Regulation and function of PAX2 in midbrain and kidney development

Research project P 13601 Pax2 in midbrain and kidney development Meinhard BUSSLINGER 28.06.1999 The transcription factor Pax2 is a key regulator of midbrain and kidney development. The midbrain is known to arise from an organizing center which is located at the midbrain-hindbrain boundary (MHB) of the embryo. The kidney develops through a series of sequential and reciprocal inductive interactions between epithelial and mesenchymal cells which are derived from the intermediate mesoderm of the embryo. Pax2 is one of the earliest genes to be expressed in the priniordia of the MHB region and the kidney. To date nothing is, however, known about upstream regulators which control the expression of Pax2 during MHB and kidney development. Moreover, the molecular mechanisms (target genes) mediating the Pax2 function are still largely unknown. 1n addition, the late function of Pax2 in MHB and kidney development has not yet been studied due to the early tissue deletion seen in homozygous Pax2 mutant embryos. To address these important questions, we will use transgenic approaches to delineate the regulatory sequences controlling the MHBand kidney-specific expression of Pax2. Biochemical and evolutionary appraoches will subsequently be used to identify the transcription factors which bind to and regulate these Pax2 enhancers. Candidate Pax2 target genes, which are expressed at the MHB and/or in the kidney, will be identified by in situ hybridization analyses of Pax2 mutant embryos. Moreover, the late function of Pax2 in MHB and kidney development will be investigated by phenotypic analysis and conditional inactivation of a hypomorphic Pax2 allele recently generated in our laboratory. Together, these experiments will provide not only important novel insights into the molecular mechanisms mediating the Pax2 function, but will also contribute to the elucidation of the regulatory cascades which govern early and late patterning of the midbrain and the kidney.

The mammalian genome consists of ~35`000 genes that need to be transcribed in their correct spatio-temporal expression pattern to allow normal development of the organism. Transcription factors are well known to control all major biological processes by activating or repressing entire gene expression programs. The nine transcription factors of the Pax protein family play an important role during embryogenesis in controlling brain development and organogenesis. The closely related Pax2, Pax5 and Pax8 proteins are essential for the development of the midbrain and cerebellum as well as for the formation of the immune system (Pax5), eye (Pax2), ear (Pax2), kidney (Pax2) and thyroid gland (Pax8). The Pax genes are particularly interesting from the medical point of view, as mutations in human Pax genes are the cause for inherited disease syndromes and the formation of different tumors. It is therefore important to study the normal and pathological function of these genes to gain insight into these Pax- related diseases. The FWF project P13601 was aimed at elucidating the role of the Pax2 gene in brain and kidney development by the use of transgenic approaches in the mouse. During embryogenesis, a signaling center (organizer) is formed at the midbrain-hindbrain boundary (MHB) that is responsible for the formation of the midbrain and cerebellum. Fgf8 is the most important secreted signal of the MHB organizer that recruits adjacent cells to participate in midbrain and cerebellum development. On the other hand, Pax2 is the first known gene to be expressed in the mid-hindbrain region of gastrula embryos. By the use of transgenic analyses, we were able to demonstrate that the early transcription factor Oct3/4 initiates the expression of the Pax2 gene in the MHB region. The analysis of Pax2-deficient embryos revealed that Pax2 controls the expression of the Fgf8 gene at the midbrain-hindbrain boundary. With these experiments we have discovered the following novel regulatory cascade that controls midbrain and cerebellum development: Oct3/4 ---- Pax2 ---- Fgf8 ---- MHB organizer ---- midbrain and cerebellum development The mammalian kidney develops in three successive steps from the initial pronephros via the mesonephros to the metanephros (adult kidney). The Pax2 and Pax8 genes are co-expressed during early kidney development. Interestingly, the pro- and mesonephros still develop normally in Pax2-deficient embryos, whereas Pax8-deficient mice form even a normal adult kidney. By analyzing Pax2,Pax8 double-mutant embryos, we could demonstrate that Pax2 and Pax8 have redundant functions in kidney development. These embryos could neither form the pronephros nor mesonephros. With these data we could show for the first time that Pax2 and Pax8 are the key regulators controlling the onset of kidney development.