Basement membrane diversification relies on two competitive secretory routes defined by Rab10 and Rab8 and modulated by dystrophin and the exocyst complex.

The basement membrane (BM) is an essential structural element of tissues, and its diversification participates in organ morphogenesis. However, the traffic routes associated with BM formation and the mechanistic modulations explaining its diversification are still poorly understood. Drosophila melanogaster follicular epithelium relies on a BM composed of oriented BM fibrils and a more homogenous matrix. Here, we determined the specific molecular identity and cell exit sites of BM protein secretory routes. First, we found that Rab10 and Rab8 define two parallel routes for BM protein secretion. When both routes were abolished, BM production was fully blocked; however, genetic interactions revealed that these two routes competed. Rab10 promoted lateral and planar-polarized secretion, whereas Rab8 promoted basal secretion, leading to the formation of BM fibrils and homogenous BM, respectively. We also found that the dystrophin-associated protein complex (DAPC) and Rab10 were both present in a planar-polarized tubular compartment containing BM proteins. DAPC was essential for fibril formation and sufficient to reorient secretion towards the Rab10 route. Moreover, we identified a dual function for the exocyst complex in this context. First, the Exo70 subunit directly interacted with dystrophin to limit its planar polarization. Second, the exocyst complex was also required for the Rab8 route. Altogether, these results highlight important mechanistic aspects of BM protein secretion and illustrate how BM diversity can emerge from the spatial control of distinct traffic routes.

SARCOPTIC MANGE IN FREE-RANGING NORTH AMERICAN PORCUPINES (ERETHIZON DORSATUM) IN NEW YORK STATE.

Sarcoptic mange causes pruritic and crusting dermatitis in a large number of mammalian species with varying population impacts. Between 2016 and 2022, 15 North American porcupines (Erethizon dorsatum) were diagnosed with sarcoptic mange at Cornell University's Janet L. Swanson Wildlife Hospital in Ithaca, New York. Disease severity varied among individuals but all shared a similar unique presentation with thick, pale tan to yellow crusts limited in distribution to the ventral, nonquilled areas of the body, including the ventral abdomen and thorax, distal limbs, and face. The thick, hard nature of the crusts resulted in additional complications in many individuals, including inability to move the jaw and cracking and fissuring of the crusts and skin over joints of the limbs. Mites were plentiful within the crusts, with some burrowing into the epidermis as deep as the stratum spinosum. Secondary bacterial and/or fungal dermatitis were common, resulting in sepsis and death in three of the porcupines. Treatment with avermectins (ivermectin and/or selamectin) for 4-5 wk was successful in 12 cases in combination with other supportive care measures, including subcutaneous fluids, antimicrobials, and analgesics. Porcupines were hospitalized for an average of 18 d (ranging from 7 to 50 d) prior to transfer to a licensed wildlife rehabilitator for continued treatment and eventual release back into the wild.

NANOG initiates epiblast fate through the coordination of pluripotency genes expression.

The epiblast is the source of all mammalian embryonic tissues and of pluripotent embryonic stem cells. It differentiates alongside the primitive endoderm in a "salt and pepper" pattern from inner cell mass (ICM) progenitors during the preimplantation stages through the activity of NANOG, GATA6 and the FGF pathway. When and how epiblast lineage specification is initiated is still unclear. Here, we show that the coordinated expression of pluripotency markers defines epiblast identity. Conversely, ICM progenitor cells display random cell-to-cell variability in expression of various pluripotency markers, remarkably dissimilar from the epiblast signature and independently from NANOG, GATA6 and FGF activities. Coordination of pluripotency markers expression fails in Nanog and Gata6 double KO (DKO) embryos. Collectively, our data suggest that NANOG triggers epiblast specification by ensuring the coordinated expression of pluripotency markers in a subset of cells, implying a stochastic mechanism. These features are likely conserved, as suggested by analysis of human embryos.

Prevalence of anticoagulant rodenticide exposure in red-tailed hawks (Buteo jamaicensis) and utility of clotting time assays to detect coagulopathy.

Anticoagulant rodenticides (ARs) continue to be used across the United States as a method for controlling pest rodent species. As a consequence, wild birds of prey are exposed to these toxicants by eating poisoned prey items. ARs prevent the hepatic recycling of vitamin K and thereby impede the post-translational processing of coagulation factors II, VII, IX, and X that are required for procoagulant complex assembly. Through this mechanism of action, ARs cause hemorrhage and death in their target species. Various studies have documented the persistence of these contaminants in birds of prey but few have attempted to use affordable and accessible diagnostic tests to diagnose coagulopathy in free-ranging birds of prey. In our study free-ranging red-tailed hawks were found to be exposed to difethialone and brodifacoum. Eleven of sixteen (68%) livers tested for AR exposure had detectable residues. Difethialone was found in 1/16 (6%), and brodifacoum was detected in 10/16 (62%) liver samples that were tested for rodenticide residues. Difethialone was found at a concentration of 0.18 ug/g wet weight and brodifacoum concentrations ranged from 0.003-0.234 ug/g wet weight. Two out of 34 (6%) RTHA assessed for blood rodenticide had brodifacoum in serum with measured concentrations of 0.003 and 0.006 ug/g. The range of clotting times in the prothrombin time (PT) and Russell's viper venom time assays for control RTHA were 16.7 to 39.7 s and 11.5 to 91.8 s, respectively. One study bird was diagnosed with clinical AR intoxication with a brodifacoum levels in blood of 0.006 and 0.234 ug/g wet weight in blood and liver respectively, a packed cell volume (PCV) of 19%, and PT and RVVT times of >180 s. No correlation was found between PT and RVVT in the control or free-range RTHA, and there was no relationship found between the presence of liver anticoagulant residues and clotting times in the PT and RVVT.

Oriented basement membrane fibrils provide a memory for F-actin planar polarization via the Dystrophin-Dystroglycan complex during tissue elongation.

How extracellular matrix contributes to tissue morphogenesis is still an open question. In the Drosophila ovarian follicle, it has been proposed that after Fat2-dependent planar polarization of the follicle cell basal domain, oriented basement membrane (BM) fibrils and F-actin stress fibers constrain follicle growth, promoting its axial elongation. However, the relationship between BM fibrils and stress fibers and their respective impact on elongation are unclear. We found that Dystroglycan (Dg) and Dystrophin (Dys) are involved in BM fibril deposition. Moreover, they also orient stress fibers, by acting locally and in parallel to Fat2. Importantly, Dg-Dys complex-mediated cell-autonomous control of F-actin fiber orientation relies on the preceding BM fibril deposition, indicating two distinct but interdependent functions. Thus, the Dg-Dys complex works as a crucial organizer of the epithelial basal domain, regulating both F-actin and BM. Furthermore, BM fibrils act as a persistent cue for the orientation of stress fibers that are the main effector of elongation.

flam piRNA precursors channel from the nucleus to the cytoplasm in a temporally regulated manner along Drosophila oogenesis.

BACKGROUND: PIWI-interacting RNAs (piRNAs) are the effectors of transposable element silencing in the reproductive apparatus. In Drosophila ovarian somatic cells, piRNAs arise from long RNA precursors presumably processed within cytoplasmic Yb-bodies. RESULTS: Here we show that the nucleo-cytoplasmic traffic of piRNA precursors encoded by the flamenco locus is subjected to a spatio-temporal regulation. Precursor RNAs first gather in a single nuclear focus, Dot COM, close to the nuclear periphery, and transit through the membrane before being delivered to the cytoplasmic Yb-bodies. Early in oogenesis, flamenco transcripts are rapidly transferred to the cytoplasm making their initial nuclear gathering in Dot COM too transient to be visualized. As oogenesis proceeds, the cytoplasmic delivery steadily decreases concomitantly with the decrease in the protein levels of Armi and Yb, two components of the Yb-bodies. Both events lead to a reduction of Yb-body assembly in late stages of oogenesis, which likely results in a drop in piRNA production. CONCLUSION: Our findings show a spatio-temporal regulation of the piRNA biogenesis in the follicle cells of Drosophila ovaries, that involves coordinated control of both piRNA precursors and components of the piRNA processing machinery. This newly unveiled regulation establishes another level of complexity in the production of piRNAs and suggests a stage-dependent involvement of the piRNA biogenesis in the mechanism of transposable elements silencing along oogenesis.

Export of piRNA precursors by EJC triggers assembly of cytoplasmic Yb-body in Drosophila.

PIWI-interacting RNAs (piRNAs) are effectors of transposable element (TE) silencing in the reproductive apparatus. In Drosophila ovarian somatic cells, piRNAs arise from longer single-stranded RNA precursors that are processed in the cytoplasm presumably within the Yb-bodies. piRNA precursors encoded by the flamenco (flam) piRNA cluster accumulate in a single focus away from their sites of transcription. In this study, we identify the exportin complex containing Nxf1 and Nxt1 as required for flam precursor nuclear export. Together with components of the exon junction complex (EJC), it is necessary for the efficient transfer of flam precursors away from their site of transcription. Indeed, depletion of these components greatly affects flam intra-nuclear transit. Moreover, we show that Yb-body assembly is dependent on the nucleo-cytoplasmic export of flam transcripts. These results suggest that somatic piRNA precursors are thus required for the assembly of the cytoplasmic transposon silencing machinery.

Distinct features of the piRNA pathway in somatic and germ cells: from piRNA cluster transcription to piRNA processing and amplification.

Transposable elements (TEs) are major components of genomes. Their mobilization may affect genomic expression and be a threat to genetic stability. This is why they have to be tightly regulated by a dedicated system. In the reproductive tissues of a large range of organisms, they are repressed by a subclass of small interfering RNAs called piRNAs (PIWI interacting RNAs). In Drosophila melanogaster, piRNAs are produced both in the ovarian germline cells and in their surrounding somatic cells. Accumulating evidence suggests that germinal and somatic piRNA pathways are far more different than previously thought. Here we review the current knowledge on piRNA production in both these cell types, and explore their similarities and differences.

Gata6, Nanog and Erk signaling control cell fate in the inner cell mass through a tristable regulatory network.

During blastocyst formation, inner cell mass (ICM) cells differentiate into either epiblast (Epi) or primitive endoderm (PrE) cells, labeled by Nanog and Gata6, respectively, and organized in a salt-and-pepper pattern. Previous work in the mouse has shown that, in absence of Nanog, all ICM cells adopt a PrE identity. Moreover, the activation or the blockade of the Fgf/RTK pathway biases cell fate specification towards either PrE or Epi, respectively. We show that, in absence of Gata6, all ICM cells adopt an Epi identity. Furthermore, the analysis of Gata6(+/-) embryos reveals a dose-sensitive phenotype, with fewer PrE-specified cells. These results and previous findings have enabled the development of a mathematical model for the dynamics of the regulatory network that controls ICM differentiation into Epi or PrE cells. The model describes the temporal dynamics of Erk signaling and of the concentrations of Nanog, Gata6, secreted Fgf4 and Fgf receptor 2. The model is able to recapitulate most of the cell behaviors observed in different experimental conditions and provides a unifying mechanism for the dynamics of these developmental transitions. The mechanism relies on the co-existence between three stable steady states (tristability), which correspond to ICM, Epi and PrE cells, respectively. Altogether, modeling and experimental results uncover novel features of ICM cell fate specification such as the role of the initial induction of a subset of cells into Epi in the initiation of the salt-and-pepper pattern, or the precocious Epi specification in Gata6(+/-) embryos.

Spatio-temporal requirements for transposable element piRNA-mediated silencing during Drosophila oogenesis.

During Drosophila oogenesis, transposable element (TE) repression involves the Piwi-interacting RNA (piRNA) pathway which ensures genome integrity for the next generation. We developed a transgenic model to study repression of the Idefix retrotransposon in the germline. Using a candidate gene KD-approach, we identified differences in the spatio-temporal requirements of the piRNA pathway components for piRNA-mediated silencing. Some of them (Aub, Vasa, Spn-E) are necessary in very early stages of oogenesis within the germarium and appear to be less important for efficient TE silencing thereafter. Others (Piwi, Ago3, Mael) are required at all stages of oogenesis. Moreover, during early oogenesis, in the dividing cysts within the germarium, Idefix anti-sense transgenes escape host control, and this is associated with very low piwi expression. Silencing of P-element-based transgenes is also strongly weakened in these cysts. This region, termed the 'Piwiless pocket' or Pilp, may ensure that new TE insertions occur and are transmitted to the next generation, thereby contributing to genome dynamics. In contrast, piRNA-mediated silencing is strong in germline stem cells in which TE mobilization is tightly repressed ensuring the continued production of viable germline cysts.

Fluorescent mRNA labeling through cytoplasmic FISH.

RNA in situ hybridization (ISH) has been widely used in cell and developmental biology research to study gene expression. Classical ISH protocols use colorimetric staining approaches, such as the assay with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP), which do not allow the implementation of multiple probe analyses and do not enable investigators to achieve cellular resolution. Here we describe a protocol to determine the presence of target cytoplasmic RNA via cytoplasmic fluorescence ISH (cFISH), an approach that renders possible the visualization of specific RNA strands from the whole tissue down to the cell. This fluorescence technique, adapted here for use in mouse embryos, enables researchers to implement multiple labeling by combining several RNA probes and/or antibodies in immuno-cFISH. Depending on the options chosen, the protocol can be completed within 2 or 3 d.

"Dot COM", a nuclear transit center for the primary piRNA pathway in Drosophila.

The piRNA pathway protects genomes by silencing mobile elements. Despite advances in understanding the processing events that generate piRNAs for silencing, little is known about how primary transcripts are transported from their genomic clusters to their processing centers. Using a model of the Drosophila COM/flamenco locus in ovarian somatic cells, we identified a prominent nuclear structure called Dot COM, which is enriched in long transcripts from piRNA clusters but located far from their transcription sites. Remarkably, transcripts from multiple clusters accumulate at Dot COM, which is often juxtaposed with Yb-bodies, the cytoplasmic processing centers for cluster transcripts. Genetic evidence suggests that the accumulation of precursor transcripts at Dot COM represents one of the most upstream events in the piRNA pathway. Our results provide new insights into the initial steps of the piRNA pathway, and open up a new research area important for a complete understanding of this conserved pathway.

KOPS: DNA motifs that control E. coli chromosome segregation by orienting the FtsK translocase.

Bacterial chromosomes are organized in replichores of opposite sequence polarity. This conserved feature suggests a role in chromosome dynamics. Indeed, sequence polarity controls resolution of chromosome dimers in Escherichia coli. Chromosome dimers form by homologous recombination between sister chromosomes. They are resolved by the combined action of two tyrosine recombinases, XerC and XerD, acting at a specific chromosomal site, dif, and a DNA translocase, FtsK, which is anchored at the division septum and sorts chromosomal DNA to daughter cells. Evidences suggest that DNA motifs oriented from the replication origin towards dif provide FtsK with the necessary information to faithfully distribute chromosomal DNA to either side of the septum, thereby bringing the dif sites together at the end of this process. However, the nature of the DNA motifs acting as FtsK orienting polar sequences (KOPS) was unknown. Using genetics, bioinformatics and biochemistry, we have identified a family of DNA motifs in the E. coli chromosome with KOPS activity.

RuvAB-directed branch migration of individual Holliday junctions is impeded by sequence heterology.

The Holliday junction, the key intermediate of recombination, is generated by strand exchange resulting in a covalent connection between two recombining DNA molecules. Translocation of a Holliday junction along DNA, or branch migration, progressively exchanges one DNA strand for another and determines the amount of information that is transferred between two recombining partners. In Escherichia coli, the RuvAB protein complex promotes rapid and unidirectional branch migration of Holliday junctions. We have studied translocation of Holliday junctions using a quantitative biochemical system together with a 'single-molecule' branch migration assay. We demonstrate that RuvAB translocates the junctions through identical DNA sequences in a processive manner with a broad distribution of individual branch migration rates. However, when the complex encounters short heterologous sequences, translocation of the Holliday junctions is impeded. We conclude that translocation of the junctions through a sequence heterology occurs with a probability of bypass being determined both by the length of the heterologous region and the lifetime of the stalled RuvAB complex.

Single-particle tracking for DNA tether length monitoring.

We describe a simple single-particle tracking approach for monitoring the length of DNA molecules in tethered particle motion experiments. In this method, the trajectory of a submicroscopic bead tethered by a DNA molecule to a glass surface is determined by videomicroscopy coupled to image analysis. The amplitude of motion of the bead is measured by the standard deviation of the distribution of successive positions of the bead in a given time interval. We were able to describe theoretically the variation of the equilibrium value of the amplitude of the bead motion with the DNA tether length for the entire applicable DNA length range (up to approximately 3500 bp). The sensitivity of the approach was illustrated by the evidence obtained for conformational changes introduced into a Holliday junction by the binding of the Escherichia coli RuvA protein. An advantage of this method is that the trajectory of the tethered bead, rather than its averaged motion, is measured, allowing analysis of the conformational dynamics of DNA chains at the single-molecule level.