The phenotypic spectrum associated with OTX2 mutations in humans.

Objective: The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development. Design: We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action. Methods: We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. Results: Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. Conclusions: OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.

Otx2b mutant zebrafish have pituitary, eye and mandible defects that model mammalian disease.

Combined pituitary hormone deficiency (CPHD) is a genetically heterogeneous disorder caused by mutations in over 30 genes. The loss-of-function mutations in many of these genes, including orthodenticle homeobox 2 (OTX2), can present with a broad range of clinical symptoms, which provides a challenge for predicting phenotype from genotype. Another challenge in human genetics is functional evaluation of rare genetic variants that are predicted to be deleterious. Zebrafish are an excellent vertebrate model for evaluating gene function and disease pathogenesis, especially because large numbers of progeny can be obtained, overcoming the challenge of individual variation. To clarify the utility of zebrafish for the analysis of CPHD-related genes, we analyzed the effect of OTX2 loss of function in zebrafish. The otx2b gene is expressed in the developing hypothalamus, and otx2bhu3625/hu3625 fish exhibit multiple defects in the development of head structures and are not viable past 10 days post fertilization (dpf). Otx2bhu3625/hu3625 fish have a small hypothalamus and low expression of pituitary growth hormone and prolactin (prl). The gills of otx2bhu3625/hu3625 fish have weak sodium influx, consistent with the role of prolactin in osmoregulation. The otx2bhu3625/hu3625 eyes are microphthalmic with colobomas, which may underlie the inability of the mutant fish to find food. The small pituitary and eyes are associated with reduced cell proliferation and increased apoptosis evident at 3 and 5 dpf, respectively. These observations establish the zebrafish as a useful tool for the analysis of CPHD genes with variable and complex phenotypes.

Identification of a promoter element mediating kisspeptin-induced increases in GnRH gene expression in sheep.

Gonadotropin-releasing hormone (GnRH) plays an important role in regulating the activities of other components downstream of the hypothalamic-pituitary-gonadal (HPG) axis and maintaining the normal reproductive cycle of animals. However, the molecular mechanisms by which GnRH synthesis and secretion are regulated in sheep remains unclear. In this study, a series of eight recombinant vectors with deletion fragments were constructed and cotransfected with pGL3-Basic and pRL-SV40 into sheep hypothalamic neuronal cells. After treatment with 1 nM kisspeptin, the core promoter of the sheep GnRH gene was identified to be in the region of -1912 bp to -1461 bp by dual-luciferase reporter assay. Bioinformatics analysis showed that there was a binding site for the transcription factor Otx-2 in the core promoter region (-1786 to -1770 bp) that was highly conserved among different species. The expression patterns of Kiss-1, Otx-2 and GnRH in the sheep hypothalamus were the same, and the expression of Kiss-1, Otx-2 and GnRH was significantly higher in the breeding season than in nonbreeding season (P < 0.01). In addition, when hypothalamic neurons were cultured in vitro with kisspeptin, kisspeptin induced the expression of GnRH and Otx-2. In conclusion, these results provide evidence that the core promoter region (-1786 to -1770 bp) of the GnRH gene is involved in the regulation of hypothalamic activity by kisspeptin and that binding of the transcription factor Otx-2 mediates this activation.

Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells.

To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene.

Otx1 promotes basal dendritic growth and regulates intrinsic electrophysiological and synaptic properties of layer V pyramidal neurons in mouse motor cortex.

The transcription factor Otx1 is specifically expressed in layer V pyramidal cells (L5PCs) in the cerebral cortex. Otx1 null mutant mice have a defect in the developmental axon pruning of L5PCs and show epileptic seizures. However, the role of Otx1 in electrophysiology, morphology and synaptology of the cortical neurons has not been fully investigated. This study examines the influences of Otx1 on neuronal properties of L5PCs by loss- and gain-of-function approaches. Mice with an Otx1-null mutation had decreased structural measurements of basal dendrites in L5PCs. In contrast, the size of basal dendrites was increased in the Otx1-over-expressed pyramidal cells (PCs) in L2/3 where the gene normally does not express. PCs showed burst and non-burst firing patterns of action potentials. The proportion of burst firing neurons was reduced in the Otx1 mutant but increased in the neurons over-expressing Otx1. Although the burst firing population decreased, the proportion of those bursting neurons with a low threshold increased in the Otx1 mutant mice. Moreover, excitatory facilitating synaptic connections formed between L5PCs were predominant in the Otx1 mutant mice, which greatly contrasted with the predominant depressing synaptic connections in the controls. Taken together, it suggests an enhanced activity of neuronal network in the cortex of Otx1 mutant mice. These data indicate that the Otx1 expression is essential for the normal development of dendritic morphology, intrinsic electrophysiology and synaptic dynamics of L5PCs. This study provides new insights into molecular mechanisms underlying the spatial and temporal regulation of neuronal and synaptic properties of L5PCs, and improves our understanding on the generation of epileptic seizures.

Deletion of Otx2 in GnRH neurons results in a mouse model of hypogonadotropic hypogonadism.

GnRH is the central regulator of reproductive function responding to central nervous system cues to control gonadotropin synthesis and secretion. GnRH neurons originate in the olfactory placode and migrate to the forebrain, in which they are found in a scattered distribution. Congenital idiopathic hypogonadotropic hypogonadism (CIHH) has been associated with mutations or deletions in a number of genes that participate in the development of GnRH neurons and expression of GnRH. Despite the critical role of GnRH in mammalian reproduction, a comprehensive understanding of the developmental factors that are responsible for regulating the establishment of mature GnRH neurons and the expression of GnRH is lacking. orthodenticle homeobox 2 (OTX2), a homeodomain protein required for the formation of the forebrain, has been shown to be expressed in GnRH neurons, up-regulated during GnRH neuronal development, and responsible for increased GnRH promoter activity in GnRH neuronal cell lines. Interestingly, mutations in Otx2 have been associated with human hypogonadotropic hypogonadism, but the mechanism by which Otx2 mutations cause CIHH is unknown. Here we show that deletion of Otx2 in GnRH neurons results in a significant decrease in GnRH neurons in the hypothalamus, a delay in pubertal onset, abnormal estrous cyclicity, and infertility. Taken together, these data provide in vivo evidence that Otx2 is critical for GnRH expression and reproductive competence.

Evaluation of retinoic acid therapy for OTX2-positive medulloblastomas.

The homeobox transcription factor OTX2 plays an essential role during embryonic brain development. It is normally silenced in the adult brain, but is overexpressed by genomic amplification or other mechanisms in the majority of medulloblastomas (MBs). Retinoic acids (RAs) can suppress OTX2 expression and inhibit MB growth. In this study, 9-cis RA most potently inhibited MB cell growth. 9-cis RA functions through the downregulation of OTX2 expression, which subsequently induces neuronal differentiation of OTX2-expressing cells. Treatment with 9-cis RA reduced the growth of D425 flank xenograft tumors in mice. In an intracranial model, however, MB tumors showed resistance to 9-cis RA treatment, and we implicated fibroblast growth factor (FGF) as a potential mediator of resistance to RA therapy. These findings suggest a mechanism for RA-mediated anti-tumor effect on OTX2-positive MB cells and indicate that therapeutic targeting of OTX2 might be effective if FGF pathway-mediated resistance can be overcome.

Eye-specific expression of an ancestral jellyfish PaxB gene interferes with Pax6 function despite its conserved Pax6/Pax2 characteristics.

Pax transcription factors are evolutionarily conserved regulators of eye development and can be distinguished on the basis of three functional domains: two DNA-binding domains (the paired domain and the paired-type homeodomain), and the octapeptide motif. PaxB of the eyed cubozoan jellyfish, Tripedalia cystophora, is characterized by a Pax2-like paired domain and octapeptide, and a Pax6-like homeodomain. In mice, functionally distinct Pax6 and Pax2 proteins have unique as well as redundant roles in eye morphogenesis. Here, we show that expression of the jellyfish PaxB gene in mouse embryonic eye tissues impairs normal development of lens and retina. Our data show that PaxB misexpression leads to a downregulation of endogenous Pax6 protein in the prospective lens and in subsets of cells within the inner nuclear layer of transgenic retina. In addition to Pax6 downregulation, the expression of PaxB leads to an almost complete loss of amacrine cells in the adult transgenic retina, a phenotype that differs from a loss-of-function of the Pax6 gene. The present data suggest that PaxB, due to its Pax2-like paired domain and Pax-6 like homeodomain, disturbs the transcriptional network regulated by Pax6 in the developing lens and retina. Taken together, our data suggest that molecular properties of individual mouse Pax2 and Pax6 proteins are essential determinants of mouse eye development and cannot be substituted for by jellyfish PaxB which possesses elements of vertebrate Pax2 and Pax6.

Dmbx1 is essential in agouti-related protein action.

Dmbx1 is a paired-class homeodomain transcription factor. We show here that mice deficient in Dmbx1 exhibit severe leanness associated with hypophagia and hyperactivity and that isolation of a Dmbx1(-/-) mouse from its cohabitants induces self-starvation, sometimes leading to death, features similar to those of anorexia nervosa in humans. Interestingly, overexpression of agouti in Dmbx1(-/-) mice failed to induce aspects of the A(y)/a phenotype, including hyperphagia, obesity, and diabetes mellitus. In Dmbx1(-/-) mice, administration of agouti-related protein increased cumulative food intake for the initial 6 h but significantly decreased it over 24- and 48-h periods. In addition, Dmbx1 was shown to be expressed at embryonic day 15.5 in the lateral parabrachial nucleus, the rostral nucleus of the tractus solitarius, the dorsal motor nucleus of the vagus, and the reticular nucleus in the brainstem, all of which receive melanocortin signaling, indicating involvement of Dmbx1 in the development of the neural network for the signaling. Thus, Dmbx1 is essential for various actions of agouti-related protein and plays a role in normal regulation of energy homeostasis and behavior.

Otx1l, Otx2 and Irx1b establish and position the ZLI in the diencephalon.

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, our understanding of the mechanisms responsible for positioning the ZLI along the neural axis is poor. Here we show 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. We therefore propose that the ZLI is induced within the competence area established by Otx1l/2, and is posteriorly restricted by Irx1b.

GABA immunoreactivity in the developing rat thalamus and Otx2 homeoprotein expression in migrating neurons.

We investigated the expression of gamma-aminobutyric acid (GABA) in the developing rat thalamus by immunohistochemistry, using light, confocal and electron microscopy. We also examined the relationship between the expression of the homeoprotein Otx2, a transcription factor implicated in brain regionalization, and the radial and non-radial migration of early generated thalamic neurons, identified by the neuronal markers calretinin (CR) and GABA. The earliest thalamic neurons generated between embryonic days (E) 13 and 15 include those of the reticular nucleus, entirely composed by GABAergic neurons. GABA immunoreactivity appeared at E14 in immature neurons and processes laterally to the neuroepithelium of the diencephalic vesicle. The embryonic and perinatal periods were characterized by the presence of abundant GABA-immunoreactive fibers, mostly tangentially oriented, and of growth cones. At E15 and E16, GABA was expressed in radially and non-radially oriented neurons in the region of the reticular thalamic migration, between the dorsal and ventral thalamic primordia, and within the dorsal thalamus. At these embryonic stages, some CR- and GABA-immunoreactive migrating-like neurons, located in the migratory stream and in the dorsal thalamus, expressed the homeoprotein Otx2. In the perinatal period, the preponderance of GABAergic neurons was restricted to the reticular nucleus and several GABAergic fibers were still detectable throughout the thalamus. The immunolabeling of fibers progressively decreased and was no longer visible by postnatal day 10, when the adult configuration of GABA immunostaining was achieved. These results reveal the spatio-temporal features of GABA expression in the developing thalamus and suggest a novel role of Otx2 in thalamic cell migration.

Ontogenetic expression of the Otx2 and Crx homeobox genes in the retina of the rat.

Otx2 and Crx are vertebrate orthologs of the orthodenticle family of homeobox genes, which are involved in retinal development. In this study, the temporal expression patterns of Otx2 and Crx in the rat retina during embryonic and postnatal stages of development were analyzed in detail. This confirmed the presence of Otx2 mRNA in both the embryonic retinal pigment epithelium and the developing neural retina. During development, the expression of Otx2 persists in the pigment epithelium, whereas Otx2 expression of the neural retina becomes progressively restricted to the outer nuclear layer and the outer part of the inner nuclear layer. Immunohistochemistry revealed that Otx2 protein is also present in cell bodies of the ganglion cell layer, which does not contain the Otx2 transcript, suggesting that Otx2 protein is synthesized in cell bodies of the bipolar neurons and then transported to and taken up by cells in the ganglion cell layer. Crx is also highly expressed in the outer nuclear layer starting at E17 and postnatally in the inner nuclear layer. The onset of expression of Crx lags behind that of Otx2 consistent with evidence that Otx2 activates Crx transcription. These expression patterns are consistent with evidence that Otx2 and Crx function during retinal development and extend the period of probable functionality to the adult. In this regard, these results provide an enhanced and expanded temporal and spatial framework for understanding the multiple roles of Otx2 and Crx in the developing and mature mammalian retina.

Otx2 controls identity and fate of glutamatergic progenitors of the thalamus by repressing GABAergic differentiation.

GABAergic and glutamatergic neurons modulate inhibitory and excitatory networks in the CNS, and their impairment may cause neurological and psychiatric disorders. Thus, understanding the molecular mechanisms that control neurotransmitter phenotype and identity of excitatory and inhibitory progenitors has considerable relevance. Here we investigated the consequence of Otx2 (orthodenticle homolog) ablation in glutamatergic progenitors of the dorsal thalamus (referred to as thalamus). We report that Otx2 is cell-autonomously required in these progenitors to repress GABAergic differentiation. Our data indicate that Otx2 may prevent GABAergic fate switch by repressing the basic helix-loop-helix gene Mash1 (mammalian achaete-schute homolog) in progenitors expressing Ngn2 (neurogenin homolog). The lack of Otx2 also resulted in the activation of Pax3 (paired box gene), Pax7, and Lim1 (Lin-11/Isl-1/Mec-3), three genes normally coexpressed with Mash1 and GABAergic markers in the pretectum, thus suggesting that thalamic progenitors lacking Otx2 exhibit marker similarities with those of the pretectum. Furthermore, Otx2 ablation gave rise to a marked increase in proliferating activity of thalamic progenitors and the formation of hyperplastic cell masses. Thus, this study provides evidence for a novel and crucial role of Otx2 in the molecular mechanism by which identity and fate of glutamatergic precursors are established in the thalamus. Our data also support the concept that proper assignment of identity and fate of neuronal precursors occurs through the suppression of alternative differentiation programs.