The functional role of the Meis/Prep-binding elements in Pax6 locus during pancreas and eye development

Abstract

Pax6 is an essential transcription factor for lens, lacrimal gland and pancreas development. Previous transgenic analyses have identified several Pax6 regulatory elements, but their functional significance and binding factors remain largely unknown. In this study, we generated two genomic truncations to delete three elements that were previously shown to bind to the Meis/Prep family homeoproteins. One 3.1 kb deletion (Pax6 ΔDP/ΔDP) removed two putative pancreatic enhancers and a previously identified ectodermal enhancer, while a 450 bp sub-deletion (Pax6ΔPE/ΔPE) eliminated only the promoter-proximal pancreatic enhancer. Immunohistochemistry and quantitative RT-PCR showed that the Pax6ΔPE/ΔPE pancreata had a significant decrease in Pax6, glucagon, and insulin expression, while no further reductions were observed in the Pax6ΔDP/ΔDP mice, indicating that only the 450 bp region is required for pancreatic development. In contrast, Pax6ΔDP/ΔDP, but not Pax6ΔPE/ΔPE mice, developed stunted lacrimal gland and lens hypoplasia which was significantly more severe than that reported when only the ectodermal enhancer was deleted. This result suggested that the ectodermal enhancer must cooperate with its neighboring sequences to regulate the Pax6 ectodermal expression. Finally, we generated conditional knockouts of Prep1 in lens and pancreas, but surprisingly, did not observe any developmental defects. Together, these results provide functional evidence for the independent and synergistic roles of the Pax6 upstream enhancers, and they suggest the potential redundancy of Meis/Prep protein in Pax6 regulation.

An Allelic Series at the Paired Box Gene 6 (Pax6) Locus Reveals the Functional Specificity of Pax Genes

“…Pax6 Targeting Vector Construction-The Pax6 targeting vector was generated using the recombineering method (34,35). Briefly, a minitargeting vector containing a Neo selection cassette and a Pax6(5a) full-length cDNA (IMAGE clone number 4008490) was used to replace the Pax6 genomic sequence from exons 4 to 13 contained in a 129S6/SvEvTac Bac clone (BACPAC Resources Center at Children's Hospital Oakland Research Institute, catalogue number RP22-55A14).…”

“…Antisense probes were generated from cDNAs for Crx ( Quantitative Real Time RT-PCR-To compare the expression levels of knock-in Pax6(5a) and Pax2 in embryonic brain at E14.5, they were individually correlated with the endogenous Pax6 expression level in Pax6 5a/ϩ and Pax6 Pax2/ϩ mutants by quantitative real time RT-PCR as described previously (34). PCR primers used were as follows: Pax6(5a)/Pax6: F, GCGC-AGACGGCATGTATGATA; R, GGGTTGCCCTGGTACT-GAAG; Pax2: F, AAGCCCGGAGTGATTGGTG; R, CAGGC-GAACATAGTCGGGTT (acquired from Harvard PrimerBank).…”

Meis2 is a Pax6 co-factor in neurogenesis and dopaminergic periglomerular fate specification in the adult olfactory bulb

“…It remains to be determined, however, whether Meis2, Pax6 and Dlx2 act as a trimeric protein complex or as individual Meis-Pax, Meis-Dlx and Pax-Dlx dimers and whether other proteins can also associate with them. It is also interesting to note that Pax6 is a Meis target gene in the retina, lens and pancreas (Carbe et al, 2012;Heine et al, 2008;Zhang et al, 2002;Zhang et al, 2006). Conversely, Pax6 was recently reported to bind to upstream regulatory regions of the meis1 gene in the embryonic zebrafish retina (Royo et al, 2012).…”

Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development

“…The Ugdh flox targeting vector was constructed using the recombineering method from a 12.7 kb genomic fragment (Liu et al, 2003;Carbe et al, 2012), which was retrieved from a C57BL/6 bacterial artificial chromosome (BAC) clone (RP23-477N9, BACPAC Resources Center at Children's Hospital Oakland Research Institute, CA, USA). It contains a neomycin (Neo) selection cassette surrounded by two frt sites and exon 6 of the Ugdh gene flanked by two loxP sites (Fig.…”