Evolution and development of the mammalian dentition: Insights from the marsupial Monodelphis domestica

To understand developmental mechanisms of evolutionary change, we must first know how different morphologies form. The vast majority of our knowledge on the developmental genetics of tooth formation derives from studies in mice, which have relatively derived mammalian dentitions. The marsupial Monodelphis domestica has a more plesiomorphic heterodont dentition with incisors, canines, premolars, and molars on both the upper and the lower jaws, and a deciduous premolar. The complexity of the M. domestica dentition ranges from simple, unicusped incisors to conical, sharp canines to multicusped molars. We examine the development of the teeth in M. domestica, with a specific focus on the enamel knot, a signaling center in the embryonic tooth that controls shape. We show that the tooth germs of M. domestica express fibroblast growth factor (FGF) genes and Sprouty genes in a manner similar to wild‐type mouse molar germs, but with a few key differences. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Uterine epithelial remodelling during pregnancy in the marsupial Monodelphis domestica (Didelphidae): Implications for mammalian placental evolution

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.     

On the prenatal initiation of T cell development in the opossum Monodelphis domestica

Thymus‐dependent lymphocytes (T cells) are a critical cell lineage in the adaptive immune system of all jawed vertebrates. In eutherian mammals the initiation of T cell development takes place prenatally and the offspring of many species are born relatively immuno‐competent. Marsupials, in contrast, are born in a comparatively altricial state and with a less well developed immune system. As such, marsupials are valuable models for studying the peri‐ and postnatal initiation of immune system development in mammals. Previous results supported a lack of prenatal T cell development in a variety of marsupial species. In the gray short‐tailed opossum, Monodelphis domestica, however, there was evidence that αβT cells were present on postnatal day 1 and likely initiated development prenatally. Demonstrated here is the presence of CD3ε⁺ lymphocytes in late‐stage embryos at a site in the upper thoracic cavity, the site of an early developing thymus. CD3ε⁺ cells were evident as early as 48 h prior to parturition. In day 14 embryos, where there is clear organogenesis, CD3ε⁺ cells were only found at the site of the early thymus, consistent with no extra‐thymic sites of T cell development in the opossum. These observations are the first evidence of prenatal T cell lineage commitment in any marsupial.     

Postnatal development of the fore- and hindlimbs in the grey short-tailed opossum,Monodelphis domestica

Marsupials are good experimental animals for developmental studies as their offspring are born at a stage comparable to embryonic stages of eutherian species. The South American opossum, Monodelphis domestica, is particularly useful because of its small size and easy maintenance. This study was carried out to compare development of opossum fore- and hindlimbs during postnatal life, using light microscopy and whole mount alizarin staining. At birth, well-developed mobile forelimbs show cartilage models of bones and myotubular striated muscle fibres. However, hindlimbs are relatively underdeveloped paddle-like outgrowths. Two days later mesodermal condensations form models of the future hindlimb bones and mononucleate myoblast aggregates are present; by 6 days post partum (dpp) the hindlimb has reached a stage of development similar to that of the forelimb at birth. At this stage, periosteal buds have invaded forelimb long bones and nuclei in forelimb muscle fibres have become displaced to the periphery. The 16 dpp hindlimb shows long bones invaded by periosteal buds and closely packed, striated muscle fibres. Epiphyseal plates are now seen in the forelimb long bones and forelimb muscle fibres show mature characteristics. Musculoskeletal development is well correlated with the functional demands of the limbs during postnatal development in the opossum, which provides an excellent model for investigations into the genes and molecules controlling limb development.     

Passively Acquired Immunity in the Newborn of a Marsupial (Monodelphis domestica)

ABSTRACT: A colony of fully pedigreed Monodelphis domestica has been used to investigate the maternal-fetal relationship in this unique marsupial species. To determine how immunity is transferred from mothers to young in M. domestica, we hyperimmunized females with sheep red blood cells (SRBC) before and during gestation. Offspring from these females were collected at various times after birth, and saline extracts of the neonates were assayed for hemolysins against SRBC. Antibodies were present in extracts of newborn that had been allowed to suckle their mothers; none were detected in extracts of infants that were not allowed to suckle. Antibodies were present in the milk of immunized mothers, but were not detected in the milk of nonimmunized mothers. The titer of antibodies in the extracts of newborns generally increased proportionately to the time that the newborn had been allowed to suckle. We conclude that the transfer of passive immunity from mothers of M. domestica to their offspring occurs primarily via the milk.     

Efferent connections of the main olfactory bulb in the opossum (Monodelphis domestica): a characterization of the olfactory entorhinal cortex in a marsupial

Olfactory projections have been investigated for decades, but few reports using modern, sensitive neural tracers are available. In marsupials, only lesion-degeneration studies exist and they are restricted to the genera Didelphis and Trichosurus. Some of the territories described as olfactory-recipient such as the upper portion of the rhinal fissure and the vomeronasal amygdala are, however, controversial. Also, the characterization of the olfactory portion of the entorhinal cortex is far from clear in acallosal mammals. The present report investigates, using biotinylated dextran-amine, the olfactory connections in the short-tailed opossum (Monodelphis domestica) and characterizes the olfactory portion of the entorhinal cortex in non-placental mammals. The data indicate that olfactory projections do not reach the upper portion of the rhinal fissure, but partially end in the vomeronasal amygdala, i.e., the medial and posteromedial cortical amygdaloid nuclei; thus, although olfactory and vomeronasal system have largely segregated outputs, areas of overlap should be restudied. The olfactory portion of the entorhinal cortex is much larger than previously described, extending up to the occipital pole of the cerebral hemisphere. Collectively, these data contribute to our understanding of the organization of the hippocampal formation in marsupials.     

It is time to beelieve the CD1a hype!

Conventional T cells have historically been linked to exacerbating allergy. By efficiently generating primarily T_H2 cells, allergens skew the immune response to produce IL‐4, IL‐13, and IgE. Previously, CD1a‐responsive T cells were shown to functionally respond to bee and wasp venom allergens. In this issue of the European Journal of Immunology, Subramaniam et al. [Eur. J. Immunol. 2016. 46 : 242–252] show that more functionally active CD1a‐restricted cells are present in bee venom‐allergic patients than in healthy patients. Additionally, the authors show that these cells are not as frequently found in individuals receiving venom immunotherapy. Consequently, this study implicates CD1a‐reactive cells as the primary responders to venom allergy, which considerably regulate the downstream immune response.     

A study on the polymorphism of human MHC class I-related MR1 gene and identification of an MR1-like pseudogene

Human MR1 is a recently discovered, ubiquitously transcribed gene very similar to the HLA class I loci and of unknown function. Mouse and rat MR1 sequences have also been described showing high similarity with the human gene. The goal of this work was to investigate if human MR1 was polymorphic. We have found that DNA sequences of MR1-specific polymerase chain reaction (PCR) products obtained from samples of diverse ethnic origin were invariant except in one case in which two silent mutations were detected. We also found an MR1-like sequence displaying significant differences with the previously described, the most remarkable of which is a STOP codon in the alpha2 domain indicating that is a pseudogene.     

尖锐湿疣表皮CD1a和CD207的表达

目的:了解尖锐湿疣(CA)表皮中CD1a和CD207的表达情况及其意义.方法:采用寡核苷酸芯片对6例CA表皮和6例正常表皮中的CD1a和CD207的mRNA表达进行检测,应用半定量RT-PCR验证CD1a和CD207的mRNA表达,采用Western blot法检测6例CA表皮和6例正常表皮中的CD1a和CD207的蛋白表达.结果:基因芯片检测到CD1a和CD207的mRNA在CA表皮中表达下调,半定量RT-PCR证实CD1a和CD207的mRNA在CA表皮中表达下调,Western blot法检测到CD1a和CD207蛋白在CA表皮中的显著下调表达.结论:CA表皮中CD1a和CD207的表达下调,提示CA表皮中LC可能出现了数目的减少及功能的受损.     

An analysis of the gene complement of a marsupial, Monodelphis domestica: evolution of lineage-specific genes and giant chromosomes

The newly sequenced genome of Monodelphis domestica not only provides the out-group necessary to better understand our own eutherian lineage, but it enables insights into the innovative biology of metatherians. Here, we compare Monodelphis with Homo sequences from alignments of single nucleotides, genes, and whole chromosomes. Using PhyOP, we have established orthologs in Homo for 82% (15,250) of Monodelphis gene predictions. Those with single orthologs in each species exhibited a high median synonymous substitution rate (d(S) = 1.02), thereby explaining the relative paucity of aligned regions outside of coding sequences. Orthology assignments were used to construct a synteny map that illustrates the considerable fragmentation of Monodelphis and Homo karyotypes since their therian last common ancestor. Fifteen percent of Monodelphis genes are predicted, from their low divergence at synonymous sites, to have been duplicated in the metatherian lineage. The majority of Monodelphis-specific genes possess predicted roles in chemosensation, reproduction, adaptation to specific diets, and immunity. Using alignments of Monodelphis genes to sequences from either Homo or Trichosurus vulpecula (an Australian marsupial), we show that metatherian X chromosomes have elevated silent substitution rates and high G+C contents in comparison with both metatherian autosomes and eutherian chromosomes. Each of these elevations is also a feature of subtelomeric chromosomal regions. We attribute these observations to high rates of female-specific recombination near the chromosomal ends and within the X chromosome, which act to sustain or increase G+C levels by biased gene conversion. In particular, we propose that the higher G+C content of the Monodelphis X chromosome is a direct consequence of its small size relative to the giant autosomes.     

Conserved and divergent expression patterns of markers of axial development in the laboratory opossum,Monodelphis domestica

Background: Previous comparative studies suggest that the requirement for Nodal in epiblast and hypoblast development is unique to mammalians. Expression of anterior visceral endoderm (AVE) genes in the visceral endoderm and of their orthologs in the hypoblast may be unique to mammalians and avians, and is absent in the reptilian hypoblast. Axis formation in reptiles is signaled by the formation of the posterior marginal epiblast (PME), which expresses a series of primitive streak genes. To assess the phylogenetic origin of Nodal and AVE gene expression and axis formation in amniotes, we examined marker gene expression in gray short‐tailed opossum, a metatherian. Results: Nodal was expressed in neither epiblast nor hypoblast of opossum embryos. No AVE genes were expressed in the opossum hypoblast. Attainment of polarity in the embryonic disk was signaled by Nodal, Wnt3a, Fgf8, and Bra expression in the PME at 8.5 days post‐coitus. Conclusions: Nodal expression in epiblast or hypoblast may be unique to eutherians. AVE gene expression in visceral endoderm and hypoblast may have been independently acquired in eutherian and avian lineages. PME formation appears to be the event that signals axis formation in reptilian and metatherian embryos, and thus may be an ancestral characteristic of basal amniotes. Developmental Dynamics 245:1176–1188, 2016. © 2016 Wiley Periodicals, Inc.     

Evidence for an early appearance of modern post-switch isotypes in mammalian evolution; cloning of IgE,IgG and IgA from the marsupial Monodelphis domestica

In birds, reptiles and amphibians the IgY isotype exhibits the functional characteristics of both of IgG and IgE. Hence, the gene for IgY most likely duplicated some time during early mammalian evolution and formed the ancestor of present day IgG and IgE. To address the question of when IgY duplicated and formed two functionally distinct isotypes, and to study when IgG and IgA lost their second constant domains, we have examined the Ig expression in a non-placental mammal, the marsupial Monodelphis domestica (grey short-tailed opossum). Screening of an opossum spleen cDNA library revealed the presence of all three isotypes in marsupials. cDNA clones encoding the entire constant regions of opossum IgE (ϵ chain), IgG (γ chain) and IgA (α chain) were isolated, and their nucleotide sequences were determined. A comparative analysis of the amino acid sequences for IgY, IgA, IgE and IgG from various animal species showed that opossum IgE, IgG and IgA on the phylogenetic tree form branches clearly separated from their eutherian counterparts. However, they still conform to the general structure found in eutherian IgE, IgG and IgA. Our findings indicate that all the major evolutionary changes in the Ig isotype repertoire, and in basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the evolutionary separation of marsupials and placental mammals.     

Ethnic differences in CD1E,but not CD1A,gene polymorphisms between Sub-Saharan Africans,West Asians and Europeans

The five closely linked CD1A-E genes encode the human CD1 family of proteins. Few studies of the allele frequencies of these genes in African populations have been published so far. This study aimed to genotype CD1A and CD1E variants and to compare their frequencies in Sub-Saharan Africans from Gabon and Ivory Coast, and Non-Africans from Syria and France.A restriction analysis of DNA fragments generated by PCR was performed to detect CD1A and CD1E alleles in 105 subjects from Gabon, 169 subjects from Ivory Coast, 107 subjects from Syria and 181 subjects from France.The frequencies of the CD1E*02 allele were high among Sub-Saharan Africans (87%) and low in West Asians (44%) and Europeans (36%), whereas the contrary was obtained for the CD1E*01 allele (7%, 55% and 64% respectively). Frequencies of CD1A alleles were similar between all groups, the CD1A*02 allele was most prevalent (91%).The high frequency of the CD1E*02 allele in Sub-Saharan Africans suggest that future work should investigate the relationship between CD1 polymorphism and infectious diseases.     

CD1a and CD1b surface expression is independent from de novo synthesized glycosphingolipids

CD1 molecules resemble classical MHC molecules in structure, bind self and bacterial glycolipids and present them to T cells. Whether the CD1 antigen-binding groove becomes filled during maturation and traffic to the cell surface is an important and still unsolved biological question. As most cell types synthesize complex glycosphingolipids (GSL), which also stimulate CD1-restricted T cells, it could be possible that these ligands associate with nascent CD1 molecules. Here, we show that treatment of cells with drugs blocking at different levels the de novo and salvage pathways of GSL synthesis does not prevent surface expression of CD1a and CD1b. Furthermore, transfection of CD1A and CD1B genes in a mutant cell line unable to synthesize glucosylceramides and galactosylceramides showed normal surface expression of both CD1 molecules. Lack of GSL did not induce intracellular CD1 accumulation as indicated by confocal microscopy. The same results were obtained by transfecting the Lec series of mutants, which are deficient in sugar addition to glycolipids and glycoproteins. These findings demonstrate that endogenous de novo synthesized GSL are not mandatory for CD1a and CD1b negotiating surface expression.     

Skeletal fiber types and spindle distribution in limb and jaw muscles of the adult and neonatal opossum,Monodelphis domestica

The South American opossum, Monodelphis domestica, is very immature at birth, and we wished to assess its potential for studies of jaw muscle development. Given the lack of prior information about any Monodelphis fiber types or spindles, our study aimed to identify for the first time fiber types in both adult and neonatal muscles and the location of spindles in the jaw muscles. Fiber types were identified in frozen sections of adult and 6-day-old jaw and limb muscles by using myosin ATPase and metabolic enzyme histochemistry and by immunostaining for myosin isoforms. The distribution of fiber types and muscle spindles throughout the jaw-closer muscles was identified by immunostaining of sections of methacarnoy-fixed, wax-embedded heads. Most muscles contained one slow (type I) and two fast fiber types (equivalent to types IIA and IIX), which were similar to those in eutherian muscle, and an additional (non-IIB) fast type. In jaw-closer muscles, the main extrafusal fiber type was IIM (characteristic of these muscles in some eutherians), and almost all spindles were concentrated in four restricted areas: one in masseter and three in temporalis. Six-day neonatal muscles were very immature, but future spindle-rich areas were revealed by immunostaining and corresponded in position to the adult areas. Extrafusal and spindle fiber types in Monodelphis share many similarities with eutherian mammalian muscle. This finding, along with the immaturity of myosin isoform expression observed 6 days postnatally, indicates that Monodelphis could provide a valuable model for studying early developmental events in the jaw-closer muscles and their spindles. Anat. Rec. 251:548–562, 1998. © 1998 Wiley-Liss, Inc.     

Molecular characterisation and expression of CD4 in two distantly related marsupials: the gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii)

The gene and corresponding cDNA for CD4 in the gray short-tailed opossum, Monodelphis domestica, and the cDNA sequence for CD4 in the tammar wallaby, Macropus eugenii, have been characterised. The opossum CD4 homolog reveals conserved synteny, preserved genomic organisation and analogous structural arrangement to human and mouse CD4. Opossum and tammar CD4 exhibit typical eutherian CD4 features including the highly conserved p56(lck) binding motif in the cytoplasmic region and the invariant cysteine residues in extracellular domains 1 and 4. Interestingly, the marsupial CD4 sequences substitute a tryptophan for the first cysteine in domain 2 negating the formation of a disulphide bond as seen in other eutherian CD4 sequences except human and mouse. Overall the marsupial CD4 sequences share amino acid identity of 59% to each other and 37-41% with eutherian mammals. However, in contrast to eutherian homologs, the marsupial CD4 sequences were found to be truncated at the terminal end of the cytoplasmic tail. This is the first report confirming the presence of CD4 in a marsupial and describing its key features.     

A feline homologue of CD1 is defined using a feline-specific monoclonal antibody

The characteristics of a feline homologue of CD1, defined by a murine monoclonal antibody, Fel.5F4 (I_gG1), are described. This antibody precipitated a 49 kDa protein from biotinylated feline thymocyte extract in conjunction with a 14 kDa protein, consistent with β2 microglobulin subunit. The tissue distribution of this antigen was restricted to cortical thymocytes and antigen presenting cells of the thymic medulla, epidermis (Langerhans cells), dermis and occasional dendritic cells in the mantle and periarteriolar lymphoid areas of the spleen. Although flow cytometry demonstrated a continuous distribution of antigen expression on thymocytes, antigen density was found to decrease with age, consistent with physiological thymic involution. Thymocytes with high density expression of this antigen were predominantly restricted to cells with dual expression of CD4 and CD8 as defined by feline specific murine monoclonal antibodies Fel.7B12 (I_gG1) and Fel.10E9 (I_gG1) respectively. The tissue distribution of this CD1 homologue indicates that it is a member of the classic thymic CD1 family. This feline homologue of CD1 was distinct from CD1c by virtue of its lack of expression in peripheral blood and splenic mantle zone B cells. An unequivocal distinction could not be made between CD la and CD 1b based on tissue distribution due to species variation in expression of these CD1 molecules. Although, the biochemical characteristics of this feline CD1 homologue more closely match with CD 1a. The pattern of tissue expression and biochemical characteristics of the feline CD1 antigen appear largely similar to those described for human and other species.     

IL-1 acts on antigen-presenting cells to enhance the in vivo proliferation of antigen-stimulated naive CD4 T cells via a CD28-dependent mechanism that does not involve increased expression of CD28 ligands

The basis of the adjuvant effect of IL-1 on the clonal expansion of Ag-specific CD4 T cells was measured in vivo using the TCR transgenic T cell adoptive transfer method. Injection of Ag plus IL-1 caused naive CD4 T cells to proliferate to a greater extent than did injection of Ag alone. The T cells had to express CD28 to experience the beneficial effect of IL-1 whereas the host had to express the type I IL-1R, raising the possibility that IL-1's adjuvant properties were related to induction of CD28 ligands on APC. Surprisingly, however, expression of CD28 ligands on DC and B cells was not affected by IL-1. Therefore, the adjuvant properties of IL-1 depend on a basal level of CD28 signaling but cannot be explained by enhanced CD28 signaling due to CD28 ligand induction.     

尖锐湿疣组织CD1a、上皮细胞钙粘素分子及其mRNA的表达和朗格汉斯细胞的电镜观察

目的:研究尖锐湿疣(Condyloma accuminatum,CA)组织中朗格汉斯细胞(Langerhans cell,LC)数目、形态和功能改变及其分子机制.方法:免疫组化SP法和逆转录RT-PCR法分别检测LC表面标记分子CD1a、粘附分子上皮细胞钙粘素(E-cadherin)及其mRNA表达,透射电镜观察LC的超微结构,并以正常包皮组织作为对照.结果:与正常对照组(21.59±10.48)相比,CA组织中CD1a+LC数目(10.66 ±11.71)减少(P＜0.05),胞体缩小,树突减少.CD1a mRNA表达(0.4066±0.2671)低于正常(0.744 4±0.3667)(P＜0.01);CA组织中E-cadherin染色变淡,平均积分值(2.36±1.41)低于正常对照(7.67±1.64)(P＜0.01),E-cadherin mRNA表达(0.173 7±0.108 3)较正常对照(0.378 6±0.146 0)相比显著降低(P＜0.01);CD1a+细胞数目与E-cadherin表达强度之间存在正相关关系(r=0.940 4,P＜0.05)、CD1a mRNA与E-cadherin mRNA的表达呈正相关(r=0.838 1,P＜0.05);CA组织LC胞质内细胞器稀少,Birbeck颗粒少见.结论:CA组织中存在着LC的缺失和抗原提呈功能异常,可能导致HPV持续性感染,LC的减少可能与E-cadherin表达下降导致LC在表皮内难以滞留有关.