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Recurrent missense mutations of the PIK3CA oncogene are among the most frequent drivers of human cancers. These often lead to constitutive activation of its product p110α, a phosphatidylinositol 3-kinase (PI3K) catalytic subunit. In addition to causing a broad range of cancers, the H1047R mutation is also found in affected tissues of a distinct set of congenital tumors and malformations. Collectively termed PIK3CA-related disorders (PRDs), these lead to overgrowth of brain, adipose, connective and musculoskeletal tissues and/or blood and lymphatic vessel components. Vascular malformations are frequently observed in PRD, due to cell-autonomous activation of PI3K signaling within endothelial cells. These, like most muscle, connective tissue and bone, are derived from the embryonic mesoderm. However, important organ systems affected in PRDs are neuroectodermal derivatives. To further examine their development, we drove the most common post-zygotic activating mutation of Pik3ca in neural crest and related embryonic lineages. Outcomes included macrocephaly, cleft secondary palate and more subtle skull anomalies. Surprisingly, Pik3ca-mutant subpopulations of neural crest origin were also associated with widespread cephalic vascular anomalies. Mesectodermal neural crest is a major source of non-endothelial connective tissue in the head, but not the body. To examine the response of vascular connective tissues of the body to constitutive Pik3ca activity during development, we expressed the mutation by way of an Egr2 (Krox20) Cre driver. Lineage tracing led us to observe new lineages that had normally once expressed Krox20 and that may be co-opted in pathogenesis, including vascular pericytes and perimysial fibroblasts. Finally, Schwann cell precursors having transcribed either Krox20 or Sox10 and induced to express constitutively active PI3K were associated with vascular and other tumors. These murine phenotypes may aid discovery of new candidate human PRDs affecting craniofacial and vascular smooth muscle development as well as the reciprocal paracrine signaling mechanisms leading to tissue overgrowth.

Pituitary development is coordinated by the timely interaction of several signaling pathways and transcription factors. The expression of lhx3 precedes that of transcription factors like tbx19, prop1, gata2 or sf1 and requires earlier expression of others like pitx1 and pitx2 and pathways like sonic hedgehog. The adult pituitary contains SOX2/SOX9 positive pituitary stem cells and progenitors leading to a differentiated pituitary, and in vitro differentiation of embryonic stem cells into mature pituitary cells was shown to recapitulate anterior pituitary development. Conditional inactivation of isl-1 in thyrotrophs induces central hypothyroidism, showing that this LIM domain factor is necessary for thyrotroph function. Large scale genomic techniques identified new pathogenic gene variants in constitutional hypopituitarism, an indirect evidence of their role in pituitary development or function: a variant of GPR161 was identified by whole exome sequencing in GH deficiency; a variant of ARNT2 was associated with constitutional panhypopituitarism, diabetes insipidus and microcephaly. Allelic variations of NFKB2 were associated with pituitary deficiency (mainly ACTH deficiency) and variable immune deficiency (VID), described as DAVID syndrome. IGSF1 alterations were identified in a complex X-linked phenotype with thyrotroph deficiency, macro-orchidism, delayed puberty, transient GH or ACTH deficiency, and low prolactin levels. The first variants of the potassium channel gene KCNQ1 were reported in families with GH deficiency, gingival fibromatosis and arrhythmia. Thanks to new genome wide techniques, more causative genes have been identified over the past 6 years than over the 2 decades following the first identification of the role of pou1f1 in human hypopituitarism. Demonstrating the role of these new genes will require an appropriate cell model that may derive from novel stem cell techniques. Such approaches will help better understand pituitary development and functions.

International audience; Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA's family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co-and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That's the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest. This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

Circadian clocks regulate rhythmic gene expression levels by means of mRNA oscillations that are mainly driven by post-transcriptional regulation. We identified a new post-transcriptional mechanism, which involves nuclear bodies called paraspeckles. Major components of paraspeckles including the long noncoding RNA Neat1, which is the structural component, and its major protein partners, as well as the number of paraspeckles, follow a circadian pattern in pituitary cells. Paraspeckles are known to retain within the nucleus RNAs containing inverted repeats of Alu sequences. We showed that a reporter gene in which these RNA duplex elements were inserted in the 3'-UTR region displayed a circadian expression. Moreover, circadian endogenous mRNA associated with paraspeckles lost their circadian pattern when paraspeckles were disrupted. This work not only highlights a new paraspeckle-based post-transcriptional mechanism involved in circadian gene expression but also provides the list of all mRNA associated with paraspeckles in the nucleus of pituitary cells.

International audience; In primary cultures of rat pituitary cells and in a pituitary sommatolactotroph cell line (GH4C1), endogenous core-clock- as well as hormone-genes such as prolactin displayed a rhythmic expression pattern, fitted by a sinusoidal equation in which the period value was close to the circadian one. This is consistent with the presence of a functional circadian oscillator in pituitary cells whose importance was ascertained in GH4C1 cell lines stably expressing a dominant negative mutant of BMAL1. In these cells, both endogenous core-clock- and prolactin-genes no more displayed a circadian pattern. Some genes we recently identified as mouse pituitary BMAL1-regulated genes in a DNA-microarray study, lost their circadian pattern in these cells, suggesting that BMAL1 controlled these genes locally in the pituitary. The intra-pituitary circadian oscillator could then play a role in the physiology of the gland that would not be seen anymore as a structure only driven by hypothalamic rhythmic control.

Paraspeckles are nuclear bodies form around the long non-coding RNA, Neat1, and RNA-binding proteins. While their role is not fully understood, they are believed to control gene expression at a post-transcriptional level by means of the nuclear retention of mRNA containing in their 3’-UTR inverted repeats of Alu sequences (IRAlu). In this study, we found that, in pituitary cells, all components of paraspeckles including four major proteins and Neat1 displayed a circadian expression pattern. Furthermore the insertion of IRAlu at the 3’-UTR of the EGFP cDNA led to a rhythmic circadian nuclear retention of the egfp mRNA that was lost when paraspeckles were disrupted whereas insertion of a single antisense Alu had only a weak effect. Using real-time video-microscopy, these IRAlu were further shown to drive a circadian expression of EGFP protein. This study shows that paraspeckles, thanks to their circadian expression, control circadian gene expression at a post-transcriptional level. DOI: http://dx.doi.org/10.7554/eLife.14837.001, eLife digest Many biological features of animals, including body temperature and hormone levels, follow daily rhythms that repeat every 24 hours. These so-called circadian rhythms are driven by an internal body clock and are essential for the organism to adapt to the daily cycle of light and dark. Circadian rhythms also take place inside individual cells – for example, the amount of a given protein in a cell often rises and falls over each 24-hour period. To generate these daily fluctuations, the processes used to make proteins based on the instructions encoded within a gene must be carefully controlled. Genes are first copied or ‘transcribed' into intermediate molecules called messenger RNAs (mRNAs). These mRNA molecules must then travel out of the cell’s nucleus before they can be de-coded to produce proteins. This means that daily fluctuations in mRNA and protein levels could occur because the rate at which the DNA is transcribed fluctuates or because controlling the steps that occur after transcription. However it is not clear how much these post-transcriptional steps contribute to circadian rhythms inside cells. Recently, structures called paraspeckles were seen inside the nucleus. These structures are made from a long RNA molecule that does not code for a protein, and a number of proteins that can bind mRNA molecules. Paraspeckles are thought to prevent certain mRNAs from leaving the nucleus and therefore stop them from being decoded to make proteins. Torres et al. have now investigated whether paraspeckles may play a role in circadian rhythms. Torres et al. looked at the long non-coding RNA and several proteins that are known to be components of paraspeckles in cells taken from the pituitary glands of rats using a variety of techniques. These cells were chosen because they were known to have a working circadian clock. The analysis showed that the levels of these components, as well as the number of paraspeckles within the nucleus, changed over the course of a daily cycle. Torres et al. then confirmed that mRNAs containing a sequence that is known to recruit mRNAs to paraspeckes (the IRAlu sequence) could be also retained in the nucleus or released with a circadian rhythm. This pattern was lost when the paraspeckles were disrupted. These findings suggest that daily fluctuations in protein levels can be post-transcriptionally controlled by paraspeckles rhythmically retaining mRNAs in the nucleus. Future studies could explore whether it may be possible to control circadian rhythms by targeting the paraspeckles, which could help to improve conditions where the internal body clock goes wrong. DOI: http://dx.doi.org/10.7554/eLife.14837.002

Au cours des deux dernieres decennies, la connaissance des mecanismes regissant l’ontogenese de la glande hypophysaire s’est grandement amelioree. Cependant, notre comprehension des deficits hypophysaire combine multiple (CPHD) d’origine genetique n’a pas suivi la meme evolution. En effet, malgre les avancees technologiques (CGH array, sequencage a haut debit) permettant l’identification de nouvelles mutations chez les patients, nous ne beneficions pas de modele adequat permettant de valider le caractere pathogene des variants alleliques rares identifies. Recemment, en nous inspirant des techniques developpees dans le laboratoire de Y. Sasai pour differencier in vitro des cellules antehypophysaires a partir de cellules souches embryonnaires, nous avons obtenu des cellules somatotropes et corticotropes a partir de cellules souches pluripotentes induites humaines (iPSC). Dans ces cultures tridimensionnelles sous forme d’organoides, nous avons observe une expression sequentielle de genes impliques dans le developpement hypophysaire, similaire a celle observee lors du developpement embryonnaire. Ce nouvel outil devrait s’averer precieux dans l’identification de nouvelles mutations, la validation de leur pathogenicite. Il devrait egalement etre utile pour le criblage pharmacologique, et permettre d’envisager de nouveau traitements bases sur la therapie cellulaire.

International audience; The daily temporal organization of rhythmic functions in mammals, which requires synchronization of the circadian clock to the 24-h light-dark cycle, is believed to involve adjustments of the mutual phasing of the cellular oscillators that comprise the time-keeper within the suprachiasmatic nucleus of the hypothalamus (SCN). Following from a previous study showing that the SCN undergoes day/night rearrangements of its neuronal-glial network that may be crucial for intercellular phasing, we investigated the contribution of glutamatergic synapses, known to play major roles in SCN functioning, to such rhythmic plastic events. Neither expression levels of the vesicular glutamate transporters nor numbers of glutamatergic terminals showed nycthemeral variations in the SCN. However, using quantitative imaging after combined immunolabelling, the density of synapses on neurons expressing vasoactive intestinal peptide, known as targets of the retinal input, increased during the day and both glutamatergic and non-glutamatergic synapses contributed to the increase (+36%). This was not the case for synapses made on vasopressin-containing neurons, the other major source of SCN efferents in the non-retinorecipient region. Together with electron microscope observations showing no differences in the morphometric features of glutamatergic terminals during the day and night, these data show that the light synchronization process in the SCN involves a selective remodelling of synapses at sites of photic integration. They provide a further illustration of how the adult brain may rapidly and reversibly adapt its synaptic architecture to functional needs.

International audience; Extracellular regulated kinase (ERK) signalling is believed to play roles in various aspects of circadian clock mechanisms. In this study, we show in rat that the nuclear versus cytoplasmic intracellular distribution of the phosphorylated forms of ERK1/2 (P-ERK1/2) in the central clock, namely the suprachiasmatic nucleus (SCN), is proportionally constant across the light/dark cycle while the spatial distribution and neurochemical phenotype of cells expressing these activated forms are time-regulated according to a daily rhythm and light-regulated. P-ERK1/2 was exclusively found in neuronal elements. At daytime, it was detected throughout the dorsoventral extent of the SCN, partly within neurons synthesizing either arginine-vasopressin or vasoactive intestinal peptide (VIP). At night time, it was segregated in the ventrolateral aspect of the nucleus, within a cluster of cells 45% of which were gastrin-releasing peptide (GRP) neurons with or without co-localization with VIP. After a light pulse at night, expression of P-ERK1/2 increased in GRP neurons but also appeared in a population of neurons that stained for VIP only. These data show that the GRP neurons are closely associated with ERK1/2 activation at night and point to the importance of ERK1/2 signalling not only in intra-SCN transmission of photic information but also in maintenance of neuronal rhythms in the SCN.

La rythmicite circadienne de l’activite de l’adenohypophyse est une composante essentielle de la physiologie de la glande parce que toutes les hormones qu’elle secrete presentent un profil rythmique, pulsatile et/ou circadien. Dans le domaine des rythmes circadiens, il apparait aujourd’hui que les regulations s’exercant au niveau post-transcriptionnel jouent un role preponderant. Dans l’adenohypophyse, nous avons identifie un mecanisme post-transcriptionnel original a la base de la rythmicite d’expression de certains ARNm. Ce mecanisme repose sur la retention nucleaire rythmique d’ARNm par des structures appelees paraspeckles dont l’element structurant est un long ARN non-codant, Neat1 (Nuclear-Enriched-Abundant-Transcript1) egalement connu sous le nom de Men1 (multiple-endocrine-neoplasia1). Nous avons montre que, dans les cellules hypophysaires, tous les composants moleculaires de ces paraspeckles, non seulement Neat1 mais aussi les principaux composants proteiques, presentent un profil d’expression circadienne. Du reste, l’expression de Neat1 est egalement rythmique dans d’autres oscillateurs y compris dans l’horloge circadienne centrale, les noyaux suprachiasmatiques de l’hypothalamus. Nous avons montre que certains ARNm qui sont associes aux differents composants des paraspeckles et dont la rythmicite circadienne est controlee au niveau post-transcriptionnel perdent leur expression circadienne en presence de siRNA dirigees contre Neat1 qui entrainent une destruction des paraspeckles. Le role grandissant que l’on attribue aujourd’hui aux longs ARN non-codants dans la pathogenie de nombreuses maladies et la caracterisation d’un role physiologique joue par un long ARN non-codant dans l’adenohypophyse ouvrent de nouvelles perspectives de strategie possible d’identification de candidats moleculaires participant a l’etiologie de pathologies hypophysaires en particulier de deficits hypophysaires chez l’homme.