Lampreys, the jawless vertebrates, contain three Pax6 genes with distinct expression in eye, brain and pancreas

10.1038/s41598-019-56085-8Scientific Reports911955

Functional Redundancy of Two Pax-Like Proteins in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia

The protozoan Giardia lamblia differentiates from a pathogenic trophozoite into an infectious cyst to survive outside of the host. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately induced. Pax family transcription factors are involved in a variety of developmental processes in animals. Nine Pax proteins have been found to play an important role in tissue and organ development in humans. To understand the progression from primitive to more complex eukaryotic cells, we tried to identify putative pax genes in the G. lamblia genome and found two genes, pax1 and pax2, with limited similarity. We found that Pax1 may transactivate the encystation-induced cwp genes and interact with AT-rich initiatior elements that are essential for promoter activity and transcription start site selection. In this study, we further characterized Pax2 and found that, like Pax1, Pax2 was present in Giardia nuclei and it may specifically bind to the AT-rich initiator elements of the encystation-induced cwp1-3 and myb2 genes. Interestingly, overexpression of Pax2 increased the cwp1-3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of nuclear localization, DNA-binding activity, and transactivation activity of Pax2. These results are similar to those found in the previous Pax1 study. In addition, the profiles of gene expression in the Pax2 and Pax1 overexpressing cells significantly overlap in the same direction and ERK1 associated complexes may phosphorylate Pax2 and Pax1, suggesting that Pax2 and Pax1 may be downstream components of a MAPK/ERK1 signaling pathway. Our results reveal functional redundancy between Pax2 and Pax1 in up-regulation of the key encystation-induced genes. These results illustrate functional redundancy of a gene family can occur in order to increase maintenance of important gene function in the protozoan organism G. lamblia

An Amphioxus Gli Gene Reveals Conservation of Midline Patterning and the Evolution of Hedgehog Signalling Diversity in Chordates

Background. Hedgehog signalling, interpreted in receiving cells by Gli transcription factors, plays a central role in the development of vertebrate and Drosphila embryos. Many aspects of the signalling pathway are conserved between these lineages, however vertebrates have diverged in at least one key aspect: they have evolved multiple Gli genes encoding functionally-distinct proteins, increasing the complexity of the hedgehog-dependent transcriptional response. Amphioxus is one of the closest living relatives of the vertebrates, having split from the vertebrate lineage prior to the widespread gene duplication prominent in early vertebrate evolution. Principal findings. We show that amphioxus has a single Gli gene, which is deployed in tissues adjacent to sources of hedgehog signalling derived from the midline and anterior endoderm. This shows the duplication and divergence of the Gli family, and hence the origin of vertebrate Gli functional diversity, was specific to the vertebrate lineage. However we also show that the single amphioxus Gli gene produces two distinct transcripts encoding different proteins. We utilise three tests of Gli function to examine the transcription regulatory capacities of these different proteins, demonstrating one has activating activity similar to Gli2, while the other acts as a weak repressor, similar to Gli3. Conclusions. These data show that the vertebrates and amphioxus have evolved functionally-similar repertoires of Gli proteins using parallel molecular routes; vertebrates via gene duplication and divergence, and amphioxus via alternate splicing of a single gene. Our results demonstrate that similar functional complexity of intercellular signalling can be achieved via different evolutionary pathways

3T3 Cell Lines Stably Expressing Pax6 or Pax6(5a) – A New Tool Used for Identification of Common and Isoform Specific Target Genes

Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities

Cloning and developmental expression of amphioxus Dachschund

The nuclear factor dachshund (dac) is a key regulator of eye and leg development in Drosophila.We have cloned a Dachshund homologue from an invertebrate chordate amphioxus (Branchiostoma floridae). Sequence comparison reveals a high degree of similarity of amphioxus Dachshund (AmphiDach) to the known vertebrate and Drosophila dachshund genes. AmphiDach is first expressed in the prospective paraxial mesoderm at the gastrula stage. At the early neurula stage, expression is detected in developing somites and anterior endoderm, but in late neurula transcripts are present exclusively in the most posterior region of the cerebral vesicle and the anterior pharynx endoderm. Then, in larva, AmphiDach is localized in photoreceptive neurons of the frontal eye, infundibular organ, and endostyle as well as in Hesse organs and in nerve cells scattered along the nerve cord. Comparison of Dach expression in amphioxus and vertebrates suggests that such patterns are relatively similar (because they are expressed in somites, photoreceptor cells and CNS), even if expression of AmphiDach in the endostyle has no counterparts in its vertebrate homolog, the thyroid

Fluorescence properties of new substituted thieno [3, 2-b] indole derivatives and their electrosynthesized oligomers.

International audienc

Synthesis and spectral properties of new fluorescent substituted thieno [3,2 b] indole derivatives

International audienceThe synthesis and optical properties of three new fluorescent alkoxy-substituted thieno[3,2-b]indole (TI) derivatives, including 7-methoxy thieno[3,2-b]indole (7-MeOTI), 6,7- methylenedioxythieno[3,2-b]indole (6,7-MDTI) and 6,7-dihexyloxythieno[3,2-b]indole, (6,7-DHTI), were investigated. Electronic absorption spectra, fluorescence excitation and emission spectra, fluorescence quantum yields (ΦF), lifetimes (τF), and other photophysical parameters of the three TI derivatives were measured in DMSO solutions at room temperature. Theoretical electronic absorption and fluorescence spectra were also calculated by means of a molecular orbital (MO) method. For all three alkoxy-TI derivatives, the fluorescence emission maximum wavelength was significantly red shifted relative to un-substituted TI, which was attributed to delocalization of the fused hetero-aromatic ring π electronic system by the electron-donating alkoxy group(s). ΦF values varied from 0.12 to 0.17, according to the compound. τF were short, in the range 0.56-1.13 ns