Using archived and biocollection samples towards deciphering the DNA virus diversity associated with rodent species in the families cricetidae and heteromyidae.

Rodentia is the most speciose order of mammals, and they are known to harbor a wide range of viruses. Although there has been significant research on zoonotic viruses in rodents, research on the diversity of other viruses has been limited, especially for rodents in the families Cricetidae and Heteromyidae. In fecal and liver samples of nine species of rodents, we identify 346 distinct circular DNA viral genomes. Of these, a large portion are circular, single-stranded DNA viruses in the families Anelloviridae (n = 3), Circoviridae (n = 5), Genomoviridae (n = 7), Microviridae (n = 297), Naryaviridae (n = 4), Vilyaviridae (n = 15) and in the phylum Cressdnaviricota (n = 13) that cannot be assigned established families. We also identified two large bacteriophages of 36 and 50 kb that are part of the class Caudoviricetes. Some of these viruses are clearly those that infect rodents, however, most of these likely infect various organisms associated with rodents, their environment or their diet.

Regeneration enhancers: a field in development.

The ability to regenerate tissues and organs following damage is not equally distributed across metazoans, and even highly related species can vary considerably in their regenerative capacity. Studies of animals with high regenerative potential have shown that factors expressed during normal development are often reactivated upon damage and required for successful regeneration. As such, regenerative potential may not be dictated by the presence or absence of the necessary genes, but whether such genes are appropriately activated following injury. The identification of damage-responsive enhancers that regulate regenerative gene expression in multiple species and tissues provides possible mechanistic insight into this phenomenon. Enhancers that are reused from developmental programs, and those that are potentially unique to regeneration, have been characterized individually and at a genome-wide scale. A better understanding of the regulatory events that, direct and in some cases limit, regenerative capacity is an important step in developing new methods to manipulate and augment regeneration, particularly in tissues that do not have this ability, including those of humans.

Necrosis-induced apoptosis promotes regeneration in Drosophila wing imaginal discs.

Regeneration is a complex process that requires a coordinated genetic response to tissue loss. Signals from dying cells are crucial to this process and are best understood in the context of regeneration following programmed cell death, like apoptosis. Conversely, regeneration following unregulated forms of death, such as necrosis, have yet to be fully explored. Here, we have developed a method to investigate regeneration following necrosis using the Drosophila wing imaginal disc. We show that necrosis stimulates regeneration at an equivalent level to that of apoptosis-mediated cell death and activates a similar response at the wound edge involving localized JNK signaling. Unexpectedly, however, necrosis also results in significant apoptosis far from the site of ablation, which we have termed necrosis-induced apoptosis (NiA). This apoptosis occurs independent of changes at the wound edge and importantly does not rely on JNK signaling. Furthermore, we find that blocking NiA limits proliferation and subsequently inhibits regeneration, suggesting that tissues damaged by necrosis can activate programmed cell death at a distance from the injury to promote regeneration.

Damage-responsive, maturity-silenced enhancers regulate multiple genes that direct regeneration in Drosophila.

Like tissues of many organisms, Drosophila imaginal discs lose the ability to regenerate as they mature. This loss of regenerative capacity coincides with reduced damage-responsive expression of multiple genes needed for regeneration. We previously showed that two such genes, wg and Wnt6, are regulated by a single damage-responsive enhancer that becomes progressively inactivated via Polycomb-mediated silencing as discs mature (Harris et al., 2016). Here we explore the generality of this mechanism and identify additional damage-responsive, maturity-silenced (DRMS) enhancers, some near genes known to be required for regeneration such as Mmp1, and others near genes that we now show function in regeneration. Using a novel GAL4-independent ablation system we characterize two DRMS-associated genes, apontic (apt), which curtails regeneration and CG9752/asperous (aspr), which promotes it. This mechanism of suppressing regeneration by silencing damage-responsive enhancers at multiple loci can be partially overcome by reducing activity of the chromatin regulator extra sex combs (esc).

Localized epigenetic silencing of a damage-activated WNT enhancer limits regeneration in mature Drosophila imaginal discs.

Many organisms lose the capacity to regenerate damaged tissues as they mature. Damaged Drosophila imaginal discs regenerate efficiently early in the third larval instar (L3) but progressively lose this ability. This correlates with reduced damage-responsive expression of multiple genes, including the WNT genes wingless (wg) and Wnt6. We demonstrate that damage-responsive expression of both genes requires a bipartite enhancer whose activity declines during L3. Within this enhancer, a damage-responsive module stays active throughout L3, while an adjacent silencing element nucleates increasing levels of epigenetic silencing restricted to this enhancer. Cas9-mediated deletion of the silencing element alleviates WNT repression, but is, in itself, insufficient to promote regeneration. However, directing Myc expression to the blastema overcomes repression of multiple genes, including wg, and restores cellular responses necessary for regeneration. Localized epigenetic silencing of damage-responsive enhancers can therefore restrict regenerative capacity in maturing organisms without compromising gene functions regulated by developmental signals.

Coordinate post-transcriptional repression of Dpp-dependent transcription factors attenuates signal range during development.

Precise control of the range of signalling molecule action is crucial for correct cell fate patterning during development. For example, Drosophila ovarian germline stem cells (GSCs) are maintained by exquisitely short-range BMP signalling from the niche. In the absence of BMP signalling, one GSC daughter differentiates into a cystoblast (CB) and this fate is stabilised by Brain tumour (Brat) and Pumilio (Pum)-mediated post-transcriptional repression of mRNAs, including that encoding the Dpp transducer, Mad. However, the identity of other repressed mRNAs and the mechanism of post-transcriptional repression are currently unknown. Here, we identify the Medea and schnurri mRNAs, which encode transcriptional regulators required for activation and/or repression of Dpp target genes, as additional Pum-Brat targets, suggesting that tripartite repression of the transducers is deployed to desensitise the CB to Dpp. In addition, we show that repression by Pum-Brat requires recruitment of the CCR4 and Pop2 deadenylases, with knockdown of deadenylases in vivo giving rise to ectopic GSCs. Consistent with this, Pum-Brat repression leads to poly(A) tail shortening and mRNA degradation in tissue culture cells, and we detect a reduced number of Mad and shn transcripts in the CB relative to the GSC based on single molecule mRNA quantitation. Finally, we show generality of the mechanism by demonstrating that Brat also attenuates pMad and Dpp signalling range in the early embryo. Together our data serve as a platform for understanding how post-transcriptional repression restricts interpretation of BMPs and other cell signals in order to allow robust cell fate patterning during development.

Patterning of the Drosophila oocyte by a sequential translation repression program involving the d4EHP and Belle translational repressors.

During Drosophila development, translational control plays a crucial role in regulating gene expression, and is particularly important during pre-patterning of the maturing oocyte. A critical step in translation initiation is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the mRNA cap structure, which ultimately leads to recruitment of the ribosome. d4EHP is a translational repressor that prevents translation initiation by out-competing eIF4E on the cap structure for a subset of mRNAs. However, only two examples of mRNAs subject to d4EHP translation repression in Drosophila are known. Here we show that the belle (bel) mRNA is translationally repressed by the d4EHP protein in the Drosophila ovary. Consistent with this regulation, d4EHP overexpression in the ovary phenocopies the bel mutant. We also provide evidence that the Bel protein binds to eIF4E and may itself function as a translation repressor protein, with bruno as a potential target for Bel repression in the oocyte. Bruno is known to repress the mRNA of the key oocyte axis determinant oskar (osk) during oogenesis, and we find that an increase in the level of Bruno protein in bel mutant ovaries is associated with a reduction in Osk protein. Overall, our data suggest that a translational regulatory network exists in which consecutive translational repression events act to correctly pattern the Drosophila oocyte.

Cease and desist: modulating short-range Dpp signalling in the stem-cell niche.

Drosophila ovarian germline stem cells (GSCs) are maintained by the extracellular BMP2/4 orthologue Dpp, which is produced from the surrounding somatic niche. The Dpp signal has a short range; it induces a response in GSCs within the niche, but is rapidly extinguished in their progeny only one cell-diameter away. To ensure the correct balance between stem-cell maintenance and differentiation, several regulatory mechanisms that modulate the Dpp signal at many stages of the pathway have been described. Here, we discuss the nature of the ovarian Dpp signal and review the catalogue of mechanisms that regulate it, demonstrating how the exquisite modulation of Dpp signalling in this context can result in precise and robust control of stem-cell fate. This modulation is applicable to other stem-cell environments that use BMPs as a niche signal, and the regulatory mechanisms are conceptually relevant to several other stem-cell systems.

Brat promotes stem cell differentiation via control of a bistable switch that restricts BMP signaling.

Drosophila ovarian germline stem cells (GSCs) are maintained by Dpp signaling and the Pumilio (Pum) and Nanos (Nos) translational repressors. Upon division, Dpp signaling is extinguished, and Nos is downregulated in one daughter cell, causing it to switch to a differentiating cystoblast (CB). However, downstream effectors of Pum-Nos remain unknown, and how CBs lose their responsiveness to Dpp is unclear. Here, we identify Brain Tumor (Brat) as a potent differentiation factor and target of Pum-Nos regulation. Brat is excluded from GSCs by Pum-Nos but functions with Pum in CBs to translationally repress distinct targets, including the Mad and dMyc mRNAs. Regulation of both targets simultaneously lowers cellular responsiveness to Dpp signaling, forcing the cell to become refractory to the self-renewal signal. Mathematical modeling elucidates bistability of cell fate in the Brat-mediated system, revealing how autoregulation of GSC number can arise from Brat coupling extracellular Dpp regulation to intracellular interpretation.

Type IV collagens regulate BMP signalling in Drosophila.

Dorsal-ventral patterning in vertebrate and invertebrate embryos is mediated by a conserved system of secreted proteins that establishes a bone morphogenetic protein (BMP) gradient. Although the Drosophila embryonic Decapentaplegic (Dpp) gradient has served as a model to understand how morphogen gradients are established, no role for the extracellular matrix has been previously described. Here we show that type IV collagen extracellular matrix proteins bind Dpp and regulate its signalling in both the Drosophila embryo and ovary. We provide evidence that the interaction between Dpp and type IV collagen augments Dpp signalling in the embryo by promoting gradient formation, yet it restricts the signalling range in the ovary through sequestration of the Dpp ligand. Together, these results identify a critical function of type IV collagens in modulating Dpp in the extracellular space during Drosophila development. On the basis of our findings that human type IV collagen binds BMP4, we predict that this role of type IV collagens will be conserved.