Splicing is dynamically regulated during limb development.

Modifications to highly conserved developmental gene regulatory networks are thought to underlie morphological diversification in evolution and contribute to human congenital malformations. Relationships between gene expression and morphology have been extensively investigated in the limb, where most of the evidence for alterations to gene regulation in development consists of pre-transcriptional mechanisms that affect expression levels, such as epigenetic alterations to regulatory sequences and changes to cis-regulatory elements. Here we report evidence that alternative splicing (AS), a post-transcriptional process that modifies and diversifies mRNA transcripts, is dynamic during limb development in two mammalian species. We evaluated AS patterns in mouse (Mus musculus) and opossum (Monodelphis domestica) across the three key limb developmental stages: the ridge, bud, and paddle. Our data show that splicing patterns are dynamic over developmental time and suggest differences between the two mammalian taxa. Additionally, multiple key limb development genes, including Fgf8, are differentially spliced across the three stages in both species, with expression levels of the conserved splice variants, Fgf8a and Fgf8b, changing across developmental time. Our data demonstrates that AS is a critical mediator of mRNA diversity in limb development and provides an additional mechanism for evolutionary tweaking of gene dosage.

The mammary hair of Monodelphis domestica and homology of the mammary pilosebacous unit.

The unitary mammary gland is a synapomorphy of therian mammals and is thought to have evolved from the pilosebaceous organ in the mammalian stem lineage from which the lactogenic patch of monotremes is also derived. One of the key lines of evidence for the homology of the nipple and the lactogenic patch is that marsupials have retained a transient hair associated with developing mammary glands. However, these structures have not been documented since the early 20th-century drawings of Ernst Bresslau. In this study, we examine the developing mammary organs of Monodelphis domestica and document the presence of mammary hairs in 12-week-old females, as well as their absence after 18 weeks of age. Histochemical staining for cystine confirms the structures as keratinized hairs. Milk ducts of both juvenile and adult nipples show a division between KRT18+ luminal epithelium and KRT14+ ACTA2+ myoepithelium. These patterns match those in eutherians and suggest a conserved ductal morphology and mechanism of milk expulsion. Finally, PTHLH, a peptide hormone which promotes homeotic transformation of hairy skin into hairless nipples in the mouse, was detected in the Monodelphis milk duct during the mammary hair stage, suggesting that the mutual exclusivity of "hairless nipple" and "hair" organ identity is derived in eutherian mammals. These results reveal shared characteristics of the M. domestica nipple with both the eutherian nipple and the pilosebaceous organ, consistent with the evolutionary derivation of the mammary gland from an ancestral hair organ via developmental individualization of pilosebaceous and mammary identities.

ABC Efflux Transporters and Solute Carriers in the Early Developing Brain of a Marsupial Monodelphis domestica (South American Gray Short-Tailed Opossum).

This study used a marsupial Monodelphis domestica, which is born very immature and most of its development is postnatal without placental protection. RNA-sequencing (RNA-Seq) was used to identify the expression of influx and efflux transporters (ATP-binding cassettes [ABCs] and solute carriers [SLCs]) and metabolizing enzymes in brains of newborn to juvenile Monodelphis. Results were compared to published data in the developing eutherian rat. To test the functionality of these transporters at similar ages, the entry of paracetamol (acetaminophen) into the brain and cerebrospinal fluid (CSF) was measured using liquid scintillation counting following a single administration of the drug along with its radiolabelled tracer [3H]. Drug permeability studies found that in Monodelphis, brain entry of paracetamol was already restricted at P5; it decreased further in the first week of life and then remained stable until the oldest age group tested (P110). Transcriptomic analysis of Monodelphis brain showed that expression of transporters and their metabolizing enzymes in early postnatal (P) pups (P0, P5, and P8) was relatively similar, but by P109, many more transcripts were identified. When transcriptomes of newborn Monodelphis brain and E19 rat brain and placenta were compared, several transporters present in the rat placenta were also found in the newborn Monodelphis brain. These were absent from E19 rat brain but were present in the adult rat brain. These data indicate that despite its extreme immaturity, the newborn Monodelphis brain may compensate for the lack of placental protection during early brain development by upregulating protective mechanisms, which in eutherian animals are instead present in the placenta.

Identification of the RSX interactome in a marsupial shows functional coherence with the Xist interactome during X inactivation.

The marsupial specific RSX lncRNA is the functional analogue of the eutherian specific XIST, which coordinates X chromosome inactivation. We characterized the RSX interactome in a marsupial representative (the opossum Monodelphis domestica), identifying 135 proteins, of which 54 had orthologues in the XIST interactome. Both interactomes were enriched for biological pathways related to RNA processing, regulation of translation, and epigenetic transcriptional silencing. This represents a remarkable example showcasing the functional coherence of independently evolved lncRNAs in distantly related mammalian lineages.

Translating the Timing of Developmental Benchmarks in Short-Tailed Opossums (Monodelphisdomestica) to Facilitate Comparisons with Commonly Used Rodent Models.

INTRODUCTION: The gray short-tailed opossum, Monodelhis domestica (M. domestica), is a widely used marsupial model species that presents unique advantages for neurodevelopmental studies. Notably their extremely altricial birth allows manipulation of postnatal pups at timepoints equivalent to embryonic stages of placental mammals. A robust literature exists on the development of short-tailed opossums, but many researchers working in the more conventional model species of mice and rats may find it daunting to identify the appropriate age at which to conduct experiments. METHODS: Here, we present detailed staging diagrams taken from photographic observations of 40 individual pups, in 6 litters, over 25 timepoints across postnatal development. We also present a comparative neurodevelopmental timeline of short-tailed opossums (M. domestica), the house mouse (Mus musculus), and the laboratory rat (Rattus norvegicus) during embryonic as well as postnatal development, using timepoints taken from this study and a review of existing literature, and use this dataset to present statistical models comparing the opossum to the rat and mouse. RESULTS: One aim of this research was to aid in testing the generalizability of results found in rodents to other mammalian brains, such as the more distantly related metatherians. However, this broad dataset also allows the identification of potential heterochronies in opossum development compared to rats and mice. In contrast to previous work, we found broad similarity between the pace of opossum neural development with that of rats and mice. We also found that development of some systems was accelerated in the opossum, such as the forelimb motor plant, oral motor control, and some aspects of the olfactory system, while the development of the cortex, some aspects of the retina, and other aspects of the olfactory system are delayed compared to the rat and mouse. DISCUSSION: The pace of opossum development is broadly similar to that of mice and rats, which underscores the usefulness of this species as a compliment to the more commonly used rodents. Many features that differ the most between opossums and rats and mice were either clustered around the day of birth and were features that have functional importance for the pup immediately after or during birth, or were features that have reduced functional importance for the pup until later in postnatal development, given that it is initially attached to the mother.

Development of the terminal air spaces in the gray short-tailed opossum (Monodelphis domestica)- 3D reconstruction by microcomputed tomography.

Marsupials are born with structurally immature lungs when compared to eutherian mammals. The gray short-tailed opossum (Monodelphis domestica) is born at the late canalicular stage of lung development. Despite the high degree of immaturity, the lung is functioning as respiratory organ, however supported by the skin for gas exchange during the first postnatal days. Consequently, the majority of lung development takes place in ventilated functioning state during the postnatal period. Microcomputed tomography (µCT) was used to three-dimensionally reconstruct the terminal air spaces in order to reveal the timeline of lung morphogenesis. In addition, lung and air space volume as well as surface area were determined to assess the functional relevance of the structural changes in the developing lung. The development of the terminal air spaces was examined in 35 animals from embryonic day 13, during the postnatal period (neonate to 57 days) and in adults. At birth, the lung of Monodelphis domestica consists of few large terminal air spaces, which are poorly subdivided and open directly from short lobar bronchioles. During the first postnatal week the number of smaller terminal air spaces increases and numerous septal ridges indicate a process of subdivision, attaining the saccular stage by 7 postnatal days. The 3D reconstructions of the terminal air spaces demonstrated massive increases in air sac number and architectural complexity during the postnatal period. Between 28 and 35 postnatal days alveolarization started. Respiratory bronchioles, alveolar ducts and a typical acinus developed. The volume of the air spaces and the surface area for gas exchange increased markedly with alveolarization. The structural transformation from large terminal sacs to the final alveolar lung in the gray short-tailed opossum follows similar patterns as described in other marsupial and placental mammals. The processes involved in sacculation and alveolarization during lung development seem to be highly conservative within mammalian evolution.

Astrocytes of the Anterior Commissure Regulate the Axon Guidance Pathways of Newly Generated Neocortical Neurons in the Opossum Monodelphis domestica.

In marsupials, upper-layer cortical neurons derived from the progenitors of the subventricular zone of the lateral ventricle (SVZ) mature morphologically and send their axons to form interhemispheric connections through the anterior commissure. In contrast, eutherians have evolved a new extra callosal pathway, the corpus callosum, that interconnects both hemispheres. In this study, we aimed to examine neurogenesis during the formation of cortical upper layers, including their morphological maturation in a marsupial species, namely the opossum (Monodelphis domestica). Furthermore, we studied how the axons of upper layers neurons pass through the anterior commissure of the opossum, which connects neocortical areas. We showed that upper-layer II/III neurons were generated within at least seven days in the opossum neocortex. Surprisingly, these neurons expressed special AT-rich sequence binding protein 2 (Satb2) and neuropilin 1 interacting protein (Nrp1), which are proteins known to be essential for the formation of the corpus callosum in eutherians. This indicates that extrinsic, but not intrinsic, cues could be key players in guiding the axons of newly generated cortical neurons in the opossum. Although oligodendrocyte precursor cells were present in the neocortex and anterior commissure, newly generated upper-layer neurons sent unmyelinated axons to the anterior commissure. We also found numerous GFAP-expressing progenitor cells in both brain structures, the neocortex and the anterior commissure. However, at P12-P17 in the opossums, a small population of astrocytes was observed only in the midline area of the anterior commissure. We postulate that in the opossum, midline astrocytes allow neocortical axons to be guided to cross the midline, as this structure resembles the glial wedge required by fibers to cross the midline area of the corpus callosum in the rodent.

SOX2 and SOX9 Expression in Developing Postnatal Opossum (Monodelphis domestica) Cortex.

(1) Background: Central nervous system (CNS) development is characterized by dynamic changes in cell proliferation and differentiation. Key regulators of these transitions are the transcription factors such as SOX2 and SOX9. SOX2 is involved in the maintenance of progenitor cell state and neural stem cell multipotency, while SOX9, expressed in neurogenic niches, plays an important role in neuron/glia switch with predominant expression in astrocytes in the adult brain. (2) Methods: To validate SOX2 and SOX9 expression patterns in developing opossum (Monodelphis domestica) cortex, we used immunohistochemistry (IHC) and the isotropic fractionator method on fixed cortical tissue from comparable postnatal ages, as well as dissociated primary neuronal cultures. (3) Results: Neurons positive for both neuronal (TUJ1 or NeuN) and stem cell (SOX2) markers were identified, and their presence was confirmed with all methods and postnatal age groups (P4-6, P6-18, and P30) analyzed. SOX9 showed exclusive staining in non-neuronal cells, and it was coexpressed with SOX2. (4) Conclusions: The persistence of SOX2 expression in developing cortical neurons of M. domestica during the first postnatal month implies the functional role of SOX2 during neuronal differentiation and maturation, which was not previously reported in opossums.

Endoparasites of marsupials in fragments of the Atlantic rainforest, western Paraná State, Brazil.

Knowledge of taxonomy and biodiversity of parasites is fundamental to better understand ecosystem dynamics. The objective of this study was to describe the helminth fauna of two species of marsupials in five fragments of the Atlantic rainforest in the western region of Paraná State, Brazil. In a total of 4050 trap-nights, the animals were captured using Sherman, Tomahawk, and Pitfall traps, euthanized, necropsied, and their organs inspected for helminths. After identification of the parasites, descriptors of infection, such as prevalence, mean abundance, mean intensity, and range of intensity, were calculated. Collectively, six helminth species were observed in 18 animals. The following five species were observed in Marmosa paraguayana: Viannaia hamata (58.8%), Gracilioxyuris agilisis (52.9%), Travassostrongylus sextus (17.6%), Oncicola luehei (5.9%), and Pritchardia boliviensis (5.9%). Whereas the following two species were observed in Monodelphis dimidiata: Trichohelix tuberculata (100%) and Travassostrongylus sextus (100%). This study represents a new locality record for all helminths described herein, and a new host for four helminth species. This is the first report on the helminth fauna of Monodelphis dimidiata, expanding knowledge about marsupials in the Brazilian Atlantic Forest.

3D reconstruction of the bronchial tree of the Gray short-tailed opossum (Monodelphis domestica) in the postnatal period.

Recent didelphid marsupials resemble the assumed mammalian ancestor and are suitable to inform on the evolution of the mammalian lung. This study uses X-ray computed tomography (µCT) to three-dimensionally reconstruct the bronchial tree of the marsupial Gray short-tailed opossum (Monodelphis domestica) in order to reveal the timeline of morphogenesis during the postnatal period. The development of the bronchial tree was examined in 37 animals from embryonic day 13, during the postnatal period (neonate to 57 days) and in adults. The first appearance and the branching of lobar, segmental and sub-segmental bronchioles in the lungs were documented. Based on the reconstructions, the generation of end-branching airways, the median and maximum generation and the number of branches were calculated for each pulmonary lobe. At birth, the lung of M. domestica has a primitive appearance since it consists of a simple system of branching airways that end in a number of terminal air spaces, lobar bronchioles, and first segmental bronchioles are present. During the postnatal period, the volumes of the lung and bronchial tree steadily increase and development, differentiation, and expansion of the bronchial tree takes place. By 14 days, the fundamental bronchial tree consisting of lobar, segmental, and sub-segmental bronchioles has been established. A mature bronchial tree, including respiratory bronchioles and alveolar ducts is present by day 35. The asymmetry of the right (predominately four lobes) and the left lung (predominately two lobes), as present in M. domestica, can be considered as plesiomorphic for Mammalia. In marsupials, the process of branching morphogenesis, which takes place intrauterine in the placental fetus, is shifted to the postnatal period, but follows similar patterns as described in placentals. Lung maturation in general and the branching morphogenesis in particular seems to be highly conservative within mammalian evolution.

Transient expression of NF200 by fibers in the nasal septum and rostral telencephalon of developing opossums (Monodelphis domestica).

Marsupials are born very immature and crawl on their mother's belly to attach to teats. Sensory information is required to guide the newborn and to induce attachment to the teat. Olfaction has been classically proposed to influence neonatal behaviors, but recent studies suggest that the central olfactory structures are too immature to account for them. In the newborn opossum, we previously described a fascicle of nerve fibers expressing neurofilament-200 (NF200, a marker of fiber maturity) from the olfactory bulbs to the rostral telencephalon. The course of these fibers is compatible with that of the terminal nerve that, during development, is characterized by the presence of neurons synthetizing gonadotropin hormones (GnRH). To evaluate if these fibers are related to the terminal nerve and if they play a role in precocious behaviors in opossums, we used immunohistochemistry against NF200 and GnRH. The results show that NF200-labeled fibers are present between P0 and P11, but do not reach much further caudally than the septal region. Only a few NF200-labeled fibers were found near the olfactory and vomeronasal epitheliums and they did not penetrate the olfactory bulbs. NF200-labeled fibers follow the same path as fibers labeled for GnRH. In contrast to the latter, NF200-labeled fibers are no longer visible at P15. These results suggest that these fibers are neither from the olfactory nor from the vomeronasal nerves but may be part of the terminal nerve. Their limited caudal extension does not support a role in the sensorimotor behaviors of the newborn opossum.

Monodelphis domestica Induced Pluripotent Stem Cells Reveal Metatherian Pluripotency Architecture.

Marsupials have been a powerful comparative model to understand mammalian biology. However, because of the unique characteristics of their embryology, marsupial pluripotency architecture remains to be fully understood, and nobody has succeeded in developing embryonic stem cells (ESCs) from any marsupial species. We have developed an integration-free iPSC reprogramming method and established validated iPSCs from two inbred strains of a marsupial, Monodelphis domestica. The monoiPSCs showed a significant (6181 DE-genes) and highly uniform (r2 [95% CI] = 0.973 ± 0.007) resetting of the cellular transcriptome and were similar to eutherian ESCs and iPSCs in their overall transcriptomic profiles. However, monoiPSCs showed unique regulatory architecture of the core pluripotency transcription factors and were more like marsupial epiblasts. Our results suggest that POU5F1 and the splice-variant-specific expression of POU5F3 synergistically regulate the opossum pluripotency gene network. It is plausible that POU5F1, POU5F3 splice variant XM_016427856.1, and SOX2 form a self-regulatory network. NANOG expression, however, was specific to monoiPSCs and epiblasts. Furthermore, POU5F1 was highly expressed in trophectoderm cells, whereas all other pluripotency transcription factors were significantly downregulated, suggesting that the regulatory architecture of core pluripotency genes of marsupials may be distinct from that of eutherians.

Experimental Rickettsia typhi Infection in Monodelphis domestica: Implications for Opossums as an Amplifying Host in the Suburban Cycle of Murine Typhus.

Murine typhus is an acute undifferentiated febrile illness caused by Rickettsia typhi. In the United States, its reemergence appears to be driven by a shift from the classic rat-rat flea cycle of transmission to one involving opossums (Didelphis virginiana) and cat fleas. Little is known of the ability of opossums to act as a reservoir and amplifying host for R. typhi. Here, we use Monodelphis domestica (the laboratory opossum) as a surrogate for D. virginiana. Opossums were inoculated via the intraperitoneal (IP) or intradermal (ID) route with 1 × 106 viable R. typhi. Blood and tissues were collected on days 6, 13, 20, and 27 or if moribund. Although one ID-infected opossum died, the remainder did not appear ill, whereas half of the IP-inoculated animals succumbed to infection. Rickettsemia was demonstrated in all animals through week 2 of infection and sporadically in weeks 3 and 4. Rickettsia typhi DNA was detected in all tissues, with most animals demonstrating the presence of bacteria into weeks 3 and 4. Histopathology and immunohistochemistry demonstrated typical findings of rickettsial infection. Akin to infection in rats, the demonstration of disseminated infection, typical inflammation, and prolonged rickettsemia with relatively few clinical effects (especially in the more natural route of ID inoculation) supports the potential of opossums to act as a competent mammalian reservoir and component of the zoonotic maintenance cycle of R. typhi. Understanding the dynamics of infection within opossums may have implications for the prevention and control of murine typhus.

Prolonged Myocardial Regenerative Capacity in Neonatal Opossum.

BACKGROUND: Early neonates of both large and small mammals are able to regenerate the myocardium through cardiomyocyte proliferation for only a short period after birth. This myocardial regenerative capacity declines in parallel with withdrawal of cardiomyocytes from the cell cycle in the first few postnatal days. No mammalian species examined to date has been found capable of a meaningful regenerative response to myocardial injury later than 1 week after birth. METHODS: We examined cardiomyocyte proliferation in neonates of the marsupial opossum (Monodelphis domestica) by immunostaining at various times after birth. The regenerative capacity of the postnatal opossum myocardium was assessed after either apex resection or induction of myocardial infarction at postnatal day 14 or 29, whereas that of the postnatal mouse myocardium was assessed after myocardial infarction at postnatal day 7. Bioinformatics data analysis, immunofluorescence staining, and pharmacological and genetic intervention were applied to determine the role of AMPK (5'-AMP-activated protein kinase) signaling in regulation of the mammalian cardiomyocyte cell cycle. RESULTS: Opossum neonates were found to manifest cardiomyocyte proliferation for at least 2 weeks after birth at a frequency similar to that apparent in early neonatal mice. Moreover, the opossum heart at postnatal day 14 showed substantial regenerative capacity both after apex resection and after myocardial infarction injury, whereas this capacity had diminished by postnatal day 29. Transcriptomic and immunofluorescence analyses indicated that AMPK signaling is activated in postnatal cardiomyocytes of both opossum and mouse. Pharmacological or genetic inhibition of AMPK signaling was sufficient to extend the postnatal window of cardiomyocyte proliferation in both mouse and opossum neonates as well as of cardiac regeneration in neonatal mice. CONCLUSIONS: The marsupial opossum maintains cardiomyocyte proliferation and a capacity for myocardial regeneration for at least 2 weeks after birth. As far as we are aware, this is the longest postnatal duration of such a capacity among mammals examined to date. AMPK signaling was implicated as an evolutionarily conserved regulator of mammalian postnatal cardiomyocyte proliferation.

Genetic and genomic architecture in eight strains of the laboratory opossum Monodelphis domestica.

The gray short-tailed opossum (Monodelphis domestica) is an established laboratory-bred marsupial model for biomedical research. It is a critical species for comparative genomics research, providing the pivotal phylogenetic outgroup for studies of derived vs ancestral states of genomic/epigenomic characteristics for eutherian mammal lineages. To characterize the current genetic profile of this laboratory marsupial, we examined 79 individuals from eight established laboratory strains. Double digest restriction site-associated DNA sequencing and whole-genome resequencing experiments were performed to investigate the genetic architecture in these strains. A total of 66,640 high-quality single nucleotide polymorphisms (SNPs) were identified. We analyzed SNP density, average heterozygosity, nucleotide diversity, and population differentiation parameter Fst within and between the eight strains. Principal component and population structure analysis clearly resolve the strains at the level of their ancestral founder populations, and the genetic architecture of these strains correctly reflects their breeding history. We confirmed the successful establishment of the first inbred laboratory opossum strain LSD (inbreeding coefficient F > 0.99) and a nearly inbred strain FD2M1 (0.98 < F < 0.99), each derived from a different ancestral background. These strains are suitable for various experimental protocols requiring controlled genetic backgrounds and for intercrosses and backcrosses that can generate offspring with informative SNPs for studying a variety of genetic and epigenetic processes. Together with recent advances in reproductive manipulation and CRISPR/Cas9 techniques for Monodelphis domestica, the existence of distinctive inbred strains will enable genome editing on different genetic backgrounds, greatly expanding the utility of this marsupial model for biomedical research.

Cubilin-, megalin-, and Dab2-dependent transcription revealed by CRISPR/Cas9 knockout in kidney proximal tubule cells.

The multiligand receptors megalin (Lrp2) and cubilin (Cubn) and their endocytic adaptor protein Dab2 (Dab2) play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule (PT) cells and have complex and poorly understood roles in the development of chronic kidney disease. Here, we used RNA-sequencing and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin, cubilin, or Dab2 expression. KO of Lrp2 had the greatest transcriptional effect, and nearly all genes whose expression was affected in Cubn KO and Dab2 KO cells were also changed in Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggested changes in pathways with immunomodulatory functions that might trigger the pathological changes observed in KO mice and patients with Donnai-Barrow syndrome. In addition, differences in transcription patterns between Lrp2 and Dab2 KO cells suggested the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon the disruption of PT endocytic function. A reduction in transcripts encoding sodium-glucose cotransporter isoform 2 was confirmed in Lrp2 KO mouse kidney lysates by quantitative PCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with the studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes.NEW & NOTEWORTHY Megalin and cubilin receptors together with their adaptor protein Dab2 represent major components of the endocytic machinery responsible for efficient uptake of filtered proteins by the proximal tubule (PT). Dab2 and megalin expression have been implicated as both positive and negative modulators of kidney disease. We used RNA sequencing to knock out CRISPR/Cas9 cubilin, megalin, and Dab2 in highly differentiated PT cells to identify PT-specific changes that are directly consequent to knockout of each component.

Proteomic analysis of opossum Monodelphis domestica spinal cord reveals the changes of proteins related to neurodegenerative diseases during developmental period when neuroregeneration stops being possible.

One of the major challenges of modern neurobiology concerns the inability of the adult mammalian central nervous system (CNS) to regenerate and repair itself after injury. It is still unclear why the ability to regenerate CNS is lost during evolution and development and why it becomes very limited in adult mammals. A convenient model to study cellular and molecular basis of this loss is neonatal opossum (Monodelphis domestica). Opossums are marsupials that are born very immature with the unique possibility to successfully regenerate postnatal spinal cord after injury in the first two weeks of their life, after which this ability abbruptly stops. Using comparative proteomic approach we identified the proteins that are differentially distributed in opossum spinal tissue that can and cannot regenerate after injury, among which stand out the proteins related to neurodegenerative diseases (NDD), such as Huntington, Parkinson and Alzheimer's disease, previously detected by comparative transcriptomics on the analog tissue. The different distribution of the selected proteins detected by comparative proteomics was further confirmed by Western blot (WB), and the changes in the expression of related genes were analysed by quantitative reverse transcription PCR (qRT-PCR). Furthermore, we explored the cellular localization of the selected proteins using immunofluorescent microscopy. To our knowledge, this is the first report on proteins differentially present in developing, non-injured mammalian spinal cord tissue with different regenerative capacities. The results of this study indicate that the proteins known to have an important role in the pathophysiology of neurodegeneration in aged CNS, could also have an important phyisological role during CNS postnatal development and in neuroregeneration process.

Targeted gene disruption in a marsupial, Monodelphis domestica, by CRISPR/Cas9 genome editing.

Marsupials represent one of three extant mammalian subclasses with very unique characteristics not shared by other mammals. Most notably, much of the development of neonates immaturely born after a relatively short gestation takes place in the external environment. Among marsupials, the gray short-tailed opossum (Monodelphis domestica; hereafter "the opossum") is one of very few established laboratory models. Due to many biologically unique characteristics and experimentally advantageous features, the opossum is used as a prototype species for basic research on marsupial biology.1,2 However, in vivo studies of gene function in the opossum, and thus marsupials in general, lag far behind those of eutherian mammals due to the lack of reliable means to manipulate their genomes. In this study, we describe the successful generation of genome edited opossums by a combination of refined methodologies in reproductive biology and embryo manipulation. We took advantage of the opossum's resemblance to popular rodent models, such as the mouse and rat, in body size and breeding characteristics. First, we established a tractable pipeline of reproductive technologies, from induction of ovulation, timed copulation, and zygote collection to embryo transfer to pseudopregnant females, that warrant an essential platform to manipulate opossum zygotes. Further, we successfully demonstrated the generation of gene knockout opossums at the Tyr locus by microinjection of pronuclear stage zygotes using CRISPR/Cas9 genome editing, along with germline transmission of the edited alleles to the F1 generation. This study provides a critical foundation for venues to expand mammalian reverse genetics into the metatherian subclass.

Two New Species of Chiggers (Trombidiformes: Trombiculidae) From Brazil.

Chigger mites are parasites of terrestrial vertebrates, including humans. Here, we describe two new species belonging to the American genera Colicus Brennan and Parasecia Loomis. Both species were described on the base of museum specimens originated from Minas Gerais State, Brazil, Colicus barrosbattestiae n. sp. parasitizing the rodent, Oligoryzomys fornesi and Parasecia jacinaviciusi n. sp. parasitizing the marsupial, Monodelphis domestica.

Kinematic analysis of sensorimotor behavior during variable ladder rung walking in short-tailed opossums (Monodelphis domestica).

This protocol presents a workflow for detecting differences in kinematics between experimental conditions. It is tailored for short-tailed opossums but can be applied to any species capable of completing the ladder rung task. There are four phases of this protocol: (1) data collection, (2) pose tracking, (3) analysis of single trials, and (4) cross-condition comparisons. This pipeline implements aspects of machine learning and signal processing, allowing for rapid data analysis that provides insight into how animals perform this task. For complete details on the use and execution of this protocol, please refer to Englund et al. (2020).