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Analysis of the pEKLF/GFP mouse. (A) Schematic of the modified expression system (based on Kyba et al., 2002) after its unidirectional, single-copy integration into the single endogenous modified HPRT locus. The EKLF promoter (950 bp; red) is directly upstream of the GFP reporter (green) (Lohmann and Bieker, 2008). (B) FACS analysis of EKLF expression in macrophage. E13.5 fetal livers (n=8) from pEKLF/GFP mice (Lohmann and Bieker, 2008) were sorted for presence of macrophage (F4/80 marker) and EKLF-expressing cells (GFP marker). The percentage of double-positive F4/80 and GFP cells, compared to total F4/80+ cells, is indicated. (C) qRT-PCR analysis of macrophage genes important for erythroblastic island integrity. F4/80+ macrophage cells were FACS-sorted into EKLF/GFP+ or EKLF/GFP− singlet populations (as in supplementary material Fig. S3). RNA was isolated and used to analyze for the presence of the indicated transcripts. All values are normalized to GAPDH, and these values were then normalized to expression in F4/80+EKLF/GFP− cells (='1'). (D) FACS analysis of VCAM1 expression in EKLF-expressing cells (GFP marker) in E13.5 fetal livers from pEKLF/GFP mice (Lohmann and Bieker, 2008). The percentage of double-positive VCAM1 and GFP cells, compared to total GFP+ cells, is indicated. (E) Erythroblastic islands from E13.5 fetal livers were probed for F4/80 (red) and monitored for endogenous EKLF/GFP expression (green). Three typical islands are shown. DAPI (blue) was used as a nuclear marker.
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The erythroblastic island provides an important nutritional and survival support niche for efficient erythropoietic differentiation. Island integrity is reliant on adhesive interactions between erythroid and macrophage cells. We show that erythroblastic islands can be formed from single progenitor cells present in differentiating embryoid bodies, a...
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... the colony preparations that gave rise to EBEI were clonal (being from dispersed single cells seeded in methylcellulose), we considered the possibility that EKLF might be expressed in both the erythroid cell and macrophage. There have been hints of EKLF expression in macrophage ( Luo et al., 2004), although our analysis of adult bone marrow hematopoietic material had shown no evidence ( Frontelo et al., 2007). To address this we used the mouse strain derived from ESCs that contain a single copy of the EKLF promoter directly upstream of a GFP reporter, integrated into the HPRT locus ( Fig. 5A) (Lohmann and Bieker, 2008). This EKLF promoter region contains erythroid cell-specific DNase hypersensitive sites and is sufficient to confer tissue-specific expression on a linked reporter in transgenic mice (Chen et al., 1998;Xue et al., 2004;Yien and Bieker, 2013). Our published studies have shown the high cell specificity of GFP expression that results from this pEKLF/GFP construct and, importantly, that GFP onset mirrors EKLF onset ( Lohmann and Bieker, 2008). We isolated E13.5 fetal liver cells from pEKLF/GFP mice and monitored, by fluorescence-activated cell sorting (FACS), for overlap of GFP (i.e. EKLF expression) and the F4/80 macrophage marker. Our results show that ∼36% of F4/80+ macrophage cells express EKLF (Fig. 5B). Two analyses were performed to exclude the possibility that the macrophage GFP signal came from adherent erythroid cells or from engulfed erythroid nuclei. First, we included an additional selection for non-clumped cells with single nuclei and obtained similar results (supplementary material Fig. S2A). Second, we cultured the fetal liver cells in the absence of erythropoietin to minimize erythroid expansion, and found that 34-55% of the EKLF/GFP+ population was also F4/80+/Ter119− (supplementary material Fig. ...
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... the colony preparations that gave rise to EBEI were clonal (being from dispersed single cells seeded in methylcellulose), we considered the possibility that EKLF might be expressed in both the erythroid cell and macrophage. There have been hints of EKLF expression in macrophage ( Luo et al., 2004), although our analysis of adult bone marrow hematopoietic material had shown no evidence ( Frontelo et al., 2007). To address this we used the mouse strain derived from ESCs that contain a single copy of the EKLF promoter directly upstream of a GFP reporter, integrated into the HPRT locus ( Fig. 5A) (Lohmann and Bieker, 2008). This EKLF promoter region contains erythroid cell-specific DNase hypersensitive sites and is sufficient to confer tissue-specific expression on a linked reporter in transgenic mice (Chen et al., 1998;Xue et al., 2004;Yien and Bieker, 2013). Our published studies have shown the high cell specificity of GFP expression that results from this pEKLF/GFP construct and, importantly, that GFP onset mirrors EKLF onset ( Lohmann and Bieker, 2008). We isolated E13.5 fetal liver cells from pEKLF/GFP mice and monitored, by fluorescence-activated cell sorting (FACS), for overlap of GFP (i.e. EKLF expression) and the F4/80 macrophage marker. Our results show that ∼36% of F4/80+ macrophage cells express EKLF (Fig. 5B). Two analyses were performed to exclude the possibility that the macrophage GFP signal came from adherent erythroid cells or from engulfed erythroid nuclei. First, we included an additional selection for non-clumped cells with single nuclei and obtained similar results (supplementary material Fig. S2A). Second, we cultured the fetal liver cells in the absence of erythropoietin to minimize erythroid expansion, and found that 34-55% of the EKLF/GFP+ population was also F4/80+/Ter119− (supplementary material Fig. ...
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... for EKLF in erythroblastic island integrity E13.5 fetal livers from the pEKLF/GFP mouse were then used to enrich for erythroblastic island clusters, which were stained for F4/80 and monitored for GFP. These show a typical island structure, with a central F4/80+ macrophage surrounded by 10-15 GFP+ cells (Fig. 5E). These results demonstrate that EKLF+ cells are associated with the island macrophages when isolated directly from fetal livers, and support the previous observations derived from differentiating ...
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... than EMP/MAEA, none are appreciably expressed in the erythroid cell. The controls confirm that EKLF is preferentially expressed in the EKLF/GFP+ population (Fig. 5C). The test samples show that there is no difference in αv integrin, EMP/ MAEA, MAF or palladin expression in the two cell populations; on the other hand, DNASE2A [recently shown to be an EKLF target in macrophages (Porcu et al., 2011)] and VCAM1 are more highly expressed in EKLF/GFP+ cells (Fig. 5C). In support of this observation, surface VCAM1 protein is expressed in ∼30% of the EKLF/GFP+ cells (Fig. 5D), and VCAM1 levels are decreased in total fetal liver cell RNA from EKLF-null compared with wild-type embryos (Tallack et al., ...
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... than EMP/MAEA, none are appreciably expressed in the erythroid cell. The controls confirm that EKLF is preferentially expressed in the EKLF/GFP+ population (Fig. 5C). The test samples show that there is no difference in αv integrin, EMP/ MAEA, MAF or palladin expression in the two cell populations; on the other hand, DNASE2A [recently shown to be an EKLF target in macrophages (Porcu et al., 2011)] and VCAM1 are more highly expressed in EKLF/GFP+ cells (Fig. 5C). In support of this observation, surface VCAM1 protein is expressed in ∼30% of the EKLF/GFP+ cells (Fig. 5D), and VCAM1 levels are decreased in total fetal liver cell RNA from EKLF-null compared with wild-type embryos (Tallack et al., ...
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... than EMP/MAEA, none are appreciably expressed in the erythroid cell. The controls confirm that EKLF is preferentially expressed in the EKLF/GFP+ population (Fig. 5C). The test samples show that there is no difference in αv integrin, EMP/ MAEA, MAF or palladin expression in the two cell populations; on the other hand, DNASE2A [recently shown to be an EKLF target in macrophages (Porcu et al., 2011)] and VCAM1 are more highly expressed in EKLF/GFP+ cells (Fig. 5C). In support of this observation, surface VCAM1 protein is expressed in ∼30% of the EKLF/GFP+ cells (Fig. 5D), and VCAM1 levels are decreased in total fetal liver cell RNA from EKLF-null compared with wild-type embryos (Tallack et al., ...
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Objective
To examine the clinical characteristics and anemia-related factors in patients with newly diagnosed multiple myeloma (NDMM), as well as the effect and mechanism of erythroblastic islands (EBIs) and EBI macrophages in NDMM patients with anemia.
Methods
We collected and analyzed clinical data to find anemia-related factors. Using flow cyto...
Citations
... In the bone marrow niche, the erythroblastic island, consisting of a central resident macrophage surrounded by differentiating erythroblasts (30,38), is responsible for maintaining the continuous production of red blood cells. This microenvironment provides essential nutritional and survival support to ensure efficient erythropoiesis (39). Additionally, extramedullary erythropoiesis predominantly takes place in the red pulp of the spleen, where stress erythropoietic progenitors are primarily sustained by endothelial cells located around sinusoids and stromal cells (9). ...
Red blood cells are the predominant cellular component in human body, and their numbers increase significantly during pregnancy due to heightened erythropoiesis. CD71⁺ erythroid cells (CECs) are immature red blood cells, encompassing erythroblasts and reticulocytes, constitute a rare cell population primarily found in the bone marrow, although they are physiologically enriched in the neonatal mouse spleen and human cord blood. Presently, the mechanisms underlying the CECs expansion during pregnancy remain largely unexplored. Additionally, the mechanisms and roles associated with extramedullary hematopoiesis (EMH) of erythroid cells during pregnancy have yet to be fully elucidated. In this study, our objective was to examine the underlying mechanisms of erythroid-biased hematopoiesis during pregnancy. Our findings revealed heightened erythropoiesis and elevated CECs in both human and mouse pregnancies. The increased presence of transforming growth factor (TGF)-β during pregnancy facilitated the differentiation of CD34⁺ hematopoietic stem and progenitor cells (HSPCs) into CECs, without impacting HSPCs proliferation, ultimately leading to enhanced erythropoiesis. The observed increase in CECs during pregnancy was primarily attributed to EMH occurring in the spleen. During mouse pregnancy, splenic stromal cells were found to have a significant impact on splenic erythropoiesis through the activation of TGF-β signaling. Conversely, splenic macrophages were observed to contribute to extramedullary erythropoiesis in a TGF-β-independent manner. Our results suggest that splenic stromal cells play a crucial role in promoting extramedullary erythropoiesis and the production of CECs during pregnancy, primarily through TGF-β-dependent mechanisms.
... switching by E9.5 to definitive cells within the hepatic primordia, and then to the red pulp of the adult spleen (10). It is expressed in the erythroblastic island macrophage, an in vivo niche that supplies a supportive environment for developing red cells (11)(12)(13). Early in hematopoietic differentiation, KLF1 is expressed at low levels in the MPP and it retains an expression pattern restricted to the CMP and MEP as its transcript levels increase prior to eventual segregation to erythroid progeny (14)(15)(16). ...
EKLF/KLF1 is an essential transcription factor that plays a global role in erythroid transcriptional activation. Regulation of KLF1 is of interest, as it displays a highly restricted expression pattern, limited to erythroid cells and its progenitors. Here we use biochemical affinity purification to identify the DDX5/p68 protein as an activator of KLF1 by virtue of its interaction with the erythroid-specific DNAse hypersensitive site upstream enhancer element (EHS1). We further show that this protein associates with DEK and CTCF. We postulate that the range of interactions of DDX5/p68 with these and other proteins known to interact with this element render it part of the enhanseosome complex critical for optimal expression of KLF1 and enables the formation of a proper chromatin configuration at the Klf1 locus. These individual interactions provide quantitative contributions that, in sum, establish the high-level activity of the Klf1 promoter and suggest they can be selectively manipulated for clinical benefit.
... The expression of Klf1 on macrophages was first highlighted in human primary cells, and its involvement in the regulation of IL12-p40 expression was also demonstrated [59]. Subsequently, a further demonstration of both the expression and activity of Klf1 in central macrophages of erythroblastic islands was described in vivo [28,37]. Recently, a Klf1+ F4/80+ macrophage population in murine foetal liver cells with a peculiar pattern of expression was identified. ...
... Erythroidmacrophage interaction is perturbed in Klf1 KO mice due to the down-regulation of two adhesive systems. The lack of Klf1 results in down-regulation of Icam4 adhesion molecules in erythroid cells [2,3,12,18], whilst the Vcam1 adhesion molecule is affected by the absence of Klf1 in central macrophages ( Figure 1D) [28]. Vcam1 and Icam4 interacting receptors, α4β1-integrin expressed on erythroblasts and αv-integrin present on macrophage surfaces were not found to be Klf1 targets ( Figure 1D). ...
Krüppel-like factor 1 (KLF1) plays a crucial role in erythropoiesis. In-depth studies conducted on mice and humans have highlighted its importance in erythroid lineage commitment, terminal erythropoiesis progression and the switching of globin genes from γ to β. The role of KLF1 in haemoglobin switching is exerted by the direct activation of β-globin gene and by the silencing of γ-globin through activation of BCL11A, an important γ-globin gene repressor. The link between KLF1 and γ-globin silencing identifies this transcription factor as a possible therapeutic target for β-hemoglobinopathies. Moreover, several mutations have been identified in the human genes that are responsible for various benign phenotypes and erythroid disorders. The study of the phenotype associated with each mutation has greatly contributed to the current understanding of the complex role of KLF1 in erythropoiesis. This review will focus on some of the principal functions of KLF1 on erythroid cell commitment and differentiation, spanning from primitive to definitive erythropoiesis. The fundamental role of KLF1 in haemoglobin switching will be also highlighted. Finally, an overview of the principal human mutations and relative phenotypes and disorders will be described.
... Thus, for single cell sequencing, we decided to apply an isolation protocol using magnetic nanoparticles that usually generates healthier cells without compromising on purity ( Figure 1). To reduce erythroid contamination in the final isolated F4/80+ macrophage population, during cell homogenization, we used a peptide that specifically inhibits the ICAM4/αv interactions that form the core of erythroblast-macrophage adhesive junctions (Xue et al., 2014). The EZ-Sep magnetic nanoparticle isolation kit from Cell Signaling Technologies utilizes a selection reagent that binds to phycoerythrin (PE) conjugated to a primary antibody such as anti-F4/80. ...
... However, since the EBI macrophage forms tight adhesive interactions with the maturing erythroblasts, removing erythroblasts from isolated macrophage populations is critical to the obtention of a pure population of macrophages for analysis.Earlier efforts to isolate macrophages from hematopoietic tissues relied on using the macrophagearticle is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). surface protein F4/80 as a marker for FACS-based methods(Xue et al., 2014; ...
In vivo erythropoiesis occurs in the erythroblast island niche (EBI), comprising of a central macrophage that attaches to and aids the maturation of erythroid progenitors into mature reticulocytes. Macrophages in hematopoietic tissue such as embryonic fetal liver are heterogeneous and express the cell surface protein F4/80. Earlier methods of isolating F4/80+ macrophages from hematopoietic tissue relied on FACS sorting, but the relatively low numbers of F4/80+ cells obtained after FACS sometimes led to poor RNA quality. Additionally, since EBI macrophages are attached to erythroblasts, care must be taken to avoid contamination with bound erythroblasts. We have developed a novel method for isolating F4/80+ cells from E13.5 mouse fetal liver using magnetic nanoparticles, which can be performed on the lab bench. During cell suspension and homogenization, we also add a peptide that disrupts erythroid macrophage interactions and generates F4/80+ single cells free of erythroid contamination. Thus, our protocol generates a population enriched in F4/80+ cells that are healthy and ready for sensitive techniques such as single cell sequencing.
... This was partly due to uncertainty regarding cell surface markers that would specifically enrich for the subpopulation of macrophages that form the EBI niche (Seu et al., 2017;Tay et al., 2020). F4/80 antigen expression is associated with EBI macrophages (Sadahira et al., 1991;Manwani and Bieker, 2008) although not all F4/80+ macrophages in hematopoietic tissues necessarily form EBIs. Nevertheless, recent studies have used F4/80 in addition to surrogate markers such as pEKLF/GFP or Epor-eGFP to enrich for EBI macrophage subpopulations from primary mouse hematopoietic tissue and have determined global gene expression at the bulk (Xue et al., 2014;Li et al., 2019;Mukherjee et al., 2021) and single cell level (Mukherjee et al., 2021). These studies have uncovered novel aspects of EBI macrophage identity, heterogeneity, and physiological function while reinforcing certain existing paradigms. ...
... Although EKLF/Klf1 is mostly known for its tissue-restricted, global transcriptional role in facilitating maturation and differentiation of proerythroblasts into mature reticulocytes (Tallack and Perkins, 2010;Siatecka and Bieker, 2011;Gnanapragasam and Bieker, 2017) and in fetal to adult globin switching (Bieker, 2010;Perkins et al., 2016), cellular analyses show that EKLF is also critical for the integrity of EB islands (Xue et al., 2014). Consequently, EBIs isolated from EKLF −/− mouse fetal livers (FL) exhibit quite a few aberrations; for example, there are overall fewer F4/80+ macrophages (Mukherjee et al., 2021), and isolated EB islands have severely altered morphology compared to EKLF +/+ FL (Xue et al., 2014). ...
... Although EKLF/Klf1 is mostly known for its tissue-restricted, global transcriptional role in facilitating maturation and differentiation of proerythroblasts into mature reticulocytes (Tallack and Perkins, 2010;Siatecka and Bieker, 2011;Gnanapragasam and Bieker, 2017) and in fetal to adult globin switching (Bieker, 2010;Perkins et al., 2016), cellular analyses show that EKLF is also critical for the integrity of EB islands (Xue et al., 2014). Consequently, EBIs isolated from EKLF −/− mouse fetal livers (FL) exhibit quite a few aberrations; for example, there are overall fewer F4/80+ macrophages (Mukherjee et al., 2021), and isolated EB islands have severely altered morphology compared to EKLF +/+ FL (Xue et al., 2014). Thus, our previous understanding of EKLF's role in erythropoiesis as being restricted to the erythroid compartment required reassessment for which, firstly, EKLF expression in F4/80+ macrophages had to be directly and clearly demonstrated. ...
During definitive erythropoiesis, maturation of erythroid progenitors into enucleated reticulocytes requires the erythroblastic island (EBI) niche comprising a central macrophage attached to differentiating erythroid progenitors. Normally, the macrophage provides a nurturing environment for maturation of erythroid cells. Its critical physiologic importance entails aiding in recovery from anemic insults, such as systemic stress or acquired disease. Considerable interest in characterizing the central macrophage of the island niche led to the identification of putative cell surface markers enriched in island macrophages, enabling isolation and characterization. Recent studies focus on bulk and single cell transcriptomics of the island macrophage during adult steady-state erythropoiesis and embryonic erythropoiesis. They reveal that the island macrophage is a distinct cell type but with widespread cellular heterogeneity, likely suggesting distinct developmental origins and biological function. These studies have also uncovered transcriptional programs that drive gene expression in the island macrophage. Strikingly, the master erythroid regulator EKLF/Klf1 seems to also play a major role in specifying gene expression in island macrophages, including a putative EKLF/Klf1-dependent transcription circuit. Our present review and analysis of mouse single cell genetic patterns suggest novel expression characteristics that will enable a clear enrichment of EBI subtypes and resolution of island macrophage heterogeneity. Specifically, the discovery of markers such as Epor, and specific features for EKLF/Klf1-expressing island macrophages such as Sptb and Add2, or for SpiC-expressing island macrophage such as Timd4, or for Maf/Nr1h3-expressing island macrophage such as Vcam1, opens exciting possibilities for further characterization of these unique macrophage cell types in the context of their critical developmental function.
... Numerous research efforts have been made in identifying the possible proteins expressed by EBI macrophages, such as erythroblast macrophage protein (EMP), vascular cell adhesion molecule-1 (VCAM1), erythroid Krüppel-like factor 1 (KLF1), and DNase2α, among others (Soni et al., 2006;Porcu et al., 2011;Xue et al., 2014). However, the mechanism through which EBI macrophages regulate normal erythropoiesis and erythroid hematopoietic disorders remains unknown, owing to a lack of accurate phenotypic profiles of these macrophages. ...
Erythroblastic islands (EBIs), discovered more than 60 years ago, are specialized microenvironments for erythropoiesis. This island consists of a central macrophage with surrounding developing erythroid cells. EBI macrophages have received intense interest in the verifications of the supporting erythropoiesis hypothesis. Most of these investigations have focused on the identification and functional analyses of EBI macrophages, yielding significant progresses in identifying and isolating EBI macrophages, as well as verifying the potential roles of EBI macrophages in erythropoiesis. EBI macrophages express erythropoietin receptor (Epor) both in mouse and human, and Epo acts on both erythroid cells and EBI macrophages simultaneously in the niche, thereby promoting erythropoiesis. Impaired Epor signaling in splenic niche macrophages significantly inhibit the differentiation of stress erythroid progenitors. Moreover, accumulating evidence suggests that EBI macrophage dysfunction may lead to certain erythroid hematological disorders. In this review, the heterogeneity, identification, and functions of EBI macrophages during erythropoiesis under both steady-state and stress conditions are outlined. By reviewing the historical data, we discuss the influence of EBI macrophages on erythroid hematopoietic disorders and propose a new hypothesis that erythroid hematopoietic disorders are driven by EBI macrophages.
... it directly activates Icam4 in the erythroid compartment, and activates Vcam1 in the macrophage 78 compartment (Xue et al., 2014). Together, Icam4 and Vcam1 enable a two-pronged adhesive intercellular 79 interaction to occur with their respective integrin partners on the opposite cell type. ...
Erythroblastic islands are a specialized niche that contain a central macrophage surrounded by erythroid cells at various stages of maturation. However, identifying the precise genetic and transcriptional control mechanisms in the island macrophage remains difficult due to macrophage heterogeneity. Using unbiased global sequencing and directed genetic approaches focused on early mammalian development, we find that fetal liver macrophage exhibit a unique expression signature that differentiates them from erythroid and adult macrophage cells. The importance of EKLF/KLF1 in this identity is shown by expression analyses in EKLF-/- and in EKLF-marked macrophage cells. Single cell sequence analysis simplifies heterogeneity and identifies clusters of genes important for EKLF-dependent macrophage function and novel cell surface biomarkers. Remarkably, this singular set of macrophage island cells appears transiently during embryogenesis. Together these studies provide a detailed perspective on the importance of EKLF in establishment of the dynamic gene expression network within erythroblastic islands in the developing embryo and provide the means for their efficient isolation.
... It was particularly interesting to note that expression of Dnase2a in EBI macrophages is regulated by KLF1 [7,83], a transcription factor that was first identified as a master regulator within the erythroid lineage, with an important role in regulating the later stages of RBC production, including globin switching to the activation of erythroid-specific genes [84−87]. Adding to its critical intrinsic role, an extrinsic role for KLF1 in the macrophage compartment of the erythroblastic island niche was first implicated using a KLF1-eGFP reporter mouse strain where GFP was detected in EBI macrophages and genes associated with island integrity such as VCAM1 were expressed at a higher level in KLF1-GFP + macrophages [8,88,89]. ...
... Macrophages expressing higher levels of KLF1 were better able to promote erythroblast maturation, resulting in an increase in enucleated RBCs in the culture [8]. The use of this KLF1 expression system therefore supported previous findings of an extrinsic role for KLF1 in EBI macrophages [8,89]. ...
Erythropoiesis is one of the most demanding processes in the body, with over two million red blood cells produced every second. Multiple hereditary and acquired red blood cell disorders arise from this complex system, with existing treatments effective in managing some of these conditions but few offering a long-term cure. Finding new treatments relies on the full understanding of the cellular and molecular interactions associated with the production and maturation of red blood cells, which takes place within the erythroblastic island niche. The elucidation of processes associated within the erythroblastic island niche in health and during stress erythropoiesis has relied on in vivo modelling in mice, with complexities dissected using simple in vitro systems. Recent progress using state of the art stem cell technology and gene editing has enabled a more detailed study of the human niche. Here, we review these different models and describe how they have been used to identify and characterise the cellular and molecular pathways associated with red blood cell production and maturation. We speculate that these systems could be applied to modelling red blood cell diseases and finding new druggable targets, which would prove especially useful for patients resistant to existing treatments. These models could also aid in research into the manufacturing of red blood cells in vitro to replace donor blood transfusions, which is the most common treatment of blood disorders.
... These components are all interconnected: the complement protein network, an essential component of the innate and humoural immune system [76], participates in several non-inflammatory processes, such as coagulation [77], along with components of the coagulation and fibrinolysis systems [76]. Genes related to erythrocyte differentiation were also observed, such as the erythroid transcription factor KLF1 (also known as EKLF), which is expressed by erythro-myeloid progenitors (EMPs) [78], erythroid cells [79], and megakaryocyte-erythrocyte progenitors (MEPs) [79]. Regarding the use of the primary jaw joint, the marsupial neonate approximates the embryonic condition of mammalian ancestors [80]. ...
Background:
The white-eared opossum (Didelphis albiventris) is widely distributed throughout Brazil and South America. It has been used as an animal model for studying different scientific questions ranging from the restoration of degraded green areas to medical aspects of Chagas disease, leishmaniasis and resistance against snake venom. As a marsupial, D. albiventris can also contribute to the understanding of the molecular mechanisms that govern the different stages of organogenesis. Opossum joeys are born after only 13 days, and the final stages of organogenesis occur when the neonates are inside the pouch, depending on lactation. As neither the genome of this opossum species nor its transcriptome has been completely sequenced, the use of D. albiventris as an animal model is limited. In this work, we sequenced the D. albiventris transcriptome by RNA-seq to obtain the first catalogue of differentially expressed (DE) genes and gene ontology (GO) annotations during the neonatal stages of marsupial development.
Results:
The D. albiventris transcriptome was obtained from whole neonates harvested at birth (P0), at 5 days of age (P5) and at 10 days of age (P10). The de novo assembly of these transcripts generated 85,338 transcripts. Approximately 30% of these transcripts could be mapped against the amino acid sequences of M. domestica, the evolutionarily closest relative of D. albiventris to be sequenced thus far. Among the expressed transcripts, 2077 were found to be DE between P0 and P5, 13,780 between P0 and P10, and 1453 between P5 and P10. The enriched GO terms were mainly related to the immune system, blood tissue development and differentiation, vision, hearing, digestion, the CNS and limb development.
Conclusions:
The elucidation of opossum transcriptomes provides an out-group for better understanding the distinct characteristics associated with the evolution of mammalian species. This study provides the first transcriptome sequences and catalogue of genes for a marsupial species at different neonatal stages, allowing the study of the mechanisms involved in organogenesis.
... These components are all interconnected: the complement protein network, an essential component of the innate and humoural immune system [90], participates in several non-in ammatory processes, such as coagulation [91], along with components of the coagulation and brinolysis systems [90]. Genes related to erythrocyte differentiation were also observed, such as the erythroid transcription factor KLF1 (also known as EKLF), which is expressed by erythro-myeloid progenitors (EMPs) [92], erythroid cells [93], and megakaryocyte-erythrocyte progenitors (MEPs) [93]. ...
Background
The white-eared opossum (Didelphis albiventris) is widely distributed throughout Brazil and South America. It has been used as an animal model for studying different scientific questions ranging from the restoration of degraded green areas to medical aspects of Chagas disease, leishmaniasis and resistance against snake venom. As a marsupial, D. albiventris can also contribute to the understanding of the molecular mechanisms that govern the different stages of organogenesis. Opossum joeys are born after only 13 days, and the final stages of organogenesis occur when the neonates are inside the pouch, depending on lactation. As neither the genome of this opossum species nor its transcriptome has been completely sequenced, the use of D. albiventris as an animal model is limited. In this work, we sequenced the D. albiventris transcriptome by RNA-seq to obtain the first catalogue of differentially expressed (DE) genes and gene ontology (GO) annotations during the neonatal stages of marsupial development.ResultsThe D. albiventris transcriptome was obtained from whole neonates harvested at birth (P0), at five days of age (P5) and at ten days of age (P10). The de novo assembly of these transcripts generated 85,338 transcripts. Approximately 30% of these transcripts could be mapped against the amino acid sequences of M. domestica, the evolutionarily closest relative of D. albiventris to be sequenced thus far. Among the expressed transcripts, 2,077 were found to be DE between P0 and P5, 13,780 between P0 and P10, and 1,453 between P5 and P10. The enriched GO terms were mainly related to the immune system, blood tissue development and differentiation, vision, hearing, digestion, the CNS and limb development.Conclusions
The elucidation of opossum transcriptomes provides an out-group for better understanding the distinct characteristics associated with the evolution of mammalian species. This study provides the first transcriptome sequences and catalogue of genes for a marsupial species at different neonatal stages, allowing the study of the mechanisms involved in organogenesis.
