Pituitary development and energy homeostasis

The pituitary gland (hypophysis) constitutes a physiological link between the nervous and the endocrine sys- tem, receiving inputs from the hypothalamus of the brain, to which it responds by the secretion of hormones that regulate basic body functions and the activity of other glands. The pituitary consists of two parts, the neurohypophysis, which derives from the brain, and the adenohypophyis, which derives from placodal ectoderm The adenohypophysis contains at least seven different cell types characterized by the different hormones they produce. Since all of these cell types derive from a common primordium, the adenohypophysis serves as a good model for organogenesis and differential cell specification. Several regulators of adenohypothesis have been identified via mouse genetics, however, no systematic forwards genetics analysis had been performed in any vertebrate system as yet. We are carrying out such large-scale mutagenesis screens in zebrafish. During a first screen, investigating growth hormone expression, we identified four complementation groups with specific defects in adenohypophysis formation or patterning. In the meantime, all four mutated genes have been positionally cloned. One of them encodes the Fibroblast growth factor Fgf3, which is expressed in presumptive neurohypophseal cells in the ventral diencephalon, from where it signals to the adenohypophysis to promote adenohypophyseal cell survival. This indicates that neuro- and adenohypophysis do not only form a common organ, but also communicate with each other during their co-development. A similar, but slightly weaker phenotype is caused by mutations in the proneural gene ascl1a, which encodes a transcription factor known to be involved in neuronal specification, and which in contrast to Fgf3 acts in a cell-autonomous fashion in adenohypophyseal cells themselves. We are currently investigating to which extent Ascl1a acts a mediator of Fgf3 signals. The two remaining mutants in the adenohypophysis-specific transcription factor Pit1 and the protein phosphatase Eya1 display trans-differentiation or de-differentiation of adenohypophyseal cells, respectively, without any sign of apoptosis. Expression profiling experiments to identify target genes mediating the different effects on cell survival or cell differentiation are in progress. In addition, we carry out new screens, now looking at the expression of proopiomelanocortin (pomc). One of the newly isolated mutants carries an antimorphic mutation in Six1, a transcription factor closely interacting with eya1.

In addition to the pituitary, pomc is expressed in the arcuate nucleus, a part of the hypothalamus involved in the regulation of energy homeostasis and feeding behavior. a-MSH, the relevant cleavage product of Pomc, mediates satiety and promotes energy expenditure by binding to its receptor, MC4R, at a whole set of different neurons, such as the CRH and TRH-positive neurons, which generate hormones inducing the release of the metabolic hormones ACTH and TSH in the pituitary. a-MSH itself is antagonized by Agouti-related peptide (Agrp), which is also made in the arcuate nucleus, and which promotes appetite and blocks energy expenditure. This hypothalamic melanocortin system seems to be functional both in mammals and fish. In mouse, loss of MC4R leads to obesity, a phenotype also obtained upon Agrp overexpression in zebrafish. However, in contrast to mammals, zebrafish in addition to becoming fat display somatic growth even during adulthood. This might be accomplished by a better connection between the melanocortin system and the pituitary, which generates hormones with somatotropic effects like growth hormone. In our pomc in situ hybridization screen, we in addition to pituitary mutants aim to isolate mutants with defects in the hypothalamic melanocortin system. In addition, we screen for altered agrp expression both in untreated larvae and in larvae after several days of starvation, which normally causes an up-regulation of agrp expression. Via these screens we wish to identify novel regulators of appetite and energy homeostasis, which might help to further elucidate the neuroendocrine system that upon dysfunction in human can lead to obesity on one and to anorexia on the other hand. In addition to isolating mutants, we want to generate transgenic zebrafish in which agrp, pomc and MC4R neurons are labelled with different fluorochromes to study development and plasticity of the melanocortin system via in vivo live imaging. In addition, we will label CRH-, Somatostatin- and GHRH (Growth hormone releasing hormone) neurons to unravel the role of the melanocortin system on obesity versus somatic growth.