Diverse Cis-Regulatory Mechanisms Contribute to Expression Evolution of Tandem Gene Duplicates

The deposited article is a post-print version and has peer review. The deposited article version contains attached the supplementary materials within the pdf.Pairs of duplicated genes generally display a combination of conserved expression patterns inherited from their unduplicated ancestor and newly acquired domains. However, how the cis-regulatory architecture of duplicated loci evolves to produce these expression patterns is poorly understood. We have directly examined the gene-regulatory evolution of two tandem duplicates, the Drosophila Ly6 genes CG9336 and CG9338, which arose at the base of the drosophilids between 40 and 60 million years ago. Comparing the expression patterns of the two paralogs in four Drosophila species with that of the unduplicated ortholog in the tephritid Ceratitis capitata, we show that they diverged from each other as well as from the unduplicated ortholog. Moreover, the expression divergence appears to have occurred close to the duplication event and also more recently in a lineage-specific manner. The comparison of the tissue-specific cis-regulatory modules (CRMs) controlling the paralog expression in the four Drosophila species indicates that diverse cis-regulatory mechanisms, including the novel tissue-specific enhancers, differential inactivation, and enhancer sharing, contributed to the expression evolution. Our analysis also reveals a surprisingly variable cis-regulatory architecture, in which the CRMs driving conserved expression domains change in number, location, and specificity. Altogether, this study provides a detailed historical account that uncovers a highly dynamic picture of how the paralog expression patterns and their underlying cis-regulatory landscape evolve. We argue that our findings will encourage studying cis-regulatory evolution at the whole-locus level in order to understand how interactions between enhancers and other regulatory levels shape the evolution of gene expression.Fundação Calouste Gulbenkian/Instituto Gulbenkian de Ciência; Toulouse RIO Imaging platform; Bloomington Drosophila Stock Center (Indiana, USA); Developmental Studies Hybridoma Bank (Iowa, USA); Drosophila Genomics Resource Center (Indiana, USA); NIH grant: (2P0OD010949); Agence Nationale de la Recherche grant: (ANR-13-ISV7-0001-01); Fundação para a Ciência e a Tecnologia grants: (SFRH/BPD/75139/2010, FCT-ANR/BIA-ANM/0003/2013, FCT-EXPL/BEX-GMG/2197/2013).info:eu-repo/semantics/acceptedVersio

Boudin trafficking reveals the dynamic internalisation of specific septate junction components in <i>Drosophila</i>

<div><p>The maintenance of paracellular barriers in invertebrate epithelia depends on the integrity of specific cell adhesion structures known as septate junctions (SJ). Multiple studies in <i>Drosophila</i> have revealed that these junctions have a stereotyped architecture resulting from the association in the lateral membrane of a large number of components. However, little is known about the dynamic organisation adopted by these multi-protein complexes in living tissues. We have used live imaging techniques to show that the Ly6 protein Boudin is a component of these adhesion junctions and can diffuse systemically to associate with the SJ of distant cells. We also observe that this protein and the claudin Kune-kune are endocytosed in epidermal cells during embryogenesis. Our data reveal that the SJ contain a set of components exhibiting a high membrane turnover, a feature that could contribute in a tissue-specific manner to the morphogenetic plasticity of these adhesion structures.</p></div

Kune is endocytosed and degraded during epidermal morphogenesis.

<p>(A-H) Confocal views of the ventral epidermis corresponding to live wild type stage 14 (A,C,E,G) or stage 16 (B,D,F,H) embryos expressing mCheKune (magenta and b/w in bottom panels) and EGFPKune (A,B), YFP-Rab5 (C,D), YFP-Rab7 (E,F) or Lamp1-YFP (G,H), all shown in green (top panels) and in b/w (middle panels). Vesicles containing both tagged Kune versions (labelled with circles) are visible in both stages (A,B). mCheKune is also detected in YFP-Rab5 early endosomes (C,D), YFP-Rab7 late endosomes (E,F) and Lamp-1-YFP lysosomes (G,H). Scale bar: 10 μm.</p

Kune membrane localisation is affected in SJ mutants.

<p>(A-H) Confocal images corresponding to the ventral epidermis of live stage 16 embryos expressing mCheKune (magenta and b/w in bottom panels) and EGFPKune (A-D, shown in green and b/w in middle panels) or YFP-Rab7 (E-H, shown green and b/w in middle panels). All images were acquired using the same parameters; specific genotypes are indicated above each column. The EGFPKune membrane levels are diminished with respect to wild type controls (A) in <i>mega</i> (B), <i>bou</i> (C) and especially in <i>Nrg</i> mutants (D). A weak mCheKune membrane signal is seen in all these backgrounds. mCheKune is also detected in internal vesicles containing EGFPKune (A-D) and YFP-Rab7 (E-H). These vesicles (labelled with circles) are more abundant in the <i>bou</i> (C and G) and <i>Nrg</i> (D and H) mutant embryos. (I) The upper graph represents for each genotype the average number of mCheKune (magenta) and YFP-Rab7 vesicles (green) found in regions of 100 μm ² corresponding to the epidermis of stage 16 embryos. The bottom graph represents the average percentage of YFP-Rab7 late endosomes also positive for mCheKune. Error bars represent the standard deviations observed after pooling the data of different sample areas (n≥13). Both <i>bou</i> and <i>Nrg</i> mutants have more mCheKune and YFP-Rab7 vesicles (respectively, p<0.01 and p<0.05, Student’s t-test) and show a higher proportion of late endosomes containing mCheKune (p<0.01, Student’s t-test). (J-L) Ventral epidermis of stage 16 live embryos expressing EGFPKune (green, b/w in middle panels) and mCheBou (magenta, b/w in lower panels) under the control of <i>HhGAL4</i>. Imaging parameters are identical for all genotypes, indicated above each column. All the vesicles containing EGFPKune are also positive for exogenous mCheBou (examples are marked with circles). Scale bars: 10 μm.</p

Bou is a SJ structural component.

<p>(A,B) Transmitted electron micrographs showing the lateral membrane of contiguous epidermal cells in both wild type (A) and <i>bou</i> (B) stage 17 embryos. An electron dense material corresponding to septa is found in the intercellular space of contiguous wild type cells (A, red arrows). These structures are completely absent in the cell contacts of <i>bou</i> embryos. The epidermal cuticle is also indicated (cu). Scale bar: 25 nm. (C-J) Confocal images corresponding to stage 16 live <i>bou</i> embryos expressing mCheBou under the control of <i>HhGAL4</i> in the epidermis (C,G: planar view, D,H: transverse view), dorsal tracheal trunk (E,I) and salivary gland (F,J). The mCheBou protein (magenta in C-F, b/w in G-J) accumulates at the level of the SJ (red arrows), co-labelled with Nrg-GFP (green). Scale bars: 15 μm. (K-M) Confocal images of live stage 16 embryos showing the distribution of mCheBou in the hindgut columnar epithelium of <i>bou</i> rescued embryos (K) or double mutants <i>bou; nrv2</i> (L) and <i>mega bou</i> (M). In the double mutants the mCheBou membrane signal is uniformly distributed over the lateral membrane, instead of accumulating in its most apical part (red arrows). Scale bar: 20 μm.</p

Exogenous mCheBou is captured and endocytosed in the embryonic epidermis.

<p>(A) Confocal optical sections showing the distribution of mCheBou in the ventral epidermis of a live stage 14 <i>bou</i> rescued embryo. After photobleaching (time 0) of an area delimited by a black square, the mCheBou signal gradually reappears in the cell cortex. Cells entirely bleached at time 0 are labelled with coloured dots, to facilitate their visualisation. (B) Confocal image showing the mCheBou distribution in the ventral epidermis of a live stage 14 wild type embryo, imaged using the same parameters as the rescued embryos shown in A. Only a faint signal is seen in the cell contours (red arrow). (C,D) mCheBou distribution (green in top panels, b/w in middle panels) in the ventral epidermis of live stage 14 <i>bou</i> rescued embryos expressing ubiquitously the early (YFP-Rab5, C) or late (YFP-Rab7, D) endosome markers (shown in magenta in top panels and on b/w, bottom panels). Examples of vesicles positive for mCheBou and each marker are labelled with circles. Scale bars: 10 μm.</p

mCheBou diffuses systemically during embryonic and larval stages.

<p>(A-E) Confocal images showing the ventral epidermis of live stage 16 embryos expressing different fluorescent proteins in the <i>HhGAL4</i> domain, which is visible in segmental stripes. An extracellular diffuse signal corresponding to mCheBou (red in A,B, b/w in A’,B’) is detected in the perivitelline space (arrows), which also accumulates Secreted-GFP (green in A, b/w in A”) and GPI-GFP (green in B, b/w in B”, arrows). Neither mCD8mCherry (D) nor mCD8GFP (E) or CD63-GFP (C) are detected outside the <i>HhGAL4</i> stripes. Scale bar: 20 μm. (F-K,F’-K’) Confocal pictures corresponding to third instar larvae live explants of wing imaginal discs (F-K) and garland cells (F’-K’, visible in the bright field right panels). The different fusion proteins are produced in the <i>HhGAL4</i> domain, visible in the posterior compartment of the wing disc (F-K). mCherryBou (F,F’), Secreted-GFP (I,I’) and GPI-GFP (J,J’) are detected in the garland cells (white arrowheads) and in the extracellular space comprised between the disc peripodial membrane and the wing pouch (arrows). No signal could be detected in these locations upon expression of mCD8mCherry (G,G’), mCD8GFP (H,H’) or CD63-GFP (K,K’). Scale bars: 100 μm.</p

A highly virulent variant of HIV-1 circulating in the Netherlands

We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.74 log10 increase (i.e., a ~3.5-fold to 5.5-fold increase) in viral load compared with, and exhibited CD4 cell decline twice as fast as, 6604 individuals with other subtype-B strains. Without treatment, advanced HIV-CD4 cell counts below 350 cells per cubic millimeter, with long-term clinical consequences-is expected to be reached, on average, 9 months after diagnosis for individuals in their thirties with this variant. Age, sex, suspected mode of transmission, and place of birth for the aforementioned 109 individuals were typical for HIV-positive people in the Netherlands, which suggests that the increased virulence is attributable to the viral strain. Genetic sequence analysis suggests that this variant arose in the 1990s from de novo mutation, not recombination, with increased transmissibility and an unfamiliar molecular mechanism of virulence