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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Developing immortal cell lines from Xenopus embryos , four novel cell lines derived from Xenopus tropicalis
The diploid anuran Xenopus tropicalis has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10 and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10 and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30°C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO 2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus .
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- Award ID(s):
- 1645105
- NSF-PAR ID:
- 10394959
- Date Published:
- Journal Name:
- Open Biology
- Volume:
- 12
- Issue:
- 7
- ISSN:
- 2046-2441
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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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Abstract Scalable processes are requisite for the robust biomanufacturing of human pluripotent stem cell (hPSC)‐derived therapeutics. Toward this end, we demonstrate the xeno‐free expansion and directed differentiation of human embryonic and induced pluripotent stem cells to definitive endoderm (DE) in a controlled stirred suspension bioreactor (SSB). Based on previous work on converting hPSCs to insulin‐producing progeny, differentiation of two hPSC lines was optimized in planar cultures yielding up to 87% FOXA2+/SOX17+cells. Next, hPSCs were propagated in an SSB with controlled pH and dissolved oxygen. Cultures displayed a 10‐ to 12‐fold increase in cell number over 5–6 days with the maintenance of pluripotency (>85% OCT4+) and viability (>85%). For differentiation, SSB cultures yielded up to 89% FOXA2+/SOX17+cells or ~ 8 DE cells per seeded hPSC. Specification to DE cell fate was consistently more efficient in the bioreactor compared to planar cultures. Hence, a tunable strategy is established that is suitable for the xeno‐free manufacturing of DE cells from different hPSC lines in scalable SSBs. This study advances bioprocess development for producing a wide gamut of human DE cell‐derived therapeutics.
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Across phyla, males often produce species-specific vocalizations to attract females. Although understanding the neural mechanisms underlying behavior has been challenging in vertebrates, we previously identified two anatomically distinct central pattern generators (CPGs) that drive the fast and slow clicks of male
Xenopus laevis, using an ex vivo preparation that produces fictive vocalizations. Here, we extended this approach to four additional species,X. amieti, X. cliivi, X. petersii, and X. tropicalis, by developing ex vivo brain preparation from which fictive vocalizations are elicited in response to a chemical or electrical stimulus. We found that even though the courtship calls are species-specific, the CPGs used to generate clicks are conserved across species. The fast CPGs, which critically rely on reciprocal connections between the parabrachial nucleus and the nucleus ambiguus, are conserved among fast-click species, and slow CPGs are shared among slow-click species. In addition, our results suggest that testosterone plays a role in organizing fast CPGs in fast-click species, but not in slow-click species. Moreover, fast CPGs are not inherited by all species but monopolized by fast-click species. The results suggest that species-specific calls of the genusXenopus have evolved by utilizing conserved slow and/or fast CPGs inherited by each species.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1098/rsob.220089
