First person – Venkatram Yellapragada

Peer reviewe

FGF8-FGFR1 signaling regulates human GnRH neuron differentiation in a time- and dose-dependent manner

Fibroblast growth factor 8 (FGF8), acting through the fibroblast growth factor receptor 1 (FGFR1), has an important role in the development of gonadotropin-releasing hormone-expressing neurons (GnRH neurons). We hypothesized that FGF8 regulates differentiation of human GnRH neurons in a time-and dose-dependent manner via FGFR1. To investigate this further, human pluripotent stem cells were differentiated during 10 days of dual-SMAD inhibition into neural progenitor cells, followed either by treatment with FGF8 at different concentrations (25 ng/ml, 50 ng/ml or 100 ng/ml) for 10 days or by treatment with 100 ng/ml FGF8 for different durations (2, 4, 6 or 10 days); cells were then matured through DAPT-induced inhibition of Notch signaling for 5 days into GnRH neurons. FGF8 induced expression of GNRH1 in a dose-dependent fashion and the duration of FGF8 exposure correlated positively with gene expression of GNRH1 (PPeer reviewe

Kallmann syndrome in a patient with Weiss-Kruszka syndrome and a de novo deletion in 9q31.2

Patients with deletions on chromosome 9q31.2 may exhibit delayed puberty, craniofacial phenotype including cleft lip/palate, and olfactory bulb hypoplasia. We report a patient with congenital HH with anosmia (Kallmann syndrome, KS) and a de novo 2.38 Mb heterozygous deletion in 9q31.2. The deletion breakpoints (determined with wholegenome linked-read sequencing) were in the FKTN gene (9:108,331,353) and in a non-coding area (9:110,707,332) (hg19). The deletion encompassed six protein-coding genes (FKTN, ZNF462, TAL2, TMEM38B, RAD23B, and KLF4). ZNF462 haploinsufficiency was consistent with the patient's Weiss-Kruszka syndrome (craniofacial phenotype, developmental delay, and sensorineural hearing loss), but did not explain his KS. In further analyses, he did not carry rare sequence variants in 32 known KS genes in whole-exome sequencing and displayed no aberrant splicing of 15 KS genes that were expressed in peripheral blood leukocyte transcriptome. The deletion was 1.8 Mb upstream of a KS candidate gene locus (PALM2AKAP2) but did not suppress its expression. In conclusion, this is the first report of a patient with Weiss-Kruszka syndrome and KS. We suggest that patients carrying a microdeletion in 9q31.2 should be evaluated for the presence of KS and KS-related features.Peer reviewe

Regulation of radial glial process growth by glutamate via mGluR5/TRPC3 and neuregulin/ErbB4

Radial glial cells play an essential role through their function as guides for neuronal migration during development. Disruption of metabotropic glutamate receptor 5 (mGluR5) function retards the growth of radial glial processes in vitro. Neuregulins (NRG) are activated by proteolytic cleavage and regulate (radial) glial maintenance via ErbB3/ErbB4 receptors. We show here that blocking ErbB4 disrupts radial process extension. Soluble NRG acting on ErbB4 receptors is able to promote radial process extension in particular where process elongation has been impeded by blockade of mGluR5, the nonselective cation channel canonical transient receptor potential 3 (TRPC3), or matrix metalloproteases (MMP). NRG does not restore retarded process growth caused by ErbB4 blockade. Stimulation of muscarinic receptors restores process elongation due to mGluR5 blockade but not that caused by TRPC3, MMP or ErbB4 blockade suggesting that muscarinic receptors can replace mGluR5 with respect to radial process extension. Additionally, NRG/ErbB4 causes Ca2+ mobilization in a population of cells through cooperation with ErbB1 receptors. Our results indicate that mGluR5 promotes radial process growth via NRG activation by a mechanism involving TRPC3 channels and MMPs. Thus neurotransmitters acting on G-protein coupled receptors could play a central role in the maintenance of the radial glial scaffold through activation of NRG/ErbB4 signaling.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.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

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

Gonadotropin-Releasing Hormone Neurons From Human Pluripotent Stem Cells : From Inductive Signals To Disease Modeling

Multiple factors influence the timing and commencement of puberty in mammals and gonadotropin-releasing hormone (GnRH) is an important one of those. GnRH is a neurohormone secreted by the neurons located at the hypothalamus referred to as GnRH neurons. At the onset of puberty, increase in the pulsatile release of GnRH is a key event and pubertal onset is under neuroendocrine control regulated by the hypothalamic-pituitary-gonadal (HPG) axis. HPG axis is an interconnecting component between neural and endocrine systems essential for the regulation of fertility. In disorders such as central precocious puberty (CPP) or congenital hypogonadotropic hypogonadism (CHH), the onset and progression of puberty is altered. CPP is caused by the premature activation of the HPG axis, and many of the CHH cases are a result of aberrations in the development or function of the GnRH neurons. Mutations in multiple genes have been identified to be causing CPP or CHH. In the case of CPP, Makorin RING finer protein 3 (MKRN3) mutations are most frequent and mutations in more than 60 genes have been implicated in CHH. However, the functional validations for the majority of the CPP or CHH causing mutations are lacking. The developmental aspects of GnRH neurons in humans remain largely to be explored. Fibroblast growth factor 8 (FGF8) is a developmental morphogen, and animal studies have implicated it strongly in the development of GnRH neurons. Interestingly, mutations in FGF8 and in one of its receptors, fibroblast growth factor receptor-1 (FGFR1), cause CHH in humans. Although the functional importance of FGF8-FGFR1 signalling in GnRH neuron emergence has been demonstrated in animal models, more studies are necessary to understand the precise mechanisms occurring downstream to FGF8-FGFR1 signalling. To understand the biology of puberty and to develop novel therapeutics for pubertal alterations, it is important to study the ontogeny and function of human GnRH neurons. However, owing to their intricate locations in the brain, accessing these neurons has been difficult and is subjected to ethical issues. The functional importance and limited availability of these neurons iterates the importance of developing in vitro models to study them. Human pluripotent stem cells (hPSCs) can produce all cell lineages of the human body and are therefore an invaluable tool for developing GnRH neurons in vitro. In this context, the first hPSC-based differentiation protocol for generating GNRH1-expressing and secreting neurons was published few years ago. The differentiation protocol served to be a valuable line of research to investigate human GnRH neuron development and function. With the advent of revolutionary gene editing technologies such as CRISPR-Cas9, genes implicated in pubertal diseases could be manipulated in hPSCs, and the resulting phenotype of the cells differentiated from hPSCs could be investigated. The gene of interest can either be knocked out, induced, or tagged with fluorescent reporters to enable recording of the phenotype after such manipulations. Next-generation sequencing technologies, particularly RNA sequencing (RNA-Seq), enabled characterization of various cell types and conditions. Quantitating the mRNA molecules enables visualisation of global gene expression patterns within the cells. These global gene expression changes are potential indicators to monitor development and disease. The aim of the thesis was to combine the methods described above and advance the understanding of GnRH neuron development and function. Additionally, the aim was to develop a model to study the mechanisms underlying puberty-associated disorders. Accordingly, the role of MKRN3 was investigated in the development of the GnRH neurons and GNRH1 expression. Protein interaction partners of MKRN3 in human cells were investigated. The non-requirement of MKRN3 in the development of GnRH neurons from hPSCs was identified. It was shown that the lack of MKRN3 had no influence on the relative expression of GNRH1 but MKRN3 interacts with several proteins implicated in the timing of puberty. GnRH neurons generated in vitro expressed several genes implicated in CHH, as well as genes involved in neuronal development and function. ISL LIM homeobox 1 (ISL1) was identified as a hub gene during the generation of GnRH neurons and its expression was confirmed in human foetal GnRH neurons. Finally, upon investigating the dose- and time-dependent effects of FGF8, we noted that FGF8 indeed affected the GnRH neuron development and GNRH1 expression in a dose- and time-dependent manner. When the functional role of FGFR1 during the GnRH neuron differentiation was examined, reduction in the activity of FGFR1 significantly reduced the relative expression of GNRH1. Interestingly, FGFR1 localized to the nucleus in addition to the cell membrane in neurons (including GnRH neurons). The time-dependent effects of FGF8 on the transcriptome were characterized using RNA-Seq, and the findings suggested very early changes in the expression of key genes following exposure to FGF8 treatment during GnRH neuron differentiation. In a conclusion, the thesis (i) uncovers the dispensable role of MKRN3 in hPSC-based GnRH neuron derivation and GNRH1 expression; (ii) describes the key gene expression patterns associated with GnRH neurons; (iii) demonstrates the dose- and time-response of FGF8 on GnRH neuron development and GNRH1 expression, finally, my work reveals the importance of FGF8-FGFR1 signalling in human GnRH neuron differentiation and provides detailed transcriptomic characterization of GnRH neuron progenitors.Murrosikä käynnistyy, kun hypotalamiset GnRH-neuronit aloittavat pulssittaisen GnRH:n, eli gonadotroptiineja vapauttavan hormonin, erityksen. GnRH:n ja edelleen gonadotropiinien erityksen käynnistyminen liian varhain johtaa ennenaikaiseen murrosikään. Gonadotropiinien puutteellinen eritys taas johtaa synnynnäiseen hypogonadotrooppiseen hypogonadismiin, joka ilmenee viivästyneenä, puuttuvana tai osittaisena murrosikäkehityksenä. Molempien sairauksien taustalta on tunnistettu useita GnRH-neuronien kehitykseen tai toimintaan haitallisesti vaikuttavia mutaatiota, esimerkiksi MKRN3-, FGF8- ja FGFR1-geeneistä. GnRH-neuroneiden kehityksestä tiedetään toistaiseksi kuitenkin verrattain vähän, sillä niiden pieni lukumäärä ja sijainti tekevät niiden tutkimuksesta haastavaa. Tämän työn tavoitteena oli tutkia ja karakterisoida GnRH-neuroneiden kehitystä erilaistamalla ihmisen villityypin ja geenieditoituja kantasoluja GnRH-neuroneiksi. Erityisesti keskityttiin MKRN3-geenin ja FGF8-FGFR1-signaloinnin rooliin. Työssä hyödynnettiin moderneja tutkimusmenetelmiä kuten Crispr-Cas-geenieditointia ja RNA-sekvensointia. Ensimmäisessä osatyössä tutkittiin miten MKRN3-geenin, jonka virheiden tiedetään aiheuttavan ennenaikaista murrosikää, puuttuminen vaikuttaa GnRH-neuroneiden kehitykseen. Lisäksi tutkittiin MKRN3-proteiinin interktioita muiden proteiinien kanssa. Työn tuloksena havaittiin, että erilaistettaessa GnRH-neuroneita kantasoluista, MKRN3-geenin poistaminen Crispr-Cas-tekniikalla ei vaikuttanut neuroneiden muodostukseen tai GNRH1-geenin ilmentymiseen. Sen sijaan havaittiin, että MKRN3-proteiini interaktoi lukuisten muiden murrosikäkehitykseen vaikuttavien proteiinien kanssa. Toisessa osatyössä tutkittiin GnRH-neuroneiden kehitystä Crispr-Cas-tekniikalla kehitetyn GnRH-kantasolureportterilinjan ja RNA-sekvensoinnin avulla. Tuloksena havaittiin, että kantasoluista erilaistetut GnRH-neuronit ilmensivät lukuisia geenejä, joiden virheet on yhdistetty hypogonadotrooppiseen hypogonadismiin. Lisäksi työssä tunnistettiin ISL1-geeni yhdeksi GnRH-neuroneiden kehityksen kannalta keskeiseksi toimijaksi, ja sen ilmentyminen osoitettiin myös ihmisen sikiöaikaisissa GnRH-neuroneissa. Kolmannessa osatyössä tutkittiin FGF8-kasvutekijän roolia erilaistettaessa GnRH-neuroneita kantasoluista. Työn tuloksena havaittiin, että FGF8 vaikuttaa GnRH-neuronien kehitykseen ja GNRH1-geenin ilmentymiseen sekä annos- että aikaperusteisesti. Lisäksi työssä tutkittiin FGFR1-reseptorin aktiivisuuden vähentämisen vaikutuksia GnRH-neuroneiden erilaistumiseen, ja havaittiin että myös tämä vähensi merkittävästi GNRH1-geenin ilmentymistä. Lisäksi tutkimuksessa havaittiin, että FGFR1-reseptori lokalisoituu neuroneissa solukalvon lisäksi myös tumaan. Tämä väitöstyö tuotti uutta tietoa GnRH-neuroneiden kehityksestä ja samalla osoitti, miten kantasoluja ja moderneja tutkimusmenetelmiä voidaan hyödyntää murrosikään liittyvien sairauksien tautimallituksessa. Työssä osoitettiin, että FGF8-FGFR1-signalointi on tärkeää GnRH-neuronien kehityksessä, ja toisaalta että MKRN3 ei ole GnRH-neuroneiden erilaistumiselle välttämätön. GnRH-neuroneiden ja niiden esiasteiden RNA-sekvensoinnista saatua transkriptiodataa on mahdollista hyödyntää tulevissa translationaalisissa tutkimuksissa

Deciphering the Transcriptional Landscape of Human Pluripotent Stem Cell-Derived GnRH Neurons : The Role of Wnt Signaling in Patterning the Neural Fate

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons lay the foundation for human development and reproduction; however, the critical cell populations and the entangled mechanisms underlying the development of human GnRH neurons remain poorly understood. Here, by using our established human pluripotent stem cell-derived GnRH neuron model, we decoded the cellular heterogeneity and differentiation trajectories at the single-cell level. We found that a glutamatergic neuron population, which generated together with GnRH neurons, showed similar transcriptomic properties with olfactory sensory neuron and provided the migratory path for GnRH neurons. Through trajectory analysis, we identified a specific gene module activated along the GnRH neuron differentiation lineage, and we examined one of the transcription factors, DLX5, expression in human fetal GnRH neurons. Furthermore, we found that Wnt inhibition could increase DLX5 expression and improve the GnRH neuron differentiation efficiency through promoting neurogenesis and switching the differentiation fates of neural progenitors into glutamatergic neurons/GnRH neurons. Our research comprehensively reveals the dynamic cell population transition and gene regulatory network during GnRH neuron differentiation.Peer reviewe