The diversity of different neuronal types lays the foundation for different functions in the brain. The development of different subpopulations and special features of neurons in the central nervous system are still partly unknown. Finding answers to these developmental issues could help in the process of characterisation of cell types and mapping of neuronal networks between the brainstem nuclei in the brain. Previous studies have shown that a ventrolateral neuroepithelial domain in the anterior hindbrain, rV2, produces excitatory (glutamatergic) and inhibitory (GABAergic) neurons, which are related to monoaminergic nuclei in the brainstem (Lahti et al., 2016). In this master’s thesis project, the development of a subpopulation of neurons expressing Gsc2 transcription factor in the interpeduncular nucleus was studied. This project was based on single-cell RNA sequencing results conducted in E13.5 mice. Predicted by single-cell RNA sequencing results, Gsc2 expressing cells are GABAergic interneurons and originate from the rV2 domain of the rhombomere 1 region in the hindbrain. Co-expression pattern with another transcription factor Sall3 with Gsc2 during development was also addressed in the study. Furthermore, the role of Notch signalling in the binary cell fate decision between GABAergic and the glutamatergic fate of rV2 neurons was investigated. Validation of single-cell RNA sequencing results was performed using in situ hybridisation and immunohistochemistry methods with mice embryos at the age of E12.5 and E15.5. This study verified previously shown origin of Gsc2 expressing cells to the rhombomere 1 region and in addition, showed that Gsc2 expressing cells are GABAergic. Co-expression pattern of Gsc2 with Sall3 neither in the rV2 domain nor in the interpeduncular nucleus was seen in our results. In the rV2 domain, the depletion of Notch signalling decreased the expression of differentiating GABAergic neurons. This indicates that Notch has a role in GABAergic neurotransmitter identity during the development of brainstem neurons in mice. Based on our results, Gsc2 could be used as a lineage marker for GABAergic interneurons originating from the rhombomere 1 region and as a marker for a subpopulation of the interpeduncular nucleus. Furthermore, results from the role of Notch signalling could help in discovering the mechanisms related to the determination of neurotransmitter identity in rV2 neurons. Further investigations, in different developmental time points and with additional markers, are needed to verify these results.

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.

Intrauterine growth restriction (IUGR) is caused by dysregulation of placental metabolism. Paternally inherited IUGR mutations in the fetus influence maternal physiology via the placenta. However, it is not known whether the maternal placenta also affects the extent of IUGR in such fetuses. In cattle and other ruminants, maternal-fetal communication occurs primarily at the placentomes. We previously identified a 3 deletion in the noncoding MER1 repeat containing imprinted transcript 1 (MIMT1) gene that, when inherited from the sire, causes IUGR and late abortion in Ayshire cattle with variable levels of severity. Here, we compared the transcriptome and genomic imprinting in fetal and maternal placentome components of wild-type and MIMT1(Del/WT) fetuses before IUGR became apparent, to identify key early events. Transcriptome analysis revealed fewer differentially expressed genes in maternal than fetal MIMT1(Del/WT) placentome. AST1, within the PEG3 domain, was the only gene consistently reduced in IUGR in both fetal and maternal samples. Several genes showed an imprinting pattern associated with IUGR, of which only secernin 3 (SCRN3) and paternally expressed 3 (PEG3) were differentially imprinted in both placentome components. Loss of strictly monoallelic, allele-specific expression (similar to 80:20) of PEG3 in the maternal MIMT1(Del/WT) placenta could be associated with incomplete penetrance of MIMT1(Del). Our data show that dysregulation of the PEG3 domain is involved in IUGR, but also reveal that maternal placental tissues may affect the penetrance of the paternally inherited IUGR mutation.

Evolutionarily conserved signaling pathways mediate cell-cell communications during development. While the extracellular signal is precisely regulated to achieve dynamic morphogenetic events at the species level, it must be also flexible to generate the diversified morphologies through evolution. However, little is known about the mechanisms behind the precision and flexibility. The insect wing vein pattern can provide an excellent model to address this fundamental question, since species-specific wing vein patterns have been diversified through evolution. In this thesis, I study how evolutionarily conserved bone morphogenetic protein (BMP)-type ligand specifies diversified insect wing vein patterns using Dipteran Drosophila melanogaster and Hymenopteran sawfly Athalia rosae as models. In Drosophila, BMP-type ligand Decapentaplegic (Dpp) is expressed in the longitudinal veins (LVs) to maintain LVs and induce crossveins (CVs) fates during pupal stages. However, the distribution of Dpp remained largely unknown. Using GFP-tagged Dpp, I demonstrated that Dpp is directionally transported from LVs into the posterior crossvein (PCV) region by two extracellular BMP-binding proteins, Short gastrulation (Sog) and Crossveinless (Cv). In contrast, most of Dpp did not diffuse from LVs by Type I BMP receptor and a positive feedback mechanism. Thus the active transport and retention mechanisms allow diffusible Dpp to draw the complex wing vein patterns in Drosophila. To investigate how BMP signal instructs wing vein morphogenesis that involves apposition and cell shape changes between two wing epithelial layers, I then focused on the function of RhoGAP Crossveinless-C (Cv-C) during the PCV morphogenesis. I found that cv-c mediates PCV morphogenesis downstream of BMP signal by inactivating various Rho-type small GTPases. Interestingly, I found that cv-c is also required for Dpp transport, while Sog/Cv mediated BMP signal is guided at the ectopic wing veins caused by loss of Rho-type small GTPases. These observations identified a feed-forward mechanism coupling Dpp transport and PCV morphogenesis. To address how BMP signal specifies diversified insect wing vein patterns, I then introduced sawfly as a new model. I found that dpp is ubiquitously expressed but BMP signal reflects distinct fore- and hindwing vein patterns in sawfly. To address if Dpp transport mechanism is involved in wing vein formation, Cv/Tsg was identified from sawfly. Loss of dpp or cv/tsg by RNAi affected BMP signal and all of wing venations. These observations suggest that ubiquitously expressed Dpp is redistributed to specify distinct fore- and hindwing vein patterns in sawfly. Taken together, I found that the extracellular distribution of Dpp/BMP is tightly regulated and coordinated to achieve precise patterning and morphogenesis of the insect wing veins. Furthermore, this study raises an interesting possibility that changes in the directionality of Dpp/BMP diffusion may underlie distinct insect wing vein patterns.