The neocortex is the most recently evolved part of the mammalian brain which is involved in a repertoire of higher order brain functions, including those that separate humans from other animals. Humans have evolved an expanded neocortex over the course of evolution through a massive increase in neuron number (compared to our close relatives-­‐‑ the chimpanzees) in spite of sharing similar gestation time frames. So what do humans do differently compared to chimpanzees within the same time frame during their development? This dissertation addresses this question by comparing the developmental progression of neurogenesis between humans and chimpanzees using cerebral organoids as the model system. The usage of cerebral organoids, has enabled us to compare the development of both the human neocortex, and the chimpanzee neocortex from the very initiation of the neural phase of embryogenesis until very long periods of time. The results obtained so far suggest that the genetic programs underlying the development of the chimpanzee neocortex and the human neocortex are not very different, but rather the difference lies in the timing of the developmental progression. These results show that the chimpanzee neocortex spends lesser time in its proliferation phase, and allots lesser time to the generation of its neurons than the human neocortex. In more scientific terms, the neurogenic phase of the neocortex is shorter in chimpanzees than it is in humans. This conclusion is supported by (1) an earlier onset of gliogenesis in chimpanzees compared to humans which is indicative of a declining neurogenic phase, (2) an earlier increase in the chimpanzee neurogenic progenitors during development, compared to humans, (3) a higher number of stem cell– like progenitors in human cortices compared to chimpanzees, (4) a decline in neurogenic areas within the chimpanzee cerebral organoids over time compared to human cerebral organoids.

Developmental biology adresses how cells are organised into functional structures and eventually into a whole organism. It is crucial to understand the molecular basis for processes in development, by studying the expression and function of relevant genes and their relationship to each other. A gene function can be studied be creating loss-of-function situations, in which the change in developmental processes is examined in the absense of a functional gene product, or in gain-of-function studies, where a gene product is either intrinsically overproduced or ectopically upregulated. One approach for a loss-of-function situation is the creation of specific mutants in single genes, and the zebrafish (Danio rerio) has proven to be an excellent model organism for this purpose. In this thesis, I report on two forward genetic screens performed to find new mutants affecting brain development, in particular mutants defective in development and function of the midbrain-hindbrain boundary (MHB), an organiser region that patterns the adjacent brain regions of the midbrain and the hindbrain. In the first screen, I could identify 10 specific mutants based on morphology and the analysis of the expression patterns of lim1 and fgf8, genes functioning as early neuronal markers and as a patterning gene, respectively. Three of these mutants lacked an MHB, and by complementation studies, I identified these mutants as being defective in the spg locus. The second screen produced 35 new mutants by screening morphologically and with antibodies against acetylated Tubulin, which marks all axonal scaffolds, and anti-Opsin, which is a marker for photoreceptors in the pineal gland. According to their phenotype, I distributed the mutant lines into 4 phenotypic subgroups, of which the brain morphology group with 18 mutant lines was studied most intensively. In the last part of my thesis, I characterise one of these brain morphology mutants, broken heart. This mutant is defective in axonal outgrowth and locomotion, and shows a striking reduction of serotonergic neurons in the epiphysis and in the raphe nuclei in the hindbrain, structures involved in serotonin and melatonin production. Studies in other model organisms suggested a role of factors from the floor plate and the MHB in induction of the serotonergic neurons in the hindbrain, and using broken heart, I show that Fgf molecules such as Fgf4 and Fgf8 can restore partially the loss of serotonergic neurons in the mutant. I conclude that forward genetic screens are an invaluable tool to generate a pool of mutations in specific genes, which can be used to dissect complex processes in development such as brain development.

The term "pattern formation" refers to the process by which order unfolds in development. The present thesis deals with a particular aspect of molecular pattern formation during vertebrate embryogenesis. The model system in the focus of this study is the zebrafish, Danio rerio. In the early developmental phases of the zebrafish, Fibroblast growth factors (Fgfs) are involved in the molecular patterning of various tissues, including two regions of the brain, the forebrain and the midbrain-hindbrain region, affecting cellular processes as diverse as cell proliferation, differentiation, and axonal targeting. The goal of this study was to better understand the mechanisms by which Fgf signaling regulates pattern formation and embryogenesis. I addressed this question on several levels, investigating the extent of intracellular signaling (MAPK activation) relative to sources of Fgf expression, and the transcriptional responses of cells to Fgf signaling during embryogenesis. By a macroarray analysis, I identified putative transcriptional targets of Fgf signaling in late gastrulation, providing a set of molecules that are likely to act as functional players in relaying the patterning information encoded by Fgf signals. Among those are the secreted signaling molecules Chordin and Wnt8, as well as Isthmin, a novel secreted molecule that I found capable to interfere with anterior embryonic patterning. In addition, I identified two ETS domain transcription factors, Erm and Pea3, which constitute bona fide integrators of FgfR signaling. By gain- and loss-of-function studies, I demonstrate that transcript levels of erm and pea3 are tightly regulated by Fgf signaling. Detailed analysis of the expression patterns of erm and pea3 along with other Fgf target genes also provides evidence for a differential read-out of Fgf concentration in the embryo, consistent with a role of Fgf as a vertebrate morphogen. The discovery of novel molecular components downstream of Fgf receptor activity paves a way to characterize previously unknown or underestimated developmental roles of Fgfs in the molecular patterning of the forebrain, the eye and parts of the neural crest.