RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether.

Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.

Identification of excitatory-inhibitory links and network topology in large-scale neuronal assemblies from multi-electrode recordings.

Functional-effective connectivity and network topology are nowadays key issues for studying brain physiological functions and pathologies. Inferring neuronal connectivity from electrophysiological recordings presents open challenges and unsolved problems. In this work, we present a cross-correlation based method for reliably estimating not only excitatory but also inhibitory links, by analyzing multi-unit spike activity from large-scale neuronal networks. The method is validated by means of realistic simulations of large-scale neuronal populations. New results related to functional connectivity estimation and network topology identification obtained by experimental electrophysiological recordings from high-density and large-scale (i.e., 4096 electrodes) microtransducer arrays coupled to in vitro neural populations are presented. Specifically, we show that: (i) functional inhibitory connections are accurately identified in in vitro cortical networks, providing that a reasonable firing rate and recording length are achieved; (ii) small-world topology, with scale-free and rich-club features are reliably obtained, on condition that a minimum number of active recording sites are available. The method and procedure can be directly extended and applied to in vivo multi-units brain activity recordings.

Expression of components of the serotonergic system in the developing rat thymus.

The developing thymus of rat fetuses contains all components of the serotonergic system: receptors, enzymes of synthesis, and membrane transporters. The expression of receptors suggests the possibility of a direct influence of serotonin on thymic development. The presence of tryptophan hydroxylase (the key rate-limiting enzyme of serotonin synthesis) and aromatic l-amino acid decarboxylase indicates the ability of fetal thymic cells to synthesize serotonin. It was shown that the cells of a developing thymus can actively uptake extracellular monoamines. The results of this study suggest different functions of the intrathymic and circulating serotonin pools in the regulation of thymic development.

Whole-rat conditional gene knockout via genome editing.

Animal models with genetic modifications under temporal and/or spatial control are invaluable to functional genomics and medical research. Here we report the generation of tissue-specific knockout rats via microinjection of zinc-finger nucleases (ZFNs) into fertilized eggs. We generated rats with loxP-flanked (floxed) alleles and a tyrosine hydroxylase promoter-driven cre allele and demonstrated Cre-dependent gene disruption in vivo. Pronuclear microinjection of ZFNs, shown by our data to be an efficient and rapid method for creating conditional knockout rats, should also be applicable in other species.

PRRX1- and PRRX2-positive mesenchymal stem/progenitor cells are involved in vasculogenesis during rat embryonic pituitary development.

We have recently shown that cells positive for the paired-related homeobox transcription factors PRRX1 and PRRX2 occur in the rat pituitary, and that they are derived from two different origins: pituitary-derived cells positive for stem cell marker SOX2 and extra-pituitary-derived cells negative for SOX2. In this study, we have further characterized the PRRX1- and PRRX2-positive cells that originate from extra-pituitary cells. Immunohistochemical analyses were performed with specific antibodies against PRRX1 and PRRX2 in order to clarify their roles in pituitary vasculogenesis. PRRX1- and PRRX2-positive cells were found in Atwell's recess and at the periphery of the pituitary on embryonic day 15.5 (E15.5). Several PRRX1-positive cells then invaded the anterior lobe, together with a few PRRX2-positive cells, on E16.5. Some PRRX1-positive cells were also positive for mesenchymal stem cell marker NESTIN. Moreover, some PRRX1/NESTIN double-positive cells showed characteristics of vascular endothelial cells with an Isolectin-B4-binding capacity. PRRX1 co-localized with vascular smooth muscle cell/pericyte marker α-smooth muscle actin in the deep area of Atwell's recess. We confirmed the presence of PRRX2/NESTIN double-positive cells at an entry area in Atwell's recess and at the periphery of the pituitary, but PRRX2 did not co-localize with Isolectin B4 or α-smooth muscle actin. These data suggest that PRRX1- and PRRX2-positive mesenchymal stem/progenitor cells are present at the periphery of the embryonic pituitary and at the entry from Atwell's recess and participate in pituitary vasculogenesis by differentiation into vascular endothelial cells and pericytes, whereas the presence of PRRX2 indicates much higher stemness than PRRX1.

Quantitative analysis of somatosensory cortex development in metatherians and monotremes, with comparison to the laboratory rat.

Metatherians and monotremes are born in an immature state, followed by prolonged nurturing by maternal lactation. Quantitative analysis of isocortical sections held in the collections at the Museum für Naturkunde, Berlin was used to compare the pace of somatosensory cortex development relative to body size and pallial thickness between metatherian groups, monotremes, and the laboratory rat. Analysis indicated that the pace of pallial growth in the monotremes is much lower than that in the metatherians or laboratory rat, with an estimated 8.6-fold increase in parietal cortex thickness between 10 and 100 mm body length, compared to a 10- to 20-fold increase among the metatherians and the rat. It was found that aggregation of cortical plate neurons occurs at similar embryo size in the mammals studied (around 8-14 mm body length) and a similar pallial thickness (around 200 µm), but that proliferative zone involution occurs at a much higher body size and pallial thickness in the monotremes compared to the metatherians and the laboratory rat. The observations suggest that cortical development in the monotremes is slower and subject to different regulatory signals to the therians studied. The slow pace may be related to either generally slower metabolism in monotremes or less efficient nutrient supply to the offspring due to the lack of teats.

Efficient collection and cryopreservation of embryos in F344 strain inbred rats.

In rats, it is now possible to produce genetically engineered strains, not only as transgenic animals but also using gene knockout techniques. Reproductive technologies have been used as indispensable tools to produce and maintain these novel valuable strains. Although studies for collecting and cryopreserving embryos have been reported using outbred rats, efficient methods have not been established in inbred strains. The F344 inbred strain is important in rat breeding and has been used for the production of transgenic/knockout strains and for genome sequencing. Here we studied the optimal conditions for oocyte collection by induction of superovulation, and the development of embryos after cryopreservation in F344 rats. The response to pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was examined by injection of 150 IU/kg PMSG + 75 IU/kg hCG or 300 IU/kg PMSG + 300 IU/kg hCG. Superovulation was achieved at high efficiency by an injection of 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, superovulation in this strain showed similar high response as Wistar rats. Of 2-cell embryos cryopreserved by vitrification in a solution containing 10% propylene glycol, 30% ethylene glycol, 20% Percoll and 0.3 M sucrose, more than 90% survived after warming and 32% developed to offspring. However, the freezability of pronuclear stage embryos was extremely low. This study demonstrated that sufficient unfertilized oocytes and embryos can be collected from F344 rats by the induction of superovulation with 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, cryopreservation of 2-cell embryos using this vitrification protocol can now be applied to maintaining valuable rat strains derived from the F344 inbred strain as genetic resources.

Chorioallantoic and yolk sac placentation in Thrichomys laurentinus (Echimyidae) and the evolution of hystricognath rodents.

The evolutionary history of Hystricognathi is associated with major transformations in their placental system. Data so far indicate that key characters are independent from size dimensions in medium to very large species. To better understand the situation in smaller species, we analyzed placental development in a spiny rat, Thrichomys laurentinus. Fourteen individuals ranging from early implantation to near term were investigated by histology, immunohistochemistry, proliferation activity and electron microscopy. Placentation in Thrichomys revealed major parallels to the guinea pig and other hystricognath rodents with respect to the early and invasive implantation, the process of trophoblast invasion, the internal organization of the labyrinth and the trophospongium as well as the establishment of the complete inverted yolk sac placenta. In contrast to systematically related small-sized species, the placental regionalization in Thrichomys was characterized by a remarkable lobulated structure and associated growing processes. Reverse to former perspectives, these conditions represented ancient character states of hystricognaths. The subplacenta was temporarily supplied by both the maternal and fetal blood systems, a rare condition among hystricognaths. The extraplacental trophoblast originating from the subplacenta was partly proliferative in mid gestation. In conclusion, the presented results indicated that only minor variations occurred in small-sized hystricognath species, independent of their systematic interrelationships. Previous views were supported that placentation in hystricognaths followed an extraordinary stable pattern, although the group had distinct habitats in South America and Africa that were separated 30-40 million years ago.

Developmental control of the Nlrp6 inflammasome and a substrate, IL-18, in mammalian intestine.

The inflammasome is a multiprotein complex whose formation is triggered when a NOD-like receptor binds a pathogen ligand, resulting in activated caspase-1, which converts certain interleukins (IL-1ß, IL-18, and IL-33) to their active forms. There is currently no information on regulation of this system around the time of birth. We employed transcript profiling of fetal rat intestinal and lung RNA at embryonic days 16 (E16) and 20 (E20) with out-of-sample validation using quantitative RT-PCR. Transcript profiling and quantitative RT-PCR demonstrated that transcripts of core components of the NOD-like receptor Nlrp6 inflammasome (Nlrp6, Pycard, Caspase-1) and one of its substrates, IL-18, were increased at E20 compared with E16 in fetal intestine and not lung. Immunohistochemistry demonstrated increased Pycard in intestinal epithelium. Western blotting demonstrated that IL-18 was undetectable at E16, clearly detectable at E20 in its inactive form, and detectable postnatally in both its inactive and active form. Dramatic upregulation of IL-18 was also observed in the fetal sheep jejunum in late gestation (P = 0.006). Transcription factor binding analysis of the rat array data revealed an overrepresentation of nuclear transcription factor binding sites peroxisome proliferator-activated receptor γ (PPAR-γ) and retinoid X receptor-α and chicken ovalbumin upstream promoter transcription factor 1 in the region 1,000 bp upstream of the transcription start site. Rosiglitazone, a PPAR-γ agonist, more than doubled levels of NLRP6 mRNA in human intestinal epithelial (Caco2) cells. These observations provide the first evidence, to our knowledge, linking activity of PPAR-γ to expression of a NOD-like receptor and adds to a growing body of evidence linking pattern recognition receptors of the innate immune system and intestinal colonization.

[A new era in rat genetics]. / Une nouvelle ère pour la génétique du rat.

Rat and mice are privileged tools for scientists. However, despite obvious advantages, such as a larger size, more faithful reproduction of human diseases, and utility for physiological and cognitive studies, rats have suffered from limited genetic technologies such as targeted mutagenesis. However, the gap between rat and mouse for genetic approaches will soon disappear with the recent advances of zinc finger nucleases applicable to early-stage rat embryos and the successful derivation of germ line competent rat ES cells, almost thirty years after murine ES cells. This will lead to new opportunities and to increase our capacity to model human pathologies.

[Immunohistochemical study of Dlx1 and Msx1 expression during cephalic development of Dumbo and Wistar rats. Correlation with morphological data]. / Étude immuno-histochimique de l'expression des protéines Dlx1 et Msx1 durant la céphalogenèse des rats Wistar et Dumbo Corrélation avec les données morphologiques.

The Dumbo rat is characterized by a short snout, low ears and relative hypoplasia of maxillar and zygomatic bones. It corresponds to an autosomal recessive genotype. Previous study demonstrated a global deficit of Dlx1 and Msx1 genes expression in comparison to Wistar embryos as considered as control animals. We performed a histological study of cephalic development of Dumbo rats compared to Wistar embryos and an immunohistochemical analysis of Dlx1 and Msx1 protein expression during cephalogenesis. Our data indicate that the pattern of expression of both genes is similar in both strains, but that quantitative differences in gene expression can be the result of delayed organogenesis in Dumbo rat in comparison to Wistar. Some data about gene expressions are discussed at the light of the postulated function of Dlx1 and Msx1 in cephalic development.

Cholinergic- rather than adrenergic-induced sweating play a role in developing and developed rat eccrine sweat glands.

Both cholinergic and adrenergic stimulation can induce sweat secretion in human eccrine sweat glands, but whether cholinergic and adrenergic stimulation play same roles in rat eccrine sweat glands is still controversial. To explore the innervations, and adrenergic- and cholinergic-induced secretory response in developing and developed rat eccrine sweat glands, rat hind footpads from embryonic day (E) 15.5-20.5, postanal day (P) 1-14, P21 and adult were fixed, embedded, sectioned and subjected to immunofluorescence staining for general fiber marker protein gene product 9.5 (PGP 9.5), adrenergic fiber marker tyrosine hydroxylase (TH) and cholinergic fiber marker vasoactive intestinal peptide (VIP), and cholinergic- and adrenergic-induced sweat secretion was detected at P1-P21 and adult rats by starch-iodine test. The results showed that eccrine sweat gland placodes of SD rats were first appeared at E19.5, and the expression of PGP 9.5 was detected surrounding the sweat gland placodes at E19.5, TH at P7, and VIP at P11. Pilocarpine-induced sweat secretion was first detected at P16 in hind footpads by starch-iodine test. There was no measurable sweating when stimulated by alpha- or beta-adrenergic agonists at all the examined time points. We conclude that rat eccrine sweat glands, just as human eccrine sweat glands, co-express adrenergic and cholinergic fibers, but different from human eccrine sweat glands, cholinergic- rather than adrenergic-induced sweating plays a role in the developing and developed rat eccrine sweat glands.

Fluidity of the surface of cultured muscle fibers. Rapid lateral diffusion of marked surface antigens.

Fluorescent antibody fragments of anti-muscle plasma membrane antibody bound as small fluorescent spots when applied by micropipetting to cultured myotubes. The spots were observed to enlarge with time. The rate of enlargement of fluorescent spots was greater when fragments were applied than when divalent antibody was used. It was also greater at 23 degrees -25 degrees C than at 0 degrees -4 degrees C. With glutaraldehyde-fixed cells no increase in the size of the spots was seen. The observations are consistent with the spread of fluorescent spots due to diffusion of surface protein antigens within the plane of a fluid membrane. From measurements of spot size against time, a diffusion constant of 1-3 x 10(-9) cm(2) s(-1) can be calculated for muscle plasma membrane proteins of mol wt approximately 200,000. This value is consistent with other observations on the diffusion of surface antigens and of labeled lipid molecules in synthetic and natural membranes.

Growth of normal and transformed rat embryo fibroblasts: effects of glycolipids from Salmonella minnesota R mutants.

Addition of glycolipids obtained from Salmonella minnesota R mutants to normal, spontaneously transformed, and SV40-transformed rat embryo fibroblasts in culture results in an inhibition of growth of transformed cells but not of normal cells. In the presence of the glycolipid with the smallest carbohydrate chain length, spontaneously transformed cells stop growing when they reach confluency. Inhibition of growth of transformed cells is inversely related to the chain length of the core sugars. Glycolipid mR595 is shown to bind with the cell membrane of transformed cells and elicits an augmentation in the intracellular level of cyclic AMP. Normal cells bind relatively less glycolipid mR595 and show a lower percent of increase in cyclic AMP due to glycolipid mR595 than do transformed cells.

Autophagic degradation of glycogen in skeletal muscles of the newborn rat.

Large amounts of glycogen accumulate in rat skeletal muscle fibers during the late fetal stages and are mobilized in the first postnatal days. This glycogen depletion is relatively slow in the immature leg muscles, in which extensive deposits are still found 24 hr after birth and, to some extent, persist until the 3rd day. In the more differentiated psoas muscle and especially in the diaphragm, the glycogen stores are completely mobilized already during the early hours. Section of the sciatic nerve 3 days before birth or within the first 2 hr after delivery does not affect glycogen depletion in the leg muscles. Neonatal glycogenolysis in rat muscle fibers takes place largely by segregation and digestion of glycogen particles in autophagic vacuoles. These vacuoles: (a) are not seen in fetal muscle fibers or at later postnatal stages, but appear concomitantly with the process of glycogen depletion and disappear shortly afterwards; (b) are prematurely formed in skeletal muscles of fetuses at term treated with glucagon; (c) contain almost exclusively glycogen particles and no other recognizable cell constituents; (d) have a double or, more often, single limiting membrane and originate apparently from flattened sacs sequestering glycogen masses; (e) are generally found to contain reaction product in preparations incubated from demonstration of acid phosphatase activity. The findings emphasize the role of the lysosomal system in the physiological process of postnatal glycogen mobilization and appear relevant in the interpretation of type II glycogen storage disease.

Apoptosis in metanephric development.

During metanephric development, non-polarized mesenchymal cells are induced to form the epithelial structures of the nephron following interaction with extracellular matrix proteins and factors produced by the inducing tissue, ureteric bud. This induction can occur in a transfilter organ culture system where it can also be produced by heterologous cells such as the embryonic spinal cord. We found that when embryonic mesenchyme was induced in vitro and in vivo, many of the cells surrounding the new epithelium showed morphological evidence of programmed cell death (apoptosis) such as condensed nuclei, fragmented cytoplasm, and cell shrinking. A biochemical correlate of apoptosis is the transcriptional activation of a calcium-sensitive endonuclease. Indeed, DNA isolated from uninduced mesenchyme showed progressive degradation, a process that was prevented by treatment with actinomycin-D or cycloheximide and by buffering intracellular calcium. These results demonstrate that the metanephric mesenchyme is programmed for apoptosis. Incubation of mesenchyme with a heterologous inducer, embryonic spinal cord prevented this DNA degradation. To investigate the mechanism by which inducers prevented apoptosis we tested the effects of protein kinase C modulators on this process. Phorbol esters mimicked the effects of the inducer and staurosporine, an inhibitor of this protein kinase, prevented the effect of the inducer. EGF also prevented DNA degradation but did not lead to differentiation. These results demonstrate that conversion of mesenchyme to epithelial requires at least two steps, rescue of the mesenchyme from apoptosis and induction of differentiation.

Primary rat embryo cells transformed by one or two oncogenes show different metastatic potentials.

Second-passage rat embryo cells were transfected with a neomycin resistance gene and the activated form of the c-Ha-ras I gene, or with these two genes plus the adenovirus type 2 E1a gene. Foci of morphologically transformed cells were observed in both cases; however, the frequency of transformation was at least ten times higher with two oncogenes than with the ras gene alone. All the transformed cell lines gave rise to rapidly growing tumors when injected subcutaneously into nude mice. All but one of the cell lines transformed by the ras oncogene alone formed metastatic nodules in the lungs of animals that had been injected subcutaneously with transformed cells. When transformed cells were injected intravenously, all the ras single-gene transformants gave rise to many metastatic lung nodules. In contrast, cell lines transformed with ras and E1a did not generate metastases after subcutaneous injection and gave rise to very few metastatic lung nodules after intravenous injection. These data demonstrate that a fully malignant cell with metastatic potential, as measured in an immunodeficient animal, can be obtained from early passage embryo cells by the transfection of the ras oncogene alone.

Optimization of Pulmonary Vasculature Tridimensional Phenotyping in The Rat Fetus.

Comparative, functional, developmental, and some morphological studies on animal anatomy require accurate visualization of three-dimensional structures. Nowadays, several widely applicable methods exist for non-destructive whole-mount imaging of animal tissues. The purpose of this study was to optimize specimen preparation and develop a method for quantitative analysis of the total pulmonary vasculature in fetal rats. Tissues were harvested at E21 and fetuses fixed overnight in 4% paraformaldehyde/phosphate buffered saline. They were treated with 25% Lugol solution for 72 hours to ensure perfusion. Four different methods were used for fetal specimen preparation; isolated lung, upper torso, direct right ventricle contrast injection, and whole body with partial thoracic skin excision. The microCT scan was performed, and pulmonary vasculature was segmented. Vessels were analyzed for diameter, length, and branching. Of the four preparation methods, only whole body with partial thoracic skin excision resulted in adequate reconstruction of the pulmonary vasculature. In silico generated 3D images gathered by micro CT showed pulmonary vasculature distributed throughout the lung, which was representative of the shape and structure of the lungs. The mean number of vessels segmented in the pulmonary tree was 900 ± 24 with a mean diameter of 134.13 µm (range 40.72-265.69 µm). While up to the 30th generation of vessels could be segmented, both for arteries and veins, the majority of branching was between the 21st and 30th generations. Passive diffusion of contrast material enables quantitative analysis of the fetal pulmonary vasculature. This technique is a useful tool to analyze the characteristics and quantify the fetal pulmonary vasculature.

Expression of genome defence protein members in proliferating and quiescent rat male germ cells and the Nuage dynamics.

During epigenetic reprogramming germ cells activate alternative mechanisms to maintain the repression retrotransposons. This mechanism involves the recruitment of genome defence proteins such as MAEL, PIWIL4 and TDRD9, which associate with piRNAs and promote Line-1 silencing. MAEL, PIWIL4 and TDRD9 form the piP-bodies, which organization and dynamics vary according to the stage of germ cell epigenetic reprogramming. Although these data have been well documented in mice, it is not known how this mechanism operates in the rat. Thus, the aim of this study was to describe the distribution and interaction of MAEL, PIWIL4, TDRD9 and DAZL during rat germ cell development and check whether specific localization of these proteins is related to the distribution of Line-1 aggregates. Rat embryo gonads at 15 days post-conception (dpc), 16dpc and 19dpc were submitted to MAEL, PIWIL4, TDRD9 and DAZL immunolabelling. The gonads of 19dpc embryos were submitted to the double-labelling of MAEL/DAZL, TDRD9/MAEL and PIWIL4/MAEL. The 19dpc gonads were submitted to co-immunoprecipitation assays and fluorescent in situ hybridization for Line-1 detection. MAEL and TDRD9 showed very similar localization at all ages, whereas DAZL and PIWIL4 showed specific distribution, with PIWIL4 showing shuttling from the nucleus to the cytoplasm by the end epigenetic reprogramming. In quiescent 19dpc gonocytes all proteins colocalized in a nuage adjacent to the nucleus. DAZL interacts with PIWIL4 and MAEL, suggesting that DAZL acts with these proteins to repress Line-1. TDRD9, however, does not interact with DAZL or MAEL despite their colocalization. Line-1 aggregates were detected predominantly in the nuclear periphery, although did not show homogeneous distribution as observed for the nuage. In conclusion, the nuage in quiescent rat gonocytes show a very distinguished organization that might be related to the organization of Line-1 clusters and describe the association of DAZL with proteins responsible for Line-1 repression.

Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization.

Rats are effective model animals and have contributed to the development of human medicine and basic research. However, the application of reproductive engineering techniques to rats is not as advanced compared with mice, and genome editing in rats has not been achieved using embryos obtained by in vitro fertilization (IVF). In this study, we conducted superovulation, IVF, and knock out and knock in using IVF rat embryos. We found that superovulation effectively occurred in the synchronized oestrus cycle and with anti-inhibin antiserum treatment in immature rats, including the Brown Norway rat, which is a very difficult rat strain to superovulate. Next, we collected superovulated oocytes under anaesthesia, and offspring derived from IVF embryos were obtained from all of the rat strains that we examined. When the tyrosinase gene was targeted by electroporation in these embryos, both alleles were disrupted with 100% efficiency. Furthermore, we conducted long DNA fragment knock in using adeno-associated virus and found that the knock-in litter was obtained with high efficiency (33.3-47.4%). Thus, in this study, we developed methods to allow the simple and efficient production of model rats.