The correct development of multicellular organisms is tightly regulated by intrinsic and extrinsic factors at specific time points. Disturbance on any level of these multiple processes may result in drastic phenotypes or eventually death of the organism. The midbrain-hindbrain boundary (also termed isthmic organizer) is a region of high interest as well in early as also in later development. The isthmic region carries organizer identity by the expression and subsequent release of FGF8. False patterning events of this region in early developmental stages would therefore display dramatic results over time. As it has been shown that the midbrain-hindbrain boundary (mhb) in the zebrafish is a compartment (or lineage restriction) boundary I tried to understand the underlying molecular mechanism for its correct establishment. In this work I focused both on embryological, molecular and genetic means to characterize involved molecules and mechanisms. In the first part of the thesis I followed in vivo cell transplantation assays, having started with an unbiased one. Cells of either side the mhb were challenged with this boundary by bringing them into direct cell contact with their ectopic counterpart. In a biased approach, cells overexpressing mRNA of specific candidate genes were transplanted and their clonal distribution in host embryos was analyzed. In the second part of the thesis I started interfering with specific candidate genes by transiently knocking down their protein translation. The adhesion molecules of the Eph/ephrin class had been shown to restrict cell mixing and thereby creating compartment boundaries in other tissues, such as the hindbrain, in the zebrafish and other organisms. Additionally, we generated several stable genetic mutant lines in cooperation with the Tilling facility at the Max-Planck-Institute. The only acquired potential null mutant ephrinB2bhu2971 was analyzed and characterized further. I observed that a knock down or knock out of only one of the ephrinB2 ligands does not seem to be sufficient for a loss of compartment boundary formation. The combinatory approach of blocking translation of EphrinB2a in ephrinB2bhu2971 mutants gave very complex and interesting phenotypes, which need to be investigated further.

The correct development of multicellular organisms is tightly regulated by intrinsic and extrinsic factors at specific time points. Disturbance on any level of these multiple processes may result in drastic phenotypes or eventually death of the organism. The midbrain-hindbrain boundary (also termed isthmic organizer) is a region of high interest as well in early as also in later development. The isthmic region carries organizer identity by the expression and subsequent release of FGF8. False patterning events of this region in early developmental stages would therefore display dramatic results over time. As it has been shown that the midbrain-hindbrain boundary (mhb) in the zebrafish is a compartment (or lineage restriction) boundary I tried to understand the underlying molecular mechanism for its correct establishment. In this work I focused both on embryological, molecular and genetic means to characterize involved molecules and mechanisms. In the first part of the thesis I followed in vivo cell transplantation assays, having started with an unbiased one. Cells of either side the mhb were challenged with this boundary by bringing them into direct cell contact with their ectopic counterpart. In a biased approach, cells overexpressing mRNA of specific candidate genes were transplanted and their clonal distribution in host embryos was analyzed. In the second part of the thesis I started interfering with specific candidate genes by transiently knocking down their protein translation. The adhesion molecules of the Eph/ephrin class had been shown to restrict cell mixing and thereby creating compartment boundaries in other tissues, such as the hindbrain, in the zebrafish and other organisms. Additionally, we generated several stable genetic mutant lines in cooperation with the Tilling facility at the Max-Planck-Institute. The only acquired potential null mutant ephrinB2bhu2971 was analyzed and characterized further. I observed that a knock down or knock out of only one of the ephrinB2 ligands does not seem to be sufficient for a loss of compartment boundary formation. The combinatory approach of blocking translation of EphrinB2a in ephrinB2bhu2971 mutants gave very complex and interesting phenotypes, which need to be investigated further.

Cerebellar organization and function have been studied in numerous species of fish. Fish models such as goldfish and weakly electric fish have led to important findings about the cerebellar architecture, cerebellar circuit physiology and brain evolution. However, most of the studied fish models are not well suited for developmental and genetic studies of the cerebellum. The rapid transparent ex utero development in zebrafish allows direct access and precise visualization of all the major events in cerebellar development. The superficial position of the cerebellar primordium and cerebellum further facilitates in vivo imaging of cerebellar structures and developmental events at single cell resolution. Furthermore, zebrafish is amenable to high-throughput screening techniques and forward genetics because of its fecundity and easy keeping. Forward genetics screens in zebrafish have resulted in several isolated cerebellar mutants and substantially contributed to the understanding of the genetic networks involved in hindbrain development (Bae et al. 2009; Brand et al. 1996). Recent developments in genetic tools, including the use of site specific recombinases, efficient transgenesis, inducible gene expression systems, and the targeted genome lesioning technologies TALEN and Cas9/CRISPR has opened up new avenues to manipulate and edit the genome of zebrafish (Hans et al. 2009; Scott 2009; Housden et al. 2016; Li et al. 2016)}. These tools enable the use of genome-wide genetic approaches, such as enhancer/exon traps and cell specific temporal control of gene expression in zebrafish. Several seminal papers have used these technologies to successfully elucidate mechanisms involved in the morphogenesis, neurogenesis and cell migration in the cerebellum (Bae et al. 2009; Chaplin et al. ; Hans et al. 2009; Volkmann et al. ; Volkmann et al. 2008). In addition, the use of genetically encoded sensors and probes that allows detection and manipulation of neuronal activity using optical methods have open up new means to study the physiology and function of the cerebellum (Simmich et al. 2012; Matsui et al. 2014). Taken together, these features have allowed zebrafish to emerge as a complete model for studies of molecular, cellular and physiological mechanisms involved in cerebellar development and function at both cell and circuit level.