prep1.2 and aldh1a2 participate to a positive loop required for branchial arches development in zebrafish.

Segmentation is a key step in embryonic development. Acting in all germ layers, it is responsible for the generation of antero-posterior asymmetries. Hox genes, with their diverse expression in individual segments, are fundamental players in the determination of different segmental fates. In vertebrates, Hox gene products gain specificity for DNA sequences by interacting with Pbx, Prep and Meis homeodomain transcription factors. In this work we cloned and analysed prep1.2 in zebrafish. In-situ hybridization experiments show that prep1.2 is maternally and ubiquitously expressed up to early somitogenesis when its expression pattern becomes more restricted to the head and trunk mesenchyme. Experiments of loss of function with prep1.2 morpholinos change the shape of the hyoid and third pharyngeal cartilages while arches 4-7 and pectoral fins are absent, a phenotype strikingly similar to that caused by loss of retinoic acid (RA). In fact, we show that prep1.2 is positively regulated by RA and required for the normal expression of aldh1a2 at later stages, particularly in tissues involved in the development of the branchial arches and pectoral fins. Thus, prep1.2 and aldh1a2 are members of an indirect positive feedback loop required for pharyngeal endoderm and posterior branchial arches development. As the paralogue gene prep1.1 is more important in hindbrain patterning and neural crest chondrogenesis, we provide evidence of a functional specialization of prep genes in zebrafish head segmentation and morphogenesis.

Characterization of the regulatory region of the zebrafish Prep1.1 gene: analogies to the promoter of the human PREP1.

Prep1 is a developmentally essential TALE class homeodomain transcription factor. In zebrafish and mouse, Prep1 is already ubiquitously expressed at the earliest stages of development, with important tissue-specific peculiarities. The Prep1 gene in mouse is developmentally essential and has haploinsufficient tumor suppressor activity [1]. We have determined the human Prep1 transcription start site (TSS) by primer extension analysis and identified, within 20 bp, the transcription start region (TSR) of the zebrafish Prep1.1 promoter. The functions of the zebrafish 5' upstream sequences were analyzed both by transient transfections in Hela Cells and by injection in zebrafish embryos. This analysis revealed a complex promoter with regulatory sequences extending up to -1.8, possibly -5.0 Kb, responsible for tissue specific expression. Moreover, the first intron contains a conserved tissue-specific enhancer both in zebrafish and in human cells. Finally, a two nucleotides mutation of an EGR-1 site, conserved in all species including human and zebrafish and located at a short distance from the TSS, destroyed the promoter activity of the -5.0 Kb promoter. A transgenic fish expressing GFP under the -1.8 Kb zebrafish promoter/enhancer co-expressed GFP and endogenous Prep1.1 during embryonic development. In the adult fish, GFP was expressed in hematopoietic regions like the kidney, in agreement with the essential function of Prep1 in mouse hematopoiesis. Sequence comparison showed conservation from man to fish of the sequences around the TSS, within the first intron enhancer. Moreover, about 40% of the sequences spread throughout the 5 Kbof the zebrafish promoter are concentrated in the -3 to -5 Kb of the human upstream region.