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1.
Int J Mol Sci ; 23(20)2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36293487

RESUMO

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.


Assuntos
Células-Tronco Pluripotentes Induzidas , Monodelphis , Animais , Monodelphis/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Embrionárias , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Mamíferos , Reprogramação Celular/genética
2.
Am J Trop Med Hyg ; 107(1): 102-109, 2022 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-35895366

RESUMO

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.


Assuntos
Didelphis , Monodelphis , Infecções por Rickettsia , Rickettsia , Sifonápteros , Tifo Endêmico Transmitido por Pulgas , Animais , Didelphis/microbiologia , Camundongos , Ratos , Rickettsia/genética , Infecções por Rickettsia/microbiologia , Rickettsia typhi , Sifonápteros/microbiologia , Tifo Endêmico Transmitido por Pulgas/microbiologia
3.
Circulation ; 146(2): 125-139, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35616010

RESUMO

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.


Assuntos
Proteínas Quinases Ativadas por AMP , Coração , Monodelphis , Infarto do Miocárdio , Regeneração , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Animais Recém-Nascidos , Proliferação de Células , Coração/fisiologia , Camundongos , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo
4.
Am J Physiol Renal Physiol ; 322(1): F14-F26, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34747197

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de Superfície Celular/metabolismo , Transcrição Genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Agenesia do Corpo Caloso/genética , Agenesia do Corpo Caloso/metabolismo , Agenesia do Corpo Caloso/patologia , Animais , Proteínas Reguladoras de Apoptose/genética , Células Cultivadas , Bases de Dados Genéticas , Redes Reguladoras de Genes , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Hérnias Diafragmáticas Congênitas/genética , Hérnias Diafragmáticas Congênitas/metabolismo , Hérnias Diafragmáticas Congênitas/patologia , Humanos , Túbulos Renais Proximais/patologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Camundongos Knockout , Monodelphis , Miopia/genética , Miopia/metabolismo , Miopia/patologia , Proteinúria/genética , Proteinúria/metabolismo , Proteinúria/patologia , Receptores de Superfície Celular/genética , Erros Inatos do Transporte Tubular Renal/genética , Erros Inatos do Transporte Tubular Renal/metabolismo , Erros Inatos do Transporte Tubular Renal/patologia
5.
G3 (Bethesda) ; 12(1)2022 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-34751383

RESUMO

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.


Assuntos
Monodelphis , Animais , Genoma , Genômica , Humanos , Laboratórios , Monodelphis/genética , Filogenia
6.
Biochem Biophys Res Commun ; 587: 85-91, 2022 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-34864550

RESUMO

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.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Monodelphis/genética , Regeneração Nervosa/genética , Proteínas do Tecido Nervoso/genética , Medula Espinal/metabolismo , Transcriptoma , Animais , Animais Recém-Nascidos , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Masculino , Anotação de Sequência Molecular , Monodelphis/crescimento & desenvolvimento , Monodelphis/metabolismo , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Proteômica/métodos , Medula Espinal/crescimento & desenvolvimento , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Fatores de Tempo
7.
Curr Biol ; 31(17): 3956-3963.e4, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34293331

RESUMO

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.


Assuntos
Monodelphis , Animais , Sistemas CRISPR-Cas , Feminino , Edição de Genes , Genoma , Camundongos , Monodelphis/genética , Ratos
8.
J Med Entomol ; 58(4): 1725-1732, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-33876821

RESUMO

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.


Assuntos
Trombiculidae/classificação , Animais , Arvicolinae/parasitologia , Brasil , Monodelphis/parasitologia , Trombiculidae/anatomia & histologia
9.
STAR Protoc ; 2(2): 100421, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33870226

RESUMO

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).


Assuntos
Fenômenos Biomecânicos/fisiologia , Monodelphis/fisiologia , Caminhada/fisiologia , Animais , Comportamento Animal/fisiologia , Biologia Computacional , Feminino , Aprendizado de Máquina , Masculino , Processamento de Sinais Assistido por Computador , Gravação em Vídeo
10.
Science ; 371(6536): 1383-1388, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33766885

RESUMO

αß and γδ T cell receptors (TCRs) are highly diverse antigen receptors that define two evolutionarily conserved T cell lineages. We describe a population of γµTCRs found exclusively in non-eutherian mammals that consist of a two-domain (Vγ-Cγ) γ-chain paired to a three-domain (Vµ-Vµj-Cµ) µ-chain. γµTCRs were characterized by restricted diversity in the Vγ and Vµj domains and a highly diverse unpaired Vµ domain. Crystal structures of two distinct γµTCRs revealed the structural basis of the association of the γµTCR heterodimer. The Vµ domain shared the characteristics of a single-domain antibody within which the hypervariable CDR3µ loop suggests a major antigen recognition determinant. We define here the molecular basis underpinning the assembly of a third TCR lineage, the γµTCR.


Assuntos
Monodelphis/imunologia , Receptores de Antígenos de Linfócitos T/química , Subpopulações de Linfócitos T/imunologia , Animais , Linhagem da Célula , Regiões Determinantes de Complementaridade/química , Cristalografia por Raios X , Modelos Moleculares , Monodelphis/genética , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores de Antígenos de Linfócitos T gama-delta
11.
J Comp Neurol ; 529(5): 969-986, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32710567

RESUMO

This study investigates the response to spinal cord injury in the gray short-tailed opossum (Monodelphis domestica). In opossums spinal injury early in development results in spontaneous axon growth through the injury, but this regenerative potential diminishes with maturity until it is lost entirely. The mechanisms underlying this regeneration remain unknown. RNA sequencing was used to identify differential gene expression in regenerating (SCI at postnatal Day 7, P7SCI) and nonregenerating (SCI at Day 28, P28SCI) cords +1d, +3d, and +7d after complete spinal transection, compared to age-matched controls. Genes showing significant differential expression (log2FC ≥ 1, Padj ≤ 0.05) were used for downstream analysis. Across all time-points 233 genes altered expression after P7SCI, and 472 genes altered expression after P28SCI. One hundred and forty-seven genes altered expression in both injury ages (63% of P7SCI data set). The majority of changes were gene upregulations. Gene ontology overrepresentation analysis in P7SCI gene-sets showed significant overrepresentations only in immune-associated categories, while P28SCI gene-sets showed overrepresentations in these same immune categories, along with other categories such as "cell proliferation," "cell adhesion," and "apoptosis." Cell-type-association analysis suggested that, regardless of injury age, injury-associated gene transcripts were most strongly associated with microglia and endothelial cells, with strikingly fewer astrocyte, oligodendrocyte and neuron-related genes, the notable exception being a cluster of mostly downregulated oligodendrocyte-associated genes in the P7SCI + 7d gene-set. Our findings demonstrate a more complex transcriptomic response in nonregenerating cords, suggesting a strong influence of non-neuronal cells in the outcome after injury and providing the largest survey yet of the transcriptomic changes occurring after SCI in this model.


Assuntos
Monodelphis/fisiologia , Traumatismos da Medula Espinal/genética , Regeneração da Medula Espinal/fisiologia , Transcriptoma , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Sequência de Bases , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neuroglia/metabolismo , Neurônios/metabolismo , Especificidade de Órgãos , Especificidade da Espécie , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia
12.
Acta amaz ; 50(4): 309-312, out. - dez. 2020.
Artigo em Inglês | LILACS | ID: biblio-1146367

RESUMO

Monodelphis glirina é endêmica da Amazônia e aspectos dos seus hábitos de vida e história natural são pouco conhecidos. Dados sobre a espécie foram coletados no norte de Mato Grosso, Brasil, incluindo observações sobre seu comportamento e simpatria com congêneres. Durante 10 expedições e com um esforço de 3.680 baldes-dia, foram capturados 29 M. glirina, três M. sacie um M. emiliae. Adultos representaram 82,8% das capturas de M. glirina e a razão sexual foi três machos para cada fêmea. A captura de jovens e fêmeas lactantes foi sazonal, uma vez que ambos foram capturados apenas na estação chuvosa. Observamos três eventos de M. glirina predando roedores em pitfall traps, todos com o mesmo comportamento de alimentação. Também registramos um individuo escalando uma árvore durante a noite, incluindo um video. Nosso trabalho contribui para uma melhor compreensão da ecologia desse gênero altamente diversificado e ainda pouco conhecido. (AU)


Assuntos
Biologia , Ecossistema Amazônico , Monodelphis
13.
Int J Syst Evol Microbiol ; 70(12): 6032-6043, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33079029

RESUMO

In a search for potential causes of increased prolapse incidence in grey short-tailed opossum colonies, samples from the gastrointestinal tracts of 94 clinically normal opossums with rectal prolapses were screened for Helicobacter species by culture and PCR. Forty strains of two novel Helicobacter species which differed from the established Helicobacter taxa were isolated from opossums with and without prolapses. One of the Helicobacter species was spiral-shaped and urease-negative whereas the other Helicobacter strain had fusiform morphology with periplasmic fibres and was urease-positive. 16S rRNA gene sequence analysis revealed that all the isolates had over 99 % sequence identity with each other, and were most closely related to Helicobacter canadensis. Strains from the two novel Helicobacter species were subjected to gyrB and hsp60 gene and whole genome sequence analyses. These two novel Helicobacter species formed separate phylogenetic clades, divergent from other known Helicobacter species. The bacteria were confirmed as novel Helicobacter species based on digital DNA-DNA hybridization and average nucleotide identity analysis of their genomes, for which we propose the names Helicobacter monodelphidis sp. nov. with the type strain MIT 15-1451T (=LMG 29780T=NCTC 14189T) and Helicobacter didelphidarum sp. nov with type strain MIT 17-337T (=LMG 31024T=NCTC 14188T).


Assuntos
Cloaca/patologia , Helicobacter/classificação , Monodelphis/microbiologia , Filogenia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Cloaca/microbiologia , DNA Bacteriano/genética , Ácidos Graxos/química , Trato Gastrointestinal/microbiologia , Genes Bacterianos , Helicobacter/isolamento & purificação , Hibridização de Ácido Nucleico , Prolapso , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Texas
14.
Nature ; 586(7830): 612-617, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32814901

RESUMO

Single-cell RNA sequencing of embryos can resolve the transcriptional landscape of development at unprecedented resolution. To date, single-cell RNA-sequencing studies of mammalian embryos have focused exclusively on eutherian species. Analysis of mammalian outgroups has the potential to identify deeply conserved lineage specification and pluripotency factors, and can extend our understanding of X dosage compensation. Metatherian (marsupial) mammals diverged from eutherians around 160 million years ago. They exhibit distinctive developmental features, including late implantation1 and imprinted X chromosome inactivation2, which is associated with expression of the XIST-like noncoding RNA RSX3. Here we perform a single-cell RNA-sequencing analysis of embryogenesis and X chromosome inactivation in a marsupial, the grey short-tailed opossum (Monodelphis domestica). We resolve the developmental trajectory and transcriptional signatures of the epiblast, primitive endoderm and trophectoderm, and identify deeply conserved lineage-specific markers that pre-date the eutherian-marsupial divergence. RSX coating and inactivation of the X chromosome occurs early and rapidly. This observation supports the hypothesis that-in organisms with early X chromosome inactivation-imprinted X chromosome inactivation prevents biallelic X silencing. We identify XSR, an RSX antisense transcript expressed from the active X chromosome, as a candidate for the regulator of imprinted X chromosome inactivation. Our datasets provide insights into the evolution of mammalian embryogenesis and X dosage compensation.


Assuntos
Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/genética , Monodelphis/embriologia , Monodelphis/genética , Análise de Célula Única , Transcriptoma/genética , Inativação do Cromossomo X/genética , Animais , Linhagem da Célula/genética , Embrião de Mamíferos/embriologia , Feminino , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Masculino , Monodelphis/classificação , RNA Antissenso/genética , RNA não Traduzido/genética , Regulação para Cima , Cromossomo X/genética
15.
J Anat ; 236(6): 1126-1136, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32052440

RESUMO

Mammalian pregnancy involves remodelling of the uterine epithelium to enable placentation. In marsupials, such remodelling has probably played a key role in the transition from ancestral invasive placentation to non-invasive placentation. Identifying uterine alterations that are unique to marsupials with non-invasive placentation can thus elucidate mechanisms of marsupial placental evolution. We identified apical alterations to uterine epithelial cells prior to implantation in Monodelphis domestica, a member of the least derived living marsupial clade (Didelphidae) with invasive (endotheliochorial) placentation. We then compared these traits with those of Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae), both with non-invasive placentation, to identify which alterations to the uterine epithelium are ancestral and which facilitate secondarily evolved non-invasive placentation. In M. domestica, remodelling of the uterine epithelium involves reduced cellular heterogeneity and development of uterodome-like cells, suggesting that similar alterations may also have occurred in the marsupial common ancestor. These alterations also overlap with those of both T. vulpecula and Ma. eugenii, suggesting that the placental shift from invasive to non-invasive placentation in marsupials involves essential, conserved characteristics, irrespective of placental mode. However, unique apical alterations of both T. vulpecula and Ma. eugenii, relative to M. domestica, imply that lineage-specific alterations underpin the evolutionary shift to non-invasive placentation in marsupials.


Assuntos
Epitélio/fisiologia , Placentação/fisiologia , Prenhez/fisiologia , Útero/fisiologia , Animais , Evolução Biológica , Implantação do Embrião/fisiologia , Feminino , Monodelphis , Gravidez
16.
Ticks Tick Borne Dis ; 11(3): 101366, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31883908

RESUMO

This study aimed to evaluate the infection of the marsupial Monodelphis domestica (the gray short-tailed opossum) by Rickettsia parkeri and its role as an amplifier of the bacterium for Amblyomma ticks. Ten M. domestica males were inoculated with phosphate-buffered saline (PBS) and 106 Vero cells infected with R. parkeri. In seven animals, inoculation was intramuscular, and in three intraperitoneal. One male (control) received 1 ml of the same vehicle used for inoculation intraperitoneally. The three animals inoculated intraperitoneally were infested with uninfected A. sculptum larvae and nymphs between the 2nd and 9th day post-infection (DPI). Parasitemia was monitored from the 3rd to 9th DPI by polymerase chain reaction, using primers for 17 kDa and ompA. The animals were also clinically evaluated. Of the animals infected intramuscularly, only one was blood-positive by the 5th DPI. The three animals infected intraperitoneally were blood-positive on the 2nd, 5th, 7th, and 9th DPI. Of the ten pools of recovered engorged ticks, six had positive bands. The kidney, liver, heart, and spleen of an intramuscularly infected animal were also positive. The rectal temperature of the animals tested increased only in the first three DPI. The animals inoculated intraperitoneally showed prostration, bristled hair, and weight loss. The study found that R. parkeri was capable of infecting M. domestica, which developed rickettsemia and caused infection in xenodiagnostic ticks.


Assuntos
Amblyomma/microbiologia , Monodelphis , Infecções por Rickettsia/veterinária , Rickettsia/fisiologia , Infestações por Carrapato/transmissão , Amblyomma/crescimento & desenvolvimento , Animais , Larva/crescimento & desenvolvimento , Larva/microbiologia , Ninfa/crescimento & desenvolvimento , Ninfa/microbiologia , Infecções por Rickettsia/microbiologia , Infecções por Rickettsia/transmissão , Infestações por Carrapato/microbiologia
17.
Dev Comp Immunol ; 104: 103562, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31785265

RESUMO

Here we demonstrate that regulation of the Complement (C') components of the immune system is an ancient and conserved feature of mammalian pregnancy. Transcript levels were reduced for complement components C3 and C4 throughout pregnancy in a marsupial, Monodelphis domestica. Downstream C' component transcripts were significantly less abundant relative to non-pregnant controls at the start of pregnancy but increased during late pregnancy, in some cases peaking close to parturition. These results are consistent with observations in human pregnancy that deposition of C5 through C9 on fetal membranes is associated with labor and parturition. Complement regulators CD46 and CD59 are present at the fetomaternal interface during M. domestica pregnancy as well, implying regulation of C' effector mechanisms is necessary for maintenance of normal marsupial pregnancy. Collectively these results support regulating the complement system may have contributed to the transition from oviparity to viviparity in mammals over 165 million years ago.


Assuntos
Proteínas do Sistema Complemento/metabolismo , Trabalho de Parto/metabolismo , Monodelphis/imunologia , Gravidez/imunologia , Animais , Evolução Biológica , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/imunologia , Evolução Molecular , Feminino , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica , Imunidade Humoral , Mamíferos , Oviparidade , Parto
18.
Physiol Behav ; 211: 112659, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31465782

RESUMO

Social behavior is critical for relationship formation and is influenced by myriad environmental and individual factors. Basic and preclinical research typically relies on rodent models to identify the mechanisms that underlie behavior; however, it is important to use non-rodent models as well. A major objective of the present study was to test the hypothesis that biological sex and social experience modulate the expression of social behavior in the adult gray short-tailed opossum (Monodelphis domestica), a non-traditional model. We also investigated the non-associative learning abilities of these animals. Following a period of social isolation, animals of both sexes were paired with a non-familiar, same-sex partner for 10 min on three different occasions, with 24-hour inter-trial intervals. We are the first research group to find significant sex differences in submissive and nonsocial behaviors in Monodelphis. Females displayed significantly higher durations of nonsocial behavior that increased over trials. Males were more aggressive; their latencies to the first attack and submissive behavior decreased over trials whereas these latencies increased for females; males' duration of submissive behavior increased over trials whereas it decreased for females. A different group of subjects habituated in response to repeated presentations to neutral odors and dishabituated in response to novel odors. In addition, both males and females demonstrated the ability to form social memories in a standard individual (social) recognition test. Our results contribute to the characterization of this marsupial species, an important first step in developing it as a model of complex social behaviors.


Assuntos
Comportamento Animal/fisiologia , Aprendizagem/fisiologia , Reconhecimento Psicológico/fisiologia , Caracteres Sexuais , Comportamento Social , Agressão/fisiologia , Animais , Feminino , Masculino , Monodelphis , Fatores Sexuais
19.
Curr Biol ; 29(15): 2533-2540.e7, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31327712

RESUMO

Identifying shared quantitative features of a neural circuit across species is important for 3 reasons. Often expressed in the form of power laws and called scaling relationships [1, 2], they reveal organizational principles of circuits, make insights gleaned from model systems widely applicable, and explain circuit performance and function, e.g., visual circuits [3, 4]. The visual circuit is topographic [5, 6], wherein retinal neurons target and activate predictable spatial loci in primary visual cortex. The brain, however, contains many circuits, where neuronal targets and activity are unpredictable and distributed throughout the circuit, e.g., olfactory circuits, in which glomeruli (or mitral cells) in the olfactory bulb synapse with neurons distributed throughout the piriform cortex [7-10]. It is unknown whether such circuits, which we term distributed circuits, are scalable. To determine whether distributed circuits scale, we obtained quantitative descriptions of the olfactory bulb and piriform cortex in six mammals using stereology techniques and light microscopy. Two conserved features provide evidence of scalability. First, the number of piriform neurons n and bulb glomeruli g scale as n∼g3/2. Second, the average number of synapses between a bulb glomerulus and piriform neuron is invariant at one. Using theory and modeling, we show that these two features preserve the discriminatory ability and precision of odor information across the olfactory circuit. As both abilities depend on circuit size, manipulating size provides evolution with a way to adapt a species to its niche without designing developmental programs de novo. These principles might apply to other distributed circuits like the hippocampus.


Assuntos
Bulbo Olfatório/fisiologia , Condutos Olfatórios/fisiologia , Córtex Piriforme/fisiologia , Animais , Gatos/fisiologia , Furões/fisiologia , Cobaias/fisiologia , Camundongos/fisiologia , Monodelphis/fisiologia , Neurônios/fisiologia , Ratos/fisiologia , Sinapses/fisiologia
20.
Proc Biol Sci ; 286(1905): 20190691, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31213185

RESUMO

In human pregnancy, recognition of an embryo within the uterus is essential to support the fetus through gestation. In most marsupials, such as the opossums, pregnancy is shorter than the oestrous cycle and the steroid hormone profile during pregnancy and oestrous cycle are indistinguishable. For these reasons, it was assumed that recognition of pregnancy, as a trait, evolved in the eutherian (placental) stem lineage and independently in wallabies and kangaroos. To investigate whether uterine recognition of pregnancy occurs in species with pregnancy shorter than the oestrous cycle, we examined reproduction in the short-tailed opossum ( Monodelphis domestica), a marsupial with a plesiomorphic mode of pregnancy. We examined the morphological and gene expression changes in the uterus of females in the non-pregnant oestrous cycle and compared these to pregnancy. We found that the presence of an embryo did not alter some aspects of uterine development but increased glandular activity. Transcriptionally, we saw big differences between the uterus of pregnant and cycling animals. These differences included an upregulation of genes involved in transport, inflammation and metabolic-activity in response to the presence of a fetus. Furthermore, transcriptional differences reflected protein level differences in transporter abundance. Our results suggest that while the uterus exhibits programmed changes after ovulation, its transcriptional landscape during pregnancy responds to the presence of a fetus and upregulates genes that may be essential for fetal support. These results are consistent with endometrial recognition of pregnancy occurring in the opossum. While the effects on maternal physiology appear to differ, recognition of pregnancy has now been observed in eutherian mammals, as well as, Australian and American marsupials.


Assuntos
Monodelphis/fisiologia , Gravidez , Animais , Ciclo Estral , Feminino , Marsupiais
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