Beschreibung:
Abstract: The spinal cord is part of the central nervous system (CNS) and develops from the embryonic invagination of the neural tube, generated as a result of primary neurulation from invagination of the neuroectoderm from the surrounding ectodermal layer. The progenitors lining the central canal within the neural tube undergo extensive proliferation resulting in two waves of neurogenesis in the mouse embryo, from which a 11 major classes of interneurons and columns of motoneurons will be specified and differentiate while migrating within the spinal cord. Identity specification of these classes heavily relies on opposing morphogen gradients in a dorsoventral axis, as well as cross-repression from the neighbouring classes. Frequently, human pregnancies present neurodevelopmental defects due to the incorrect process of neural tube closure (NTC) as last step of the neurulation, with variable clinical outcomes for the foetus according to the anteroposterior location of the defect. Studies in the past few years have shown how alterations to normal patterns of defined post-translational modifications (PTM) of histone proteins inevitably lead to defective NTC. Other developmental issues affecting cell specification and migration, and therefore the function, have been associated with disrupted PTM in the spinal cord.<br>Different publications implicated that lower levels of H3K79 methylation could associate with increased occurrence of defective NTC, and previous studies in Vogel’s lab defined the relevance of the well-known methyltransferase Dot1-like protein (DOT1L, responsible for H3K79 methylation) for rostral CNS development, leaving open questions with regard of its role in the caudal CNS.<br>My thesis focused therefore on defining the role of DOT1L methyltransferase activity in neurulation and neurogenesis. Initially I used a small cohort of chick embryos to pharmacologically prevent DOT1L-mediated methylation during neurulation, after defining suitable experimental conditions for the EPZ5676-based treatment. Although an increase of neural tube defects (NTDs) was observed upon effective DOT1L-inhibition, the frequency of the defect did not suggest a strong dependency on DOT1L activity for NTC. As a second approach, I generated a new transgenic mouse line for conditional knockout of Dot1l (Dot1l-cKO) within the developing spinal cord and characterized the resulting phenotype at prenatal stages for morphology and physiological markers, as the Dot1l-cKO resulted in lethality at birth. The major morphological defects observed at E18.5 were marked cell death and defective distribution of marker for inhibitory interneurons. Many differentiating interneurons present only temporarily specific molecular marks at the beginning of neurogenesis and with differentiation more functional and shared markers are expressed. Additionally, previous studies in Vogel’s lab suggest a pivotal role of DOT1L in the balance between neural potency and differentiation, e.g. in the developing cortex or in the cerebellum. Thus, I focused the characterisation of the transgenic line at embryonic stages depicting the molecular heterogeneity of the developing spinal cord (E11.5-E12.5). An initial bulk RNA-seq for the whole lumbar spinal cord for control and Dot1l-cKO at E12.5 highlighted an expression trend among mutants for decreased expression of genes characteristic of active cell cycle, accompanied by an increased expression of those associated with differentiation and migration processes. Markers for dorsal progenitor classes were under-expressed in the mutant samples, while positive and negative alterations were observed widely throughout the spinal cord for markers of differentiated interneuron classes. To gain a higher resolution on the phenotype, I performed a thorough analysis of molecular markers between the first and the second wave of neurogenesis targeting all the dorsal populations (dI1, dI2, dI3, dI4/6, dI5) and partially some ventral ones (V0, V2, V3). Parallel analysis of progenitors and early postmitotic interneurons revealed the population- and time-specific sensitivity of interneurons to DOT1L activity, as dI1 subsets were differentially affected by the cKO – with dI1i being numerically affected by the cKO while the complementary dI1c subset presented altered cell positioning in the same condition. Dorsal interneuron classes as dI1 and dI5 presented early (E11.5) significant defective development, while dI2 and dI3 appeared to develop a phenotype at a later stage (E12.5). Although of minor interest for the research focus and with a less extensive KO domain I observed also ventral effects for cell positioning, suggesting extensive cross regulation within the developing system. This study represents the first direct description of the implication of DOT1L activity in spinal cord formation and development. Due to the exploratory nature of the study, many questions remain open and call for further analysis. However, clear directions emerge from this research work: DOT1L-mediated H3K79me2 is necessary for proper identity specification of defined interneuron classes, as well as for the cellular positioning of others. Loss of DOT1L methyltransferase activity in the developing spinal cord leads to aberrant cell positioning and underrepresentation of defined classes during neurogenesis, likely resulting in extensive cell death and incorrect patterning in late prenatal stages