Bsx Is Essential for Differentiation of Multiple Neuromodulatory Cell Populations in the Secondary Prosencephalon.

The hypothalamus is characterized by great neuronal diversity, with many neuropeptides and other neuromodulators being expressed within its multiple anatomical domains. The regulatory networks directing hypothalamic development have been studied in detail, but, for many neuron types, control of differentiation is still not understood. The highly conserved Brain-specific homeobox (Bsx) transcription factor has previously been described in regulating Agrp and Npy expression in the hypothalamic arcuate nucleus (ARC) in mice. While Bsx is expressed in many more subregions of both tuberal and mamillary hypothalamus, the functions therein are not known. Using genetic analyses in zebrafish, we show that most bsx expression domains are dependent on Nkx2.1 and Nkx2.4 homeodomain transcription factors, while a subset depends on Otp. We show that the anatomical pattern of the ventral forebrain appears normal in bsx mutants, but that Bsx is necessary for the expression of many neuropeptide encoding genes, including agrp, penka, vip, trh, npb, and nts, in distinct hypothalamic anatomical domains. We also found Bsx to be critical for normal expression of two Crh family members, crhb and uts1, as well as crhbp, in the hypothalamus and the telencephalic septal region. Furthermore, we demonstrate a crucial role for Bsx in serotonergic, histaminergic and nitrergic neuron development in the hypothalamus. We conclude that Bsx is critical for the terminal differentiation of multiple neuromodulatory cell types in the forebrain.

Conserved Genoarchitecture of the Basal Hypothalamus in Zebrafish Embryos.

Analyses of genoarchitecture recently stimulated substantial revisions of anatomical models for the developing hypothalamus in mammalian and other vertebrate systems. The prosomeric model proposes the hypothalamus to be derived from the secondary prosencephalon, and to consist of alar and basal regions. The basal hypothalamus can further be subdivided into tuberal and mamillary regions, each with distinct subregions. Albeit being a widely used model system for neurodevelopmental studies, no detailed genoarchitectural maps exist for the zebrafish (Danio rerio) hypothalamus. Here, we compare expression domains of zebrafish genes, including arxa, shha, otpa, isl1, lhx5, nkx2.1, nkx2.2a, pax6, and dlx5a, the orthologs of which delimit specific subregions within the murine basal hypothalamus. We develop the highly conserved brain-specific homeobox (bsx) gene as a novel marker for genoarchitectural analysis of hypothalamic regions. Our comparison of gene expression patterns reveals that the genoarchitecture of the basal hypothalamus in zebrafish embryos 48 hours post fertilization is highly similar to mouse embryos at E13.5. We found the tuberal hypothalamus in zebrafish embryos to be relatively large and to comprise previously ill-defined regions around the posterior hypothalamic recess. The mamillary hypothalamus is smaller and concentrates to rather medial areas in proximity to the anterior end of the neural tube floor plate. Within the basal hypothalamus we identified longitudinal and transverse tuberal and mamillary subregions topologically equivalent to those previously described in other vertebrates. However, the hypothalamic diencephalic boundary region and the posterior tuberculum still provide a challenge. We applied the updated prosomeric model to the developing zebrafish hypothalamus to facilitate cross-species comparisons. Accordingly, we applied the mammalian nomenclature of hypothalamic organization to zebrafish and propose it to replace some controversial previous nomenclature.

Bsx controls pineal complex development.

Neuroendocrine cells in the pineal gland release melatonin during the night and, in teleosts, are directly photoreceptive. During development of the pineal complex, a small number of cells migrate leftward away from the pineal anlage to form the parapineal cell cluster, a process that is crucial for asymmetrical development of the bilateral habenular nuclei. Here, we show that, throughout zebrafish embryonic development, the brain-specific homeobox (bsx) gene is expressed in all cell types of the pineal complex. We identified Bmp and Noto/Flh as major regulators of bsx expression in the pineal complex. Upon loss of Bsx through the generation of a targeted mutation, embryos fail to form a parapineal organ and develop right-isomerized habenulae. Crucial enzymes in the melatonin biosynthesis pathway are not expressed, suggesting the absence of melatonin from the pineal gland in bsx mutants. Several genes involved in rod-like or cone-like phototransduction are also abnormally expressed, indicating that Bsx has a pivotal role in the differentiation of multiple cell types in the zebrafish pineal complex.