Thomas Lamonerie

A Pituitary Cell-Restricted T Box Factor, Tpit, Activates POMC Transcription in Cooperation with Pitx Homeoproteins

The pituitary gland has provided unique insight into molecular mechanisms and regulatory factors controlling both differentiation and gene transcription. We identified Tpit, a novel T box factor only present in the two pituitary POMC-expressing lineages, the corticotrophs and melanotrophs, and apparently in no other tissue, including hypothalamic POMC neurons. In pituitary cells, Tpit activation of POMC gene transcription requires cooperation with Pitx1, the two factors binding to contiguous sites within the same regulatory element. In gain-of-function experiments, Tpit induces POMC expression in undifferentiated pituitary cells, indicating that it can initiate differentiation into POMC-expressing lineages. TPIT gene mutations were found in patients with isolated deficiency of pituitary POMC-derived ACTH, in support of an essential role of Tpit for differentiation of the pituitary POMC lineage.

The PTX family of homeodomain transcription factors during pituitary developments.

A subfamily of bicoid-related homeodomain factors was recently discovered through its involvement in transcription of pituitary-specific genes. We isolated the first member of this family, Ptxl (pituitary homeobox 1), through its DNA binding properties whereas a second related gene, Ptx2 (RIEG), was identified by positional cloning as the causative gene for Riegers syndrome. The mechanisms of Ptx action on its target genes as well as its putative roles during development are reviewed with particular emphasis on its role in pituitary function.

ES cell-derived renewable and functional midbrain dopaminergic progenitors

During early development, midbrain dopaminergic (mDA) neuronal progenitors (NPs) arise from the ventral mesencephalic area by the combined actions of secreted factors and their downstream transcription factors. These mDA NPs proliferate, migrate to their final destinations, and develop into mature mDA neurons in the substantia nigra and the ventral tegmental area. Here, we show that such authentic mDA NPs can be efficiently isolated from differentiated ES cells (ESCs) using a FACS method combining two markers, Otx2 and Corin. Purified Otx2+Corin+ cells coexpressed other mDA NP markers, including FoxA2, Lmx1b, and Glast. Using optimized culture conditions, these mDA NPs continuously proliferated up to 4 wk with almost 1,000-fold expansion without significant changes in their phenotype. Furthermore, upon differentiation, Otx2+Corin+ cells efficiently generated mDA neurons, as evidenced by coexpression of mDA neuronal markers (e.g., TH, Pitx3, Nurr1, and Lmx1b) and physiological functions (e.g., efficient DA secretion and uptake). Notably, these mDA NPs differentiated into a relatively homogenous DA population with few serotonergic neurons. When transplanted into PD model animals, aphakia mice, and 6-OHDA–lesioned rats, mDA NPs differentiated into mDA neurons in vivo and generated well-integrated DA grafts, resulting in significant improvement in motor dysfunctions without tumor formation. Furthermore, grafted Otx2+Corin+ cells exhibited significant migratory function in the host striatum, reaching >3.3 mm length in the entire striatum. We propose that functional and expandable mDA NPs can be efficiently isolated by this unique strategy and will serve as useful tools in regenerative medicine, bioassay, and drug screening.

Purkinje cells and Bergmann glia are primary targets of the TRα1 thyroid hormone receptor during mouse cerebellum postnatal development.

Thyroid hormone is necessary for normal development of the central nervous system, as shown by the severe mental retardation syndrome affecting hypothyroid patients with low levels of active thyroid hormone. The postnatal defects observed in hypothyroid mouse cerebellum are recapitulated in mice heterozygous for a dominant-negative mutation of Thra, the gene encoding the ubiquitous TRα1 receptor. Using CRE/loxP-mediated conditional expression approach, we found that this mutation primarily alters the differentiation of Purkinje cells and Bergmann glia, two cerebellum-specific cell types. These primary defects indirectly affect cerebellum development in a global manner. Notably, the inward migration and terminal differentiation of granule cell precursors is impaired. Therefore, despite the broad distribution of its receptors, thyroid hormone targets few cell types that exert a predominant role in the network of cellular interactions that govern normal cerebellum maturation.

The homeobox gene Otx2 in development and disease.

The Otx2 gene encodes a transcription factor essential for the normal development of brain, cerebellum, pineal gland, and eye. In the retina, Otx2 has essential functions from early embryogenesis to adulthood. As soon as the optic vesicle is formed, the gene is required for retinal pigment epithelium specification. Otx2 is also a key regulator of photoreceptor genesis and differentiation, and is required after birth for bipolar cells terminal maturation. Otx2 expression is maintained in the differentiated retina wherein the gene is critical for the outer retina maintenance. In the visual cortex, the gene modulates the neuronal plasticity through a paracrine mechanism. OTX2 heterozygous mutations in humans have been linked to severe ocular malformations associated with brain abnormalities and pituitary dysfunction. Recent studies have also established the OTX2 gene as an oncogene for medulloblastoma, a malignant brain tumour originating in the cerebellum.

Molecular dissection reveals decreased activity and not dominant negative effect in human OTX2 mutants

The paired-type homeodomain transcription factor Otx2 is essential for forebrain and eye development. Severe ocular malformations in humans have recently been associated with heterozygous OTX2 mutations. To document the molecular defects in human mutants, Otx2 structural characterization was carried out. A collection of deletion and point mutants was created to perform transactivation, DNA binding, and subcellular localization analyses. Transactivation was ascribed to both N- and C-termini of the protein, and DNA binding to the minimal homeodomain, where critical amino acid residues were identified. Acute nuclear localization appeared controlled by a nuclear localization sequence located within the homeodomain which acts in conjunction with a novel nuclear retention domain that we unraveled located in the central part of the protein. This region, which is poorly conserved among Otx proteins, was also endowed with dominant negative activity suggesting that it might confer unique properties to Otx2. Molecular diagnostic of human mutant OTX2 proteins discriminates hypomorphic and loss of function mutations from other mutations that may not be relevant to ocular pathology.

Temporal and spatial delineation of mouse Otx2 functions by conditional self-knockout

To identify the independent spatial and temporal activities of the essential developmental gene the Otx2, the germline mutation of which is lethal at embryonic day 8.5, we floxed one allele and substituted the other with an inducible CreER recombinase gene. This makes ‘trans’ self‐knockout possible at any developmental stage. The transient action of tamoxifen pulses allows time‐course mutation. We demonstrate efficient temporal knockout and demarcate spatio‐temporal windows in which Otx2 controls the head, brain structures and body development.

Loss of Otx2 in the Adult Retina Disrupts Retinal Pigment Epithelium Function, Causing Photoreceptor Degeneration

Photoreceptors are specialized neurons of the retina that receive nursing from the adjacent retinal pigment epithelium (RPE). Frequent in the elderly, photoreceptor loss can originate from primary dysfunction of either cell type. Despite intense interest in the etiology of these diseases, early molecular actors of late-onset photoreceptor degeneration remain elusive, mostly because of the lack of dedicated models. Conditional Otx2 ablation in the adult mouse retina elicits photoreceptor degeneration, providing a new model of late-onset neuronal disease. Here, we use this model to identify the earliest events after Otx2 ablation. Electroretinography and gene expression analyses suggest a nonautonomous, RPE-dependent origin for photoreceptor degeneration. This is confirmed by RPE-specific ablation of Otx2, which results in similar photoreceptor degeneration. In contrast, constitutive Otx2 expression in RPE cells prevents degeneration of photoreceptors in Otx2-ablated retinas. We use chromatin immunoprecipitation followed by massive sequencing (ChIP-seq) analysis to identify the molecular network controlled in vivo by Otx2 in RPE cells. We uncover four RPE-specific functions coordinated by Otx2 that underpin the cognate photoreceptor degeneration. Many direct Otx2 target genes are associated with human retinopathies, emphasizing the significance of the model. Importantly, we report a secondary genetic response after Otx2 ablation, which largely precedes apoptosis of photoreceptors, involving inflammation and stress genes. These findings thus provide novel general markers for clinical detection and prevention of neuronal cell death.

New Otx2 mRNA isoforms expressed in the mouse brain

The mouse Otx2 gene is essential throughout head and brain development, from anterior–posterior polarity determination and neuroectoderm induction to post‐natal sensory organ maturation. These numerous activities must rely on a very finely tuned regulation of expression. In order to understand the molecular control of the Otx2 gene, we set out to isolate its promoter. During this quest, we identified three remote transcription start sites, two defining two new upstream exons and one mapping within the previously reported first exon. The three transcripts differed in their 5′ non‐coding region but encoded the same protein. The transcription start nucleotides of each mRNA species have been mapped by RNase protection assays and by an RNA circularization technique. We have demonstrated that they are all used and linked to functional promoters. In addition to leader versatility, we also detected alternative splicing within the coding sequence that gives rise to a new protein endowed with an 8 amino‐acid insertion upstream of the homeodomain. Combined analysis of the relative abundance of Otx2 mRNA isoforms in representative tissues and in situ hybridization studies revealed distinct spatial and temporal, although partially overlapping, expression patterns of the mRNA isoforms. These findings provide new clues to a better understanding of the relationships between Otx2 gene architecture and its complex regulatory requirements.

A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors

BackgroundDynamic monitoring of protein expression and localization is fundamental to the understanding of biological processes. The paired-class homeodomain-containing transcription factor Otx2 is essential for normal head and brain development in vertebrates. Recent conditional knockout studies have pointed to multiple roles of this protein during late development and post-natal life. Yet, later expression and functions remain poorly characterized as specific reagents to detect the protein at any stage of development are still missing.ResultsWe generated a new mouse line harbouring an insertion of the GFP gene within the Otx2 coding sequence to monitor the gene activity while preserving most of its functions. Our results demonstrate that this line represents a convenient tool to capture the dynamics of Otx2 gene expression from early embryonic stages to adulthood. In addition, we could visualize the intracellular location of Otx2 protein. In the retina, we reinterpret the former view of protein distribution and show a further level of regulation of intranuclear protein localization, which depends on the cell type.ConclusionThe GFP-tagged Otx2 mouse line fully recapitulates previously known expression patterns and brings additional accuracy and easiness of detection of Otx2 gene activity. This opens up the way to live imaging of a highly dynamic actor of brain development and can be adapted to any mutant background to probe for genetic interaction between Otx2 and the mutated gene.