.5 Emx2-/-Pax6-/- double mutant, the caudal forebrain remained unspecified and subsequently transformed into tectum in a mirror image of the endogenous one.

cfm is a novel gene uniquely expressed in developing forebrain and midbrain, but its null mutant exhibits no obvious phenotype.

A novel gene, cfm, that is expressed uniquely during early forebrain and midbrain development was isolated, and its null mutant was generated. cfm does not have any known functional domains, but is conserved in human, chick, Xenopus and zebrafish; a site of phosphorylation by MAP kinase exists in one of the domains conserved among them. Its expression was initially found at the 5-somite stage in the future midbrain and caudal forebrain region. The expression in mesencephalon subsequently decreased, was found in a stripe in the mid mesencephalon at E9.0. The expression in diencephalon was restricted to the dorsal thalamic region by E9.5 and to epiphysis at E12.5. In mouse a cognate, cfm2, exists that is expressed uniquely in the somite just formed and the presomite to be segmented, but not in forebrain or midbrain during early development. However, the cfm null mutant was live-born without any apparent defects.

ang is a novel gene expressed in early neuroectoderm, but its null mutant exhibits no obvious phenotype.

To find genes that play roles in initial regionalization of anterior neuroectoderm, 15 novel genes were isolated that are expressed in anterior neuroectoderm at E8.0-E8.5. Moreover, to assess their functions by generation of mutant mice a conventional targeting strategy was designed, exploiting the availability of accurate long amplification PCR and BAC library that is coupled with genome information, in C57BL/6 strain. The ang is one of such genes; it has no known functional domains or no cognates, but is conserved not only in vertebrates, but also in Drosophila. Its expression was initially found throughout neuroectoderm at E7.5; subsequently the expression became high in rostral brain and caudal neuropore regions and low in hindbrain and spinal cord regions. At E12.5 the expression was found in undifferentiated neuroepithelium in ventricular zone, dorsal root ganglia and several non-neural tissues. However, ang null mutant was live-born without any apparent defects.

Zinc finger gene fez-like functions in the formation of subplate neurons and thalamocortical axons.

fez-like (fezl) is a forebrain-expressed zinc finger gene required for the formation of the hypothalamic dopaminergic and serotonergic (monoaminergic) neurons in zebrafish. To reveal its function in mammals, we analyzed the expression of the mouse orthologue of fezl and generated fezl-deficient mice by homologous recombination. Mouse fezl was expressed specifically in the forebrain from embryonic day 8.5. At mid-gestation, fezl expression was detected in subdomains of the forebrain, including the dorsal telencephalon and ventral diencephalon. Unlike the zebrafish fezl mutant too few, the fezl-deficient mice displayed normal development of hypothalamic monoaminergic neurons, but showed abnormal "hyperactive" behavior. In fezl(-/-) mice, the thalamocortical axons (TCA) were reduced in number and aberrantly projected to the cortex. These mutants had a reduced number of subplate neurons, which are involved in guiding the TCA from the dorsal thalamus, although the subplate neurons were born normally. These results suggest that fezl is required for differentiation or survival of the subplate neurons, and reduction of the subplate neurons in fezl-deficient mice leads to abnormal development of the TCA, providing a possible link between the transcriptional regulation of forebrain development and hyperactive behavior.

Emx1 and Emx2 cooperate in initial phase of archipallium development.

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

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.

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. In the present study we show that Emx1 and Emx2 double mutant neocortex is much more severely affected. Thickness of the cerebral wall was diminished with the decrease in cell number. Bromodeoxyuridine uptake in the germinal zone was 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 was greatly inhibited. The wild-type ganglionic eminence cells transplanted into Emx1/2-double mutant telencephalon did not move to the cortex. MAP2-positive neuronal bodies and RC2-positive radial glial cells emerged normally, but the laminar structure subsequently formed was completely abnormal. Furthermore, both corticofugal and corticopetal fibers were predominantly absent in the cortex. Most importantly, neither Cajal-Retzius cells nor subplate neurons were 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.