MKRN3 Interacts With Several Proteins Implicated in Puberty Timing but Does Not Influence GNRH1 Expression

Paternally-inherited loss-of-function mutations in makorin ring finger protein 3 gene (MKRN3) underlie central precocious puberty. To investigate the puberty-related mechanism(s) of MKRN3 in humans, we generated two distinct bi-allelic MKRN3 knock-out human pluripotent stem cell lines, Del 1 and Del 2, and differentiated them into GNRH1-expressing neurons. Both Del 1 and Del 2 clones could be differentiated into neuronal progenitors and GNRH1-expressing neurons, however, the relative expression of GNRH1 did not differ from wild type cells (P = NS). Subsequently, we investigated stable and dynamic protein-protein interaction (PPI) partners of MKRN3 by stably expressing it in HEK cells followed by mass spectrometry analyses. We found 81 high-confidence novel protein interaction partners, which are implicated in cellular processes such as insulin signaling, RNA metabolism and cell-cell adhesion. Of the identified interactors, 20 have been previously implicated in puberty timing. In conclusion, our stem cell model for generation of GNRH1 -expressing neurons did not offer mechanistic insight for the role of MKRN3 in puberty initiation. The PPI data, however, indicate that MKRN3 may regulate puberty by interacting with other puberty-related proteins. Further studies are required to elucidate the possible mechanisms and outcomes of these interactions.Peer reviewe

Characterization of the human GnRH neuron developmental transcriptome using a GNRH1-TdTomato reporter line in human pluripotent stem cells

Gonadotropin-releasing hormone (GnRH) neurons provide a fundamental signal for the onset of puberty and subsequent reproductive functions by secretion of gonadotropin-releasing hormone. Their disrupted development or function leads to congenital hypogonadotropic hypogonadism (CHH). To model the development of human GnRH neurons, we generated a stable GNRH1-TdTomato reporter cell line in human pluripotent stem cells (hPSCs) using CRISPR-Cas9 genome editing. RNA-sequencing of the reporter clone, differentiated into GnRH neurons by dual SMAD inhibition and FGF8 treatment, revealed 6461 differentially expressed genes between progenitors and GnRH neurons. Expression of the transcription factor ISL1, one of the top 50 most upregulated genes in the TdTomato-expressing GnRH neurons, was confirmed in 10.5 gestational week-old human fetal GnRH neurons. Among the differentially expressed genes, we detected 15 genes that are implicated in CHH and several genes that are implicated in human puberty timing. Finally, FGF8 treatment in the neuronal progenitor pool led to upregulation of 37 genes expressed both in progenitors and in TdTomato-expressing GnRH neurons, which suggests upstream regulation of these genes by FGF8 signaling during GnRH neuron differentiation. These results illustrate how hPSC-derived human GnRH neuron transcriptomic analysis can be utilized to dissect signaling pathways and gene regulatory networks involved in human GnRH neuron development.This article has an associated First Person interview with the first author of the paper.Peer reviewe

Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells

Gonadotropin-releasing hormone (GnRH) neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs) into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs) by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders.Peer reviewe

MKRN3 Interacts With Several Proteins Implicated in Puberty Timing but Does Not Influence GNRH1 Expression

Paternally-inherited loss-of-function mutations in makorin ring finger protein 3 gene (MKRN3) underlie central precocious puberty. To investigate the puberty-related mechanism(s) of MKRN3 in humans, we generated two distinct bi-allelic MKRN3 knock-out human pluripotent stem cell lines, Del 1 and Del 2, and differentiated them into GNRH1-expressing neurons. Both Del 1 and Del 2 clones could be differentiated into neuronal progenitors and GNRH1-expressing neurons, however, the relative expression of GNRH1 did not differ from wild type cells (P = NS). Subsequently, we investigated stable and dynamic protein-protein interaction (PPI) partners of MKRN3 by stably expressing it in HEK cells followed by mass spectrometry analyses. We found 81 high-confidence novel protein interaction partners, which are implicated in cellular processes such as insulin signaling, RNA metabolism and cell-cell adhesion. Of the identified interactors, 20 have been previously implicated in puberty timing. In conclusion, our stem cell model for generation of GNRH1-expressing neurons did not offer mechanistic insight for the role of MKRN3 in puberty initiation. The PPI data, however, indicate that MKRN3 may regulate puberty by interacting with other puberty-related proteins. Further studies are required to elucidate the possible mechanisms and outcomes of these interactions

Iloa, leikkiä ja yhdessä tekemistä – Varhaisvuosien fyysisen aktiivisuuden suositukset

AbstractThe first recommendations concerning physical activity of under eight-year-olds, Recommendations for physical activity in early childhood education 2005 (Guides of the Ministry of Social Affairs and Health 2005:17) have now been updated based on the initiative of the national Joy in Motion programme. The recommendations were hoped to respond to the changes in the children’s living conditions and the amount and types of physical activities over the past ten years. Another aim was to update the scientific base of the recommendations. The new Recommendations for physical activity in early childhood (2016) were prepared as part of the work of the Steering group for health-enhancing physical activity (TEHYLI). The steering group commented on and approved the recommendations compiled by the multidisciplinary national group of experts specifically invited to carry out the task. The recommendations are founded on the UN Convention on the Rights of the Child. The recommendations are based on scientific information on how the adults interacting with under eight-year-olds can facilitate the realisation of the rights of children by comprehensive support for their growth, development, health and wellbeing through physical activities. Studies have shown that physical activity promotes the child’s physical, cognitive, psychological, emotional and social development. The scientific knowledge base of the recommendations has also been published (Scientific justification for the recommendations for physical activity in early childhood 2016. Ministry of Education and Culture 2016:22) The Recommendations for physical activity in early childhood inform about the amount and type of physical activity for the under eight-year-old, roles of the physical, psychological and social environments, and planning and implementation of guided physical exercise and education on exercise as part of early childhood education. The recommendations are intended for parents, early childhood education teachers, physical exercise and healthcare professionals, and other organisations and individuals involved in promoting physical activity, health or wellbeing among under eight-year-olds. TiivistelmäEnsimmäiset suomalaiset alle kahdeksanvuotiaiden lasten liikuntaan tarkoitetut suositukset, Varhaiskasvatuksen liikunnan suositukset 2005 (Sosiaali- ja terveysministeriön oppaita 2005:17), esitettiin päivitettäviksi valtakunnallisessa Ilo kasvaa liikkuen – ohjelman kehittämistyössä. Suositusten toivottiin vastaavan viimeisen kymmenen vuoden aikana tapahtuneita muutoksia lasten elinolosuhteissa sekä liikkumisen määrässä ja laadussa. Tavoitteeksi asetettiin myös suositusten tutkimusperustan päivittäminen. Uudet Varhaisvuosien fyysisen aktiivisuuden suositukset (2016) valmisteltiin osana opetus- ja kulttuuriministeriön sekä sosiaali- ja terveysministeriön yhdessä asettaman ja koordinoiman Terveyttä ja hyvinvointia edistävän liikunnan (TEHYLI) –ohjausryhmän työtä. TEHYLI –ohjausryhmä kommentoi ja hyväksyi tehtävään erikseen kutsutun monitieteisen kansallisen asiantuntijaryhmän kokoamat suositukset. Suositusten taustalla on YK:n lapsen oikeuksien yleissopimus. Suositukset perustuvat tutkimustietoon siitä, miten alle kahdeksanvuotiaiden lasten kanssa toimivat aikuiset voivat mahdollistaa lasten oikeuksien toteutumisen tukemalla lasten kokonaisvaltaista kasvua, kehitystä, terveyttä ja hyvinvointia liikunnan avulla. Tutkimukset ovat osoittaneet, että liikunnalla voidaan tukea lapsen fyysistä, kognitiivista, psyykkistä, emotionaalista ja sosiaalista kehittymistä. Suositusten tieteelliset perusteet on julkaistu erillisenä julkaisuna (Tieteelliset perusteet varhaisvuosien fyysisen aktiivisuuden suosituksille 2016. Opetus- ja kulttuuriministeriö 2016:22) Varhaisvuosien fyysisen aktiivisuuden suositukset antavat ohjeita alle kahdeksanvuotiaiden lasten fyysisen aktiivisuuden määrästä ja laadusta, fyysisen, psyykkisen ja sosiaalisen ympäristön rooleista sekä ohjatun liikunnan ja liikuntakasvatuksen suunnittelusta ja toteuttamisesta osana varhaiskasvatusta. Varhaisvuosien fyysisen aktiivisuuden suositukset on tarkoitettu lasten vanhempien, varhaiskasvatus-, liikunta- ja terveydenhuollon ammattilaisten sekä muiden alle kahdeksanvuotiaiden lasten liikunnan, terveyden tai hyvinvoinnin edistämiseen osallistuvien yhteisöjen ja yksilöiden käyttöön. Julkaisuun viitattaessa käytetään seuraavaa lähdemerkintää: Varhaisvuosien fyysisen aktiivisuuden suositukset 2016. Iloa, leikkiä ja yhdessä tekemistä. Opetus- ja kulttuuriministeriö 2016:21