Pax6 and Pax2 are members of the Pax family of transcrip-tion factors that are both expressed in the developing visual system of zebrafish embryos. Pax6 protein is present in all cells that form the neural retina and pigment epithelium, whereas Pax2 is located primarily in cells that will give rise to the optic stalk. In this study, we have addressed the role of midline signalling in the regulation of Pax2 and Pax6 dis-tributions and in the subsequent morphogenesis of the eyes. Midline signalling is severely perturbed in cyclops mutant embryos resulting in an absence of ventral midline CNS tissue and fusion of the eyes. Mutant embryos ectopically express Pax6 in a bridge of tissue around the anterior pole of the neural keel in the position normally occupied by cells that form the optic stalks. In contrast, Pax2 protein is almost completely absent from this region in mutant embryos. Con-commitant with the changes in Pax protein distribution, cells in the position of the optic stalks differentiate as retina. These results suggest that a signal emanating from the midline, which is absent in cyclops mutant embryos, may be required to promote Pax2 and inhibit Pax6 expression in cells destined to form the optic stalks. Sonic hedgehog (Shh also known as Vhh-1 and Hhg-1) is a midline sig-nalling molecule that is absent from the neuroepithelium of cyclops mutant embryos at early developmental stages. To test the possibility that Shh might be able to regulate the spatial expression of Pax6 and Pax2 in the optic primordia, it was overexpressed in the developing CNS. The number of cells containing Pax2 was increased following shh over-expression and embryos developed hypertrophied optic stalk-like structures. Complimentary to the changes in Pax2 distribution, there were fewer Pax6-containing cells and pigment epithelium and neural retina were reduced. Our results suggest that Shh or a closely related sig-nalling molecule emanating from midline tissue in the ventral forebrain either directly or indirectly induces the expression of Pax2 and inhibits the expression of Pax6 and thus may regulate the partitioning of the optic primordia into optic stalks and retinal tissue.

The transcription factor Pax5 is essential for initiating B cell lineage commitment, but its role in maintaining commitment is unknown. Using conditional Pax5 inactivation in committed pro-B cells, we demonstrate that Pax5 is required not only to initiate its B lymphoid transcription program, but also to maintain it in early B cell development. As a consequence of Pax5 inactivation, previously committed pro-B cells regained the capacity to differentiate into macrophages in vitro and to reconstitute T cell development in vivo in RAG2 ^−/− mice. Hence, Pax5 expression is continuously required to maintain B cell lineage commitment, because its loss converts committed pro-B cells into hematopoietic progenitors with multilineage potential.

The Pax6 genes encode evolutionary conserved transcription factors that act high up in the regulatory hierarchy controlling development of central organs such as the eyes and the central nervous system. These proteins contain two DNA-binding domains. The N-terminal paired domain is separated from a paired-type homeodomain by a linker region, and a transactivation domain is located C-terminal to the homeodomain. Vertebrate Pax6 genes express a paired-less isoform of Pax6 (Pax6DeltaPD) from an internal start codon in the coding region between the paired domain and homeodomain. We now provide evidence for an interaction between the full-length isoform and Pax6DeltaPD, which enhances the transactivation activity of Pax6 from paired domain-binding sites. The paired-like homeodomain protein Rax behaved similarly to Pax6DeltaPD. Both Pax6DeltaPD and Rax bound to the homeodomain of Pax6 in vitro in the absence of specific DNA binding. Coimmunoprecipitation experiments following cotransfection confirmed the existence of complexes between Pax6 and Pax6DeltaPD, Pax6 and Rax, and Pax6DeltaPD and Rax in vivo. Interestingly, the C-terminal subdomain of the paired domain and the homeodomain can interact with each other. The paired domain can also interact with itself. Surprisingly, GST pull-down assays revealed that the homeodomains of such diverse proteins as Chx10, Six3, Lhx2, En-1, Prep1, Prox1, and HoxB1 could all bind to Pax6, and several of these enhanced Pax6-mediated transactivation upon coexpression. Since many homeodomain proteins are coexpressed with Pax6 in several tissues during development, our results indicate the existence of novel regulatory interactions that may be important for fine tuning of gene regulation.

The transcription factor Pax6 is required for normal development of the central nervous system, the eyes, nose, and pancreas. Here we show that the transactivation domain (TAD) of zebrafish Pax6 is phosphorylated in vitro by the mitogen-activated protein kinases (MAPKs) extracellular-signal regulated kinase (ERK) and p38 kinase but not by Jun N-terminal kinase (JNK). Three of four putative proline-dependent kinase phosphorylation sites are phosphorylated in vitro. Of these sites, the serine 413 (Ser⁴¹³) is evolutionary conserved from sea urchin to man. Ser⁴¹³ is also phosphorylatedin vivo upon activation of ERK or p38 kinase. Substitution of Ser⁴¹³ with alanine strongly decreased the transactivation potential of the Pax6 TAD whereas substitution with glutamate increased the transactivation. Reporter gene assays with wild-type and mutant Pax6 revealed that transactivation by the full-length Pax6 protein from paired domain-binding sites was strongly enhanced (16-fold) following co-transfection with activated p38 kinase. This enhancement was largely dependent on the Ser⁴¹³ site. ERK activation, however, produced a 3-fold increase in transactivation which was partly independent of the Ser⁴¹³ site. These findings provide a starting point for further studies aimed at elucidating a post-translational regulation of Pax6 following activation of MAPK signaling pathways.