Abstrakti
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.
Alkuperäiskieli | englanti |
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Valvoja/neuvonantaja |
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Julkaisupaikka | Helsinki |
Kustantaja | |
Painoksen ISBN | 978-951-51-8923-3 |
Sähköinen ISBN | 978-951-51-8924-0 |
Tila | Julkaistu - 2023 |
OKM-julkaisutyyppi | G5 Tohtorinväitöskirja (artikkeli) |
Lisätietoja
M1 - 116 s. + liitteetTieteenalat
- 3111 Biolääketieteet
- 1184 Genetiikka, kehitysbiologia, fysiologia