orthodenticle

EVOLUTIONARY HOMOLOGS

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Orthodenticle homologs and mammalian brain development (part 2 of 2)

How gene activity is translated into phenotype and how it can modify morphogenetic pathways is of central importance when studying the evolution of regulatory control mechanisms. Previous studies in mouse have suggested that, despite the homeodomain-restricted homology, Drosophila orthodenticle (otd) and murine Otx1 genes share functional equivalence and that translation of Otx2 mRNA in epiblast and neuroectoderm might require a cell type-specific post-transcriptional control depending on its 5' and 3' untranslated sequences (UTRs). In order to study whether OTD is functionally equivalent to OTX2 and whether synthesis of OTD in epiblast is molecularly dependent on the post-transcriptional control of Otx2 mRNA, a first mouse model (otd²) was generated in which an Otx2 region including 213 bp of the 5' UTR, exons, introns and the 3' UTR was replaced by an otd cDNA and a second mutant (otd^2FL) was generated, replacing only exons and introns of Otx2 with the otd coding sequence fused to intact 5' and 3' UTRs of Otx2. otd² and otd^2FL mRNAs were properly transcribed under the Otx2 transcriptional control, but mRNA translation in epiblast and neuroectoderm occurred only in otd^2FL mutants. Phenotypic analysis has revealed that visceral endoderm (VE)-restricted translation of otd² mRNA is sufficient to rescue Otx2 requirement for early anterior patterning and proper gastrulation but it fails to maintain forebrain and midbrain identity. Importantly, epiblast and neuroectoderm translation of otd^2FL mRNA rescues maintenance of anterior patterning as it did in a third mouse model replacing (as in otd^2FL) exons and introns of Otx2 with an Otx2 cDNA (Otx2^2c). The molecular analysis has revealed that Otx2 5' and 3' UTR sequences, deleted in the otd² mRNA, are required for nucleo-cytoplasmic export and epiblast-restricted translation. Indeed, these molecular impairments were completely rescued in otd^2FL and Otx2^2c mutants. These data provide novel in vivo evidence supporting the concept that during evolution pre-existing gene functions have been recruited into new developmental pathways by modifying their regulatory control (Acampora, 2001).

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

The most studied secondary neural organizer is the isthmic organizer, which is localized at the mid-hindbrain transition of the neural tube and controls the anterior hindbrain and midbrain regionalization. Otx2 and Gbx2 expressions are fundamental for positioning the organizer and the establishment of molecular interactions that induce Fgf8. Evidence in this study demonstrates that Otx2 and Gbx2 have an overlapping expression in the isthmic region. This area is the transversal domain where expression of Fgf8 is induced. The Fgf8 protein produced in the isthmus stabilizes and up-regulates Gbx2 expression, which, in turn, down-regulates Otx2 expression. The inductive effect of the Gbx2/Otx2 limit keeps Fgf8 expression stable and thus maintains its positive role in the expression of Pax2, En1,2 and Wnt1 (Garda, 2001).

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

Otx genes play an important role in brain development. Previous mouse models have suggested that the untranslated regions (UTRs) of Otx2 mRNA may contain regulatory element(s) required for Otx2's post-transcriptional control in epiblast and neuroectoderm. In order to study this, the 3' UTR of Otx2 was perturbed by inserting a small fragment of DNA from the lambda phage. Otx2lambda mutants exhibit proper gastrulation and normal patterning of the early anterior neural plate, but from 8.5 days of development they develop severe forebrain and midbrain abnormalities. OTX2 protein levels in Otx2lambda mutants are heavily reduced in the epiblast, axial mesendoderm and anterior neuroectoderm but not in the visceral endoderm. At the molecular level, it was found that the ability of the Otx2lambda mRNA to form efficient polyribosome complexes is impaired. Sequence analysis of the Otx2-3' UTR reveals a 140 bp long element that is present only in vertebrate Otx2 genes and conserved in identity by over 80%. These data provide experimental evidence that murine brain development requires accurate translational control of Otx2 mRNA in epiblast and neuronal progenitor cells. This leads to the hypothesis that this control might have important evolutionary implications. The establishment of this type of translational control seems to correlate with the transition to a more complicated vertebrate brain, that of gnathostomes (jawed vertebrates). Indeed, in agnathes (lamprey), only a small portion of the element was conserved and noteworthy: even though the basic organization of the rostral CNS was similar to gnathostomes, the topography and relative extent of different areas appears remarkably different (Boyl, 2001).

Reduction below a critical threshold or ectopic gain of OTX2 protein correspond to reduction or increase in the size of forebrain and midbrain territory, respectively. Otx1-/-; Otx2+/- double mutants display transformation of midbrain and posterior diencephalon in an expanded metencephalon; in hOtx12/hOtx12 embryos, the entire forebrain and midbrain are replaced with the metencephalon, while, by contrast, mice expressing Otx2 under En1 transcriptional control move the MHB posteriorly and increase the extent of the posterior midbrain. Otx2lambda mutants exhibit additional hypomorphic phenotypes: Otx2lambda/Otx2lambda mutants are similar to Otx1-/-; Otx2+/- double mutants or reveal a moderate anterior repositioning of the MHB and additional abnormalities affecting the size and the shape of forebrain, as well as the closure of the neural tube: 90% of Otx2lambda/- mutants were almost headless. Even a relatively small reduction of OTX2 protein below a critical threshold corresponding to a normal single copy is phenotypically translated into head abnormalities. Indeed, compared with Otx2+/- embryos that are viable and fertile, Otx2lambda/Otx2lambda show a 10% reduction in the OTX2 protein and 65% of them reveal head abnormalities. Therefore, these results provide evidence that Otx2 may have developed a regulatory mechanism that allows its efficient translation in epiblast and derived tissues. This positive control has permitted the availability of a critical amount of OTX2 protein sufficient and necessary to maintain and/or stabilize early forebrain and midbrain regional identities (Boyl, 2001).

Otx2 is required in the neuroectoderm for development of the forebrain region. In order to elucidate the precise role of Otx2 in forebrain development, attempts were made to generate an allelic series of Otx2 mutations by Flp- and Cre-mediated recombination for the production of conditional knock-out mice. Unexpectedly, the neo-cassette insertion created a hypomorphic Otx2 allele; consequently, the phenotype of compound mutant embryos carrying both a hypomorphic and a null allele (Otx2^frt-neo/-) was analyzed. Otx2^frt-neo/- mutant mice die at birth, displaying rostral head malformations. Molecular marker analysis demonstrated that Otx2^frt-neo/- mutant embryos appear to undergo anterior-posterior axis generation and induction of anterior neuroectoderm normally; however, these mutants subsequently fail to correctly specify the forebrain region. Since the rostral margin of the neural plate, termed the anterior neural ridge (ANR), plays crucial roles with respect to neural plate specification, expression of molecular markers for the ANR and the neural plate was examined; moreover, neural plate explant studies were performed. Analyses revealed that telencephalic gene expression does not occur in mutant embryos due to defects of the neural plate; however, the mutant ANR gives rise to normal induction activity on gene expression. These results further suggest that Otx2 dosage may be crucial in the neural plate with respect to response to inductive signals primarily from the ANR for forebrain specification (Tian, 2002).

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

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

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

Otx2 and Gbx2 are among the earliest genes expressed in the neuroectoderm, dividing it into anterior and posterior domains with a common border that marks the mid-hindbrain junction. Otx2 is required for development of the forebrain and midbrain, and Gbx2 for the anterior hindbrain. Furthermore, opposing interactions between Otx2 and Gbx2 play an important role in positioning the mid-hindbrain boundary, where an organizer forms that regulates midbrain and cerebellum development. The expression domains of Otx2 and Gbx2 are initially established independently of each other at the early headfold stage, and then their expression rapidly becomes interdependent by the late headfold stage. Since the repression of Otx2 by retinoic acid is dependent on an induction of Gbx2 in the anterior brain, molecules other than retinoic acid must regulate the initial expression of Otx2 in vivo. In contrast to previous suggestions that an interaction between Otx2- and Gbx2-expressing cells may be essential for induction of mid-hindbrain organizer factors such as Fgf8, it has been found that Fgf8 and other essential mid-hindbrain genes are induced in a correct temporal manner in mouse embryos deficient for both Otx2 and Gbx2. However, expression of these genes is abnormally co-localized in a broad anterior region of the neuroectoderm. By removing Otx2 function, development of rhombomere 3 is rescued in Gbx2^–/– embryos, showing that Gbx2 plays a permissive, not instructive, role in rhombomere 3 development. These results provide new insights into induction and maintenance of the mid-hindbrain genetic cascade by showing that a mid-hindbrain competence region is initially established independent of the division of the neuroectoderm into an anterior Otx2-positive domain and posterior Gbx2-positive domain. Furthermore, Otx2 and Gbx2 are required to suppress hindbrain and midbrain development, respectively, and thus allow establishment of the normal spatial domains of Fgf8 and other genes (Li, 2001).

The specification of distinct neuronal cell-types is controlled by inducing signals whose interpretation in distinct areas along the central nervous system provides neuronal progenitors with a precise and typical expression code of transcription factors. To gain insights into this process, the role of Otx2 was investigated in the specification of identity and fate of neuronal progenitors in the ventral midbrain. To achieve this, Otx2 was inactivated by Cre recombinase under the transcriptional control of En1. Lack of Otx2 in the ventrolateral and posterior midbrain results in a dorsal expansion of Shh expression and in a dorsal and anterior rotation of the midbrain-hindbrain boundary and Fgf8 expression. Indeed, in this mutant correct positioning of the ventral site of midbrain-hindbrain boundary and Fgf8 expression are efficiently controlled by Otx1 function, thus allowing the study of the identity and fate of neuronal progenitors of the ventral midbrain in the absence of Otx2. The results suggest that Otx2 acts in two ways: by repressing Nkx2.2 in the ventral midbrain and maintaining the Nkx6.1-expressing domain through dorsal antagonism on Shh. Failure of this control affects the identity code and fate of midbrain progenitors, which exhibit features in common with neuronal precursors of the rostral hindbrain even though the midbrain retains its regional identity and these neuronal precursors are rostral to Fgf8 expression. Dopaminergic neurons are greatly reduced in number, red nucleus precursors disappear from the ventral midbrain where a relevant number of serotonergic neurons are generated. These results indicate that Otx2 is an essential regulator of the identity, extent and fate of neuronal progenitor domains in the ventral midbrain and provide novel insights into the mechanisms by which neuronal diversity is generated in the central nervous system (Puelles, 2004).

The transcription factor Otx2 is required to determine mesencephalic versus metencephalic (cerebellum/pons) territory during embryogenesis. This function of Otx2 primarily involves positioning and maintaining the mid-hindbrain organizer at the border between midbrain and anterior hindbrain. Otx2 expression is maintained long after this organizer is established. Conditional mutants of Otx2 were generated using the Cre/loxP system to study later roles during rostral brain development. For inactivation of Otx2 in neuronal progenitor cells, Otx2^flox/flox animals were crossed with Nestin-Cre transgenic animals. In Nestin-Cre/+; Otx2^flox/flox embryos, Otx2 activity was lost from the ventral midbrain starting at embryonic day 10.5 (E10.5). In these mutant embryos, the mid-hindbrain organizer was properly positioned at E12.5, although Otx2 is absent from the midbrain. Hence, the Nestin-Cre/+; Otx2^flox/flox animals represent a novel mouse model for studying the role of Otx2 in the midbrain, independently of abnormal development of the mid-hindbrain organizer. These data demonstrate that Otx2 controls the development of several neuronal populations in the midbrain by regulating progenitor identity and neurogenesis. Dorsal midbrain progenitors ectopically expressed Math1 and generate an ectopic cerebellar-like structure. Similarly, Nkx2.2 ectopic expression ventrally into tegmentum progenitors is responsible for the formation of serotonergic neurons and hypoplasia of the red nucleus in the midbrain. In addition, a novel role was discovered for Otx2 in regulating neurogenesis of dopaminergic neurons. Altogether, these results demonstrate that Otx2 is required from E10.5 onward to regulate neuronal subtype identity and neurogenesis in the midbrain (Vernay, 2005).

The thalamic complex is the major sensory relay station in the vertebrate brain and comprises three developmental subregions: the prethalamus, the thalamus and an intervening boundary region -- the zona limitans intrathalamica (ZLI). Shh signalling from the ZLI confers regional identity of the flanking subregions of the ZLI, making it an important local signalling centre for regional differentiation of the diencephalon. However, understanding of the mechanisms responsible for positioning the ZLI along the neural axis is poor. This study shows that, before ZLI formation, both Otx1l and Otx2 (collectively referred to as Otx1l/2) are expressed in spatially restricted domains. Formation of both the ZLI and the Irx1b-positive thalamus require Otx1l/2; embryos impaired in Otx1l/2 function fail to form these areas, and, instead, the adjacent pretectum and, to a lesser extent, the prethalamus expand into the mis-specified area. Conditional expression of Otx2 in these morphant embryos cell-autonomously rescues the formation of the ZLI at its correct location. Furthermore, absence of thalamic Irx1b expression, in the presence of normal Otx1l/2 function, leads to a substantial caudal broadening of the ZLI by transformation of thalamic precursors. It is therefore proposed that the ZLI is induced within the competence area established by Otx1l/2, and is posteriorly restricted by Irx1b (Scholpp, 2007).

Meso-diencephalic dopaminergic (mdDA) neurons control voluntary movement, cognition and the reward response, and their degeneration is associated with Parkinson's disease (PD). Prospective cell transplantation therapies for PD require full knowledge of the developmental pathways that control mdDA neurogenesis. Otx2 is required for the establishment of the mesencephalic field and molecular code of the entire ventral mesencephalon (VM). This study investigated whether Otx2 is a specific determinant of mesencephalic dopaminergic (mesDA) neurogenesis by studying mouse mutants that conditionally overexpress or lack Otx2. The data show that Otx2 overexpression in the VM causes a dose-dependent and selective increase in both mesDA progenitors and neurons, which correlates with a remarkable and specific enhancement in the proliferating activity of mesDA progenitors. Consistently, lack of Otx2 in the VM specifically affects the proliferation of Sox2+ mesDA progenitors and causes their premature post-mitotic transition. Analysis of the developmental pathway that controls the differentiation of mesDA neurons shows that, in the absence of Otx2, the expression of Lmx1a and Msx1, and the proneural genes Ngn2 and Mash1 is not activated in Sox2+ mesDA progenitors, which largely fail to differentiate into Nurr1+ mesDA precursors. Furthermore, proliferation and differentiation abnormalities exhibit increasing severity along the anterior-posterior (AP) axis of the VM. These findings demonstrate that Otx2, through an AP graded effect, is intrinsically required to control proliferation and differentiation of mesDA progenitors. Thus, these data provide new insights into the mechanism of mesDA neuron specification and suggest Otx2 as a potential target for cell replacement-based therapeutic approaches in PD (Omodei, 2008).

Meis2 competes with the Groucho co-repressor Tle4 for binding to Otx2 and specifies tectal fate without induction of a secondary midbrain-hindbrain boundary organizer

The transcription factor Otx2 is expressed throughout the anterior neuroectoderm and is required for the formation of all forebrain- and midbrain-derived structures. The molecular determinants that cooperate with Otx2 to subdivide its expression domain into distinct functional units are, however, poorly understood at present. This study shows that the TALE-homeodomain protein Meis2 is expressed in the chick tectal anlage and is both necessary and sufficient for tectal development. Unlike known tectum-inducing genes, the ability of Meis2 to initiate tectal development does not involve the formation of a secondary midbrain-hindbrain boundary organizer, but instead requires direct interaction with Otx2. Using an Otx2-dependent reporter assay it was demonstrated that Meis2 competes with the Groucho co-repressor Tle4 (Grg4) for binding to Otx2 and thereby restores Otx2 transcriptional activator function. Together, these data suggest a model in which the balance between a co-repressor and a co-activator, which compete for binding to Otx2 in the mesencephalic vesicle, provides spatial and temporal control over tectal development. Controlled formation of Meis2-containing higher order protein complexes might thus serve as a general mechanism to achieve subdivision of the anterior neuroectoderm into distinct functional units during embryogenesis (Agoston, 2009).

Meis2 is a key regulator of tectal development. In contrast to other known genes involved in tectal development, Meis2 initiates tectal fate specification without inducing a secondary MHB organizer. Instead, Meis2 binds to Otx2 in the absence of DNA, competes with the co-repressor Tle4 for binding to Otx2 and thereby restores Otx2 transcriptional activator function. As discussed below, these results suggest a model in which the balance between a co-repressor and a co-activator, which compete for binding to Otx2 in the mesencephalic vesicle, provides spatial and temporal control over the onset of tectal development. These data thus argue for a novel, potentially DNA-independent function of TALE-homeodomain proteins: the controlled assembly and disassembly of transcription regulator complexes (Agoston, 2009).

Tectum development is induced when an ectopic Fgf8 source is generated in the prosencephalon through transplantation of an ectopic MHB organizer or implantation of Fgf8-releasing beads into the lateral wall of the diencephalon. In addition to Fgf8, several transcription factors can trigger tectal development upon misexpression, including Otx2, Pax2/5, En1/2 and Pax3/7. Unlike Meis2, expression of these proteins is not specific for the tectal anlage. Moreover, each of these proteins participates in the interdependent, positive maintenance loop at the MHB organizer and, consequently, induces ectopic expression of MHB marker genes, including Fgf8, when misexpressed. These molecules therefore evoke tectal development indirectly through formation of an ectopic MHB organizer. Meis2, by contrast, is unique as it can initiate tectal development without participating in MHB organizer function or maintenance. Endogenous Meis2 expression is repressed when metencephalic development is experimentally induced through activation of the Ras-MAP kinase pathway in the mesencephalon and is upregulated concomitantly to the metencephalic-to-mesencephalic fate change that occurs when Ras-MAP kinase signaling is blocked in rhombomere 1 (Vennemann, 2008; Agoston, 2009).

Meis2 expression must therefore be directly or indirectly under control of the MHB organizer. Notably, a single, transient transfection of Meis2 in the diencephalic alar plate at the 10- to 11-somite stage was sufficient to initiate long-term expression of endogenous Meis2 in transfected cells. Meis2, once induced, can therefore stabilize its own expression. Together, these results suggest a model in which regulation of tectal development by signals from the MHB is mediated via induction and subsequent auto-maintenance of Meis2 expression (Agoston, 2009).

Meis family proteins act as co-factors of other transcriptional regulators. To date, Meis-interacting proteins have been isolated from non-neuronal tissue and the posterior hindbrain, yet Meis co-factors in the developing anterior brain have remained elusive. GST pull-down experiments from tectal extracts or with in vitro translated proteins as well as co-immunoprecipitation experiments were performed with native tectal proteins to demonstrate direct interaction of Meis2 and Otx2 during early midbrain development. Using deletion constructs of Otx2, it was found that complex formation requires a short motif N-terminal of the Otx2 homeodomain. The region of the Otx2 polypeptide chain that contacts Meis2 thus differs from the tryptophan-containing hexapeptide that mediates cooperative DNA binding of Hox or myogenic bHLH proteins with TALE-homeodomain proteins. Meis family proteins can therefore interact with different protein motifs present in a variety of transcription factors (Agoston, 2009).

Employing an Otx2-dependent reporter assay, evidence was provided that Meis2 competes with the co-repressor Tle4 for binding to Otx2. Tle4 expression begins in the anterior primitive streak (thus preceding that of Meis2), is later strong in the anterior neural tube and decreases after the 20- to 25-somite stage. Tle4 binding to Otx2 was previously shown be required for the ability of Otx2 to repress Fgf8 anterior to the MHB, an important step in the formation and stabilization of the MHB organizer. Overexpression of Tle4 in the mesencephalic vesicle, in turn, disrupts normal development and lamination of the optic tecta. Together with the results presented in this study, these data might allow reconstruction of the probable temporal sequence of tectal fate specification in the embryo. In the anterior neural plate and anterior neural tube at early somite stages, Tle4 is co-expressed with Otx2 but Meis2 is missing. In the absence of Meis2, Otx2 and Tle4 can interact, prevent precocious tectal differentiation and inhibit Fgf8 expression anterior to the organizer, which stabilizes the MHB signaling center (Agoston, 2009).

Meis2 expression in the mesencephalic alar plate begins at HH11-12 (13-16 somites) and is strong from the 20- to 22-somite stage onwards, at which time the MHB organizer is established. Meis2 competes with Tle4 for binding to Otx2 in the tectal anlage, releases Otx2 from Groucho-mediated repression and thereby allows tectal development to commence (Agoston, 2009).

If correct, two predictions can be drawn from this model. First, loss of Tle4 from the diencephalic vesicle (where Tle4 is co-expressed with Otx2 at early somite stages) should lead to derepression of tectal genes. Second, precocious and ectopic expression of Meis2 in the MHB territory may destabilize the Fgf8 expression domain through premature restoration of Otx2 transcriptional activator function. Indeed, as previously demonstrated, transfection of a putative dominant-negative form of Tle4 - a truncation comprising only the first 203 amino acids of the protein - into the diencephalic vesicle causes widespread ectopic induction of En2 transcripts. In addition, when Meis2 was ectopically introduced into the MHB region at the 4- to 6-somite stage, small ectopic patches of Fgf8 transcripts anterior to the normal Fgf8 expression domain at the MHB were visible. These ectopic patches of Fgf8 expression might correspond to cells that have escaped Fgf8 downregulation by Otx2-Tle4 during the period of MHB organizer formation owing to the precocious inactivation of the Otx2-Tle4 complex by Meis2HA (Agoston, 2009).

Meis2 had to be transfected in excess to Tle4 in order to restore Otx2 transactivation in the Otx2-dependent reporter assays. This observation is consistent with the fact that between the 24- and 44-somite stages, Meis2 transcripts are abundant in the dorsal midbrain, whereas Tle4 expression is barely detectable. Tle4 thus appears to bind to Otx2 with higher affinity than does Meis2, which might allow for tight control over the tectum-inducing activity of Otx2. Recently, the spatial-temporal windows of Otx2 control over head, brain and body development were defined by Tamoxifen-induced deletion of Otx2. Interestingly, Otx2 deletion at E10.5-12.5 resulted in a mesencephalic-to-metencephalic fate change without shifting the molecular MHB. Hence, the interaction of Otx2 with Meis2 in the tectal anlage reported in this study occurs at a similar developmental stage to that at which Otx2 is required for mesencephalic fate determination but not MHB organizer positioning (Agoston, 2009).

Possible targets of Otx2/Meis2 include ephrin B1 (Efnb1) and Dbx1, both of which carry several potential consensus Bicoid- and Meis-binding sites upstream of their transcriptional start sites. Direct regulation of a midbrain-specific regulatory element of the EphA8 gene by Meis2 has also been demonstrated in mice. However, because Meis2 binding to Otx2 does not require either protein to be bound to DNA, Meis2-Otx2 interaction and restoration of Otx2 transcriptional activator function might in fact take place before both proteins have contacted the regulatory elements of downstream genes. Regulation of gene expression by putative transcription factors independent of DNA binding is not unprecedented. For instance, several Hox proteins can modulate gene expression by inhibiting the activity of CBP histone acetyltransferases (HATs) without forming DNA-binding complexes with CBP HAT. Meis family members might therefore affect gene expression by multiple, DNA-dependent and -independent mechanisms. This view is supported by the fact that despite the identification of Meis proteins as transcriptional co-factors, few direct target genes of these proteins have been reported to date (Agoston, 2009).

In summary, the results reported in this study strongly suggest that Tle4 and Meis2 compete for binding to Otx2 in the mesencephalic vesicle and that the balance between these proteins provides spatial and temporal control over the onset of tectal differentiation. Formation of spatially and temporally distinct higher order protein complexes involving Meis proteins and known regulators of neural patterning or fate determination might serve as a simple, yet versatile, mechanism to subdivide broad territories into smaller functional units during brain development (Agoston, 2009).

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