Across vertebrates, hematopoiesis takes place within designated tissues, wherein committed myeloid progenitors further differentiate toward cells with megakaryocyte/erythroid potential (MEP) or those with granulocyte/macrophage potential (GMP). While the liver periphery (LP) of the Xenopus laevis amphibian functions as a principal site of hematopoiesis and contains MEPs, cells with GMP potential are instead segregated to the bone marrow (BM) of this animal. Presently, using gene expression and western blot analyses of blood cell lineage-specific transcription factors, we confirmed that while the X. laevis LP hosts hematopoietic stem cells and MEPs, their BM contains GMPs. In support of our hypothesis that cells bearing GMP potential originate from the frog LP and migrate through blood circulation to the BM in response to chemical cues; we demonstrated that medium conditioned by the X. laevis BM chemoattracts LP and peripheral blood cells. Compared to LP and by examining a comprehensive panel of chemokine genes, we showed that the X. laevis BM possessed greater expression of a single chemokine, CXCL12, the recombinant form of which was chemotactic to LP and peripheral blood cells and appeared to be a major chemotactic component within BM-conditioned medium. In confirmation of the hepatic origin of the cells that give rise to these frogs' GMPs, we also demonstrated that the X. laevis BM supported the growth of their LP-derived cells.
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Evolution and Potential Subfunctionalization of Duplicated fms -Related Class III Receptor Tyrosine Kinase flt3 s and Their Ligands in the Allotetraploid Xenopus laevis
Abstract The fms-related tyrosine kinase 3 (Flt3) and its ligand (Flt3lg) are important regulators of hematopoiesis and dendritic cell (DC) homeostasis with unsettled coevolution. Gene synteny and deduced amino acid sequence analyses identified conserved flt3 gene orthologs across all jawed vertebrates. In contrast, flt3lg orthologs were not retrieved in ray-finned fish, and the gene locus exhibited more variability among species. Interestingly, duplicated flt3/flt3lg genes were maintained in the allotetraploid Xenopus laevis. Comparison of modeled structures of X. laevis Flt3 and Flt3lg homoeologs with the related diploid Xenopus tropicalis and with humans indicated a higher conformational divergence between the homoeologous pairs than their respective counterparts. The distinctive developmental and tissue expression patterns of Flt3 and Flt3lg homoeologs in tadpoles and adult frogs suggest a subfunctionalization of these homoeologs. To characterize Flt3 cell surface expression, X. laevis–tagged rFlt3lg.S and rFlt3lg.L were produced. Both rFlt3lg.S and rFlt3lg.L bind in vitro Flt3.S and Flt3.L and can trigger Erk1/2 signaling, which is consistent with a partial overlapping function between homoeologs. In spleen, Flt3.S/L cell surface expression was detected on a fraction of B cells and a population of MHC class IIhigh/CD8+ leukocytes phenotypically similar to the recently described dual follicular/conventional DC-like XL cells. Our result suggests that 1) Flt3lg.S and Flt3lg.L are both involved in XL cell homeostasis and that 2) XL cells have hematopoietic origin. Furthermore, we detected surface expression of the macrophage/monocyte marker Csf1r.S on XL cells as in mammalian and chicken DCs, which points to a common evolutionary origin in vertebrate DCs.
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- Award ID(s):
- 1754274
- NSF-PAR ID:
- 10406926
- Date Published:
- Journal Name:
- The Journal of Immunology
- Volume:
- 209
- Issue:
- 5
- ISSN:
- 0022-1767
- Page Range / eLocation ID:
- 960 to 969
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Background The mammalian guanine deaminase (GDA), called cypin, is important for proper neural development, by regulating dendritic arborization through modulation of microtubule (MT) dynamics. Additionally, cypin can promote MT assembly in vitro. However, it has never been tested whether cypin (or other GDA orthologs) binds to MTs or modulates MT dynamics. Here, we address these questions and characterize
Xenopus laevis Results We find that exogenously expressed human cypin and Gda both display a cytosolic distribution in primary embryonic cells. Furthermore, while expression of human cypin can promote MT polymerization,
Xenopus Gda has no effect. Additionally, we find that the tubulin‐binding collapsin response mediator protein (CRMP) homology domain is only partially conserved between cypin and Gda. This likely explains the divergence in function, as we discovered that the cypin region containing the CRMP homology and PDZ‐binding domain is necessary for regulating MT dynamics. Finally, we observed thatgda is strongly expressed in the kidneys during late embryonic development, although it does not appear to be critical for kidney development.Conclusions Together, these results suggest that GDA has diverged in function between mammals and amphibians, and that mammalian GDA plays an indirect role in regulating MT dynamics. Developmental Dynamics 248:296–305, 2019. © 2019 Wiley Periodicals, Inc.
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Abstract Egg extracts of the African clawed frog
Xenopus laevis have provided a cell‐free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog,Xenopus tropicalis , which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog,Xenopus borealis , which is intermediate in size between the two species, but more recently diverged in evolution fromX. laevis thanX. tropicalis .X. borealis eggs were slightly smaller than those ofX. laevis , and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of theX. borealis spindles differed from bothX. laevis andX. tropicalis . Extract mixing experiments revealed common scaling phenomena amongXenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate thatX. borealis spindles possess bothX. laevis andX. tropicalis features. Thus,X. borealis egg extract provides a thirdin vitro system to investigate interspecies scaling and spindle morphometric variation. -
In situ capillary microsampling with capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS) enabled the characterization of cationic metabolites in single cells in complex tissues and organisms. For deeper coverage of the metabolome and metabolic networks, analytical approaches are needed that provide complementary detection for anionic metabolites, ideally using the same instrumentation. Described here is one such approach that enables sequential cationic and anionic (dual) analysis of metabolites in the same identified cell in a live vertebrate embryo. A calibrated volume was microaspirated from the animal-ventral cell in a live 8-cell embryo of Xenopus laevis , and cationic and anionic metabolites were one-pot microextracted from the aspirate, followed by CE-ESI-MS analysis of the same extract. A laboratory-built CE-ESI interface was reconfigured to enable dual cationic–anionic analysis with ∼5–10 nM (50–100 amol) lower limit of detection and a capability for quantification. To provide robust separation and efficient ion generation, the CE-ESI interface was enclosed in a nitrogen gas filled chamber, and the operational parameters were optimized for the cone-jet spraying regime in both the positive and negative ion mode. A total of ∼250 cationic and ∼200 anionic molecular features were detected from the cell between m / z 50–550, including 60 and 24 identified metabolites, respectively. With only 11 metabolites identified mutually, the duplexed approach yielded complementary information on metabolites produced in the cell, which in turn deepened network coverage for several metabolic pathways. With scalability to smaller cells and adaptability to other types of tissues and organisms, dual cationic–anionic detection with in situ microprobe CE-ESI-MS opens a door to better understand cell metabolism.more » « less
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Summary The chromodomain family member chromodomain 1 (CHD1) has been shown to have numerous critical molecular functions including transcriptional regulation, splicing, and DNA repair. Complete loss of function of this gene is not compatible with life. On the other hand, missense and copy number variants of
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.4049/jimmunol.2200201
