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spib is required for primitive myeloid development in Xenopus.

Costa RM, Soto X, Chen Y, Zorn AM, Amaya E - Blood (2008)

Bottom Line: Furthermore, we isolated spib, an ETS transcription factor, specifically expressed in primitive myeloid precursors.Using spib antisense morpholino knockdown experiments, we show that spib is required for myeloid specification, and, in its absence, primitive myeloid cells retain hemangioblast-like characteristics and fail to migrate.Thus, we conclude that spib sits at the top of the known genetic hierarchy that leads to the specification of primitive myeloid cells in amphibians.

View Article: PubMed Central - PubMed

Affiliation: The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom. ricardo.costa@manchester.ac.uk

ABSTRACT
Vertebrate blood formation occurs in 2 spatially and temporally distinct waves, so-called primitive and definitive hematopoiesis. Although definitive hematopoiesis has been extensively studied, the development of primitive myeloid blood has received far less attention. In Xenopus, primitive myeloid cells originate in the anterior ventral blood islands, the equivalent of the mammalian yolk sac, and migrate out to colonize the embryo. Using fluorescence time-lapse video microscopy, we recorded the migratory behavior of primitive myeloid cells from their birth. We show that these cells are the first blood cells to differentiate in the embryo and that they are efficiently recruited to embryonic wounds, well before the establishment of a functional vasculature. Furthermore, we isolated spib, an ETS transcription factor, specifically expressed in primitive myeloid precursors. Using spib antisense morpholino knockdown experiments, we show that spib is required for myeloid specification, and, in its absence, primitive myeloid cells retain hemangioblast-like characteristics and fail to migrate. Thus, we conclude that spib sits at the top of the known genetic hierarchy that leads to the specification of primitive myeloid cells in amphibians.

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Primitive myeloid migration time-lapse video microscopy. (A) Experimental setup, stage-14 to -16 anterior ventral blood islands were transplanted from microruby-injected to noninjected embryos. Transplanted cells become migratory and leave the transplant to colonize the embryo in 4 to 8 hours. Throughout this period, primitive myeloid cells show different behaviors (Videos S1–S3). (B,C) Stage-18 brightfield/fluorescent composite of transplanted embryos. (D,E) Stills from supplementary movies. (Panel D and Video S2) Ventral view of primitive myeloid cells leaving the transplanted aVBI with “blebbing” behavior and low migratory speeds. (Panel E and Video S3) Primitive myeloid cells leaving the transplanted aVBI (stage 26, lateral view). At this stage, cells acquire elongated cell morphology and higher motility. Large dashed line shows the embryo contour, and the light dashed square shows the enlarged region shown in still frames. Colored arrowheads point and track the same cell. (D,E) Anterior view is shown to the left; dorsal, to the top. Time is shown in minutes. Images in panels B and C were obtained on a fluorescence stereoscope Leica MZ FLIII (Wetzlar, Germany) attached to a Sony CCD camera DXC-950 image capture system controlled by Northern Eclipse software 7.0 (Empix Imaging, Mississauga, ON). For panels D and E, the same image capture system was attached to an Olympus IX70 inverted fluorescent microscope; 0.1× MMR was used as imaging medium. (D) Total magnification 300× objective (Olympus LCPlan 20×/0.4 NA). (E) Total magnification 40× objective (Olympus UPlanFL 4×/0.13 NA). Photoshop CS2 (Adobe Systems, San Jose, CA) or ImageJ 1.38 (National Institutes of Health, Bethesda, MD) were used for image or time-lapse video processing.
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Figure 2: Primitive myeloid migration time-lapse video microscopy. (A) Experimental setup, stage-14 to -16 anterior ventral blood islands were transplanted from microruby-injected to noninjected embryos. Transplanted cells become migratory and leave the transplant to colonize the embryo in 4 to 8 hours. Throughout this period, primitive myeloid cells show different behaviors (Videos S1–S3). (B,C) Stage-18 brightfield/fluorescent composite of transplanted embryos. (D,E) Stills from supplementary movies. (Panel D and Video S2) Ventral view of primitive myeloid cells leaving the transplanted aVBI with “blebbing” behavior and low migratory speeds. (Panel E and Video S3) Primitive myeloid cells leaving the transplanted aVBI (stage 26, lateral view). At this stage, cells acquire elongated cell morphology and higher motility. Large dashed line shows the embryo contour, and the light dashed square shows the enlarged region shown in still frames. Colored arrowheads point and track the same cell. (D,E) Anterior view is shown to the left; dorsal, to the top. Time is shown in minutes. Images in panels B and C were obtained on a fluorescence stereoscope Leica MZ FLIII (Wetzlar, Germany) attached to a Sony CCD camera DXC-950 image capture system controlled by Northern Eclipse software 7.0 (Empix Imaging, Mississauga, ON). For panels D and E, the same image capture system was attached to an Olympus IX70 inverted fluorescent microscope; 0.1× MMR was used as imaging medium. (D) Total magnification 300× objective (Olympus LCPlan 20×/0.4 NA). (E) Total magnification 40× objective (Olympus UPlanFL 4×/0.13 NA). Photoshop CS2 (Adobe Systems, San Jose, CA) or ImageJ 1.38 (National Institutes of Health, Bethesda, MD) were used for image or time-lapse video processing.

Mentions: To analyze the initial steps of primitive myeloid cell migration, we performed homotypic transplantations of aVBIs from microruby-injected embryos into stage-14 and -16 host embryos. This allowed us to observe the migration of primitive myeloid cells away from the aVBI, using time-lapse fluorescence video microscopy (Figure 2). By stages 19 and 20, migration of microruby-positive cells was observed as single individual cells leaving the transplant (Figure 2D,E; Videos S1,S2, available on the Blood website; see the Supplemental Materials link at the top of the online article). The overall morphology and cell population behavior throughout the colonization process was very dynamic. It started with a “blebbing” cell behavior with low motility as cells left the aVBI, changing to a behavior with higher protrusive activity and low migratory speeds (Videos S1,S2). As the distance from the aVBI increased the cells changed to a highly motile bipolar behavior, which allowed the cells to colonize the whole embryo within 4 to 8 hours (Figure 2E; Video S3). Once that process was completed, a third phase ensued as isolated primitive myeloid cells began to patrol the embryo, coincident with less protrusive activity in the cells. Throughout these migratory behaviors, primitive myeloid cells were capable of undergoing cell divisions and some were phagocytic (data not shown). In summary, the behavior of these cells is consistent with the previously described Xenopus primitive macrophages or zebrafish primitive macrophages and neutrophils.27,31–34


spib is required for primitive myeloid development in Xenopus.

Costa RM, Soto X, Chen Y, Zorn AM, Amaya E - Blood (2008)

Primitive myeloid migration time-lapse video microscopy. (A) Experimental setup, stage-14 to -16 anterior ventral blood islands were transplanted from microruby-injected to noninjected embryos. Transplanted cells become migratory and leave the transplant to colonize the embryo in 4 to 8 hours. Throughout this period, primitive myeloid cells show different behaviors (Videos S1–S3). (B,C) Stage-18 brightfield/fluorescent composite of transplanted embryos. (D,E) Stills from supplementary movies. (Panel D and Video S2) Ventral view of primitive myeloid cells leaving the transplanted aVBI with “blebbing” behavior and low migratory speeds. (Panel E and Video S3) Primitive myeloid cells leaving the transplanted aVBI (stage 26, lateral view). At this stage, cells acquire elongated cell morphology and higher motility. Large dashed line shows the embryo contour, and the light dashed square shows the enlarged region shown in still frames. Colored arrowheads point and track the same cell. (D,E) Anterior view is shown to the left; dorsal, to the top. Time is shown in minutes. Images in panels B and C were obtained on a fluorescence stereoscope Leica MZ FLIII (Wetzlar, Germany) attached to a Sony CCD camera DXC-950 image capture system controlled by Northern Eclipse software 7.0 (Empix Imaging, Mississauga, ON). For panels D and E, the same image capture system was attached to an Olympus IX70 inverted fluorescent microscope; 0.1× MMR was used as imaging medium. (D) Total magnification 300× objective (Olympus LCPlan 20×/0.4 NA). (E) Total magnification 40× objective (Olympus UPlanFL 4×/0.13 NA). Photoshop CS2 (Adobe Systems, San Jose, CA) or ImageJ 1.38 (National Institutes of Health, Bethesda, MD) were used for image or time-lapse video processing.
© Copyright Policy - creativecommons
Related In: Results  -  Collection

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Figure 2: Primitive myeloid migration time-lapse video microscopy. (A) Experimental setup, stage-14 to -16 anterior ventral blood islands were transplanted from microruby-injected to noninjected embryos. Transplanted cells become migratory and leave the transplant to colonize the embryo in 4 to 8 hours. Throughout this period, primitive myeloid cells show different behaviors (Videos S1–S3). (B,C) Stage-18 brightfield/fluorescent composite of transplanted embryos. (D,E) Stills from supplementary movies. (Panel D and Video S2) Ventral view of primitive myeloid cells leaving the transplanted aVBI with “blebbing” behavior and low migratory speeds. (Panel E and Video S3) Primitive myeloid cells leaving the transplanted aVBI (stage 26, lateral view). At this stage, cells acquire elongated cell morphology and higher motility. Large dashed line shows the embryo contour, and the light dashed square shows the enlarged region shown in still frames. Colored arrowheads point and track the same cell. (D,E) Anterior view is shown to the left; dorsal, to the top. Time is shown in minutes. Images in panels B and C were obtained on a fluorescence stereoscope Leica MZ FLIII (Wetzlar, Germany) attached to a Sony CCD camera DXC-950 image capture system controlled by Northern Eclipse software 7.0 (Empix Imaging, Mississauga, ON). For panels D and E, the same image capture system was attached to an Olympus IX70 inverted fluorescent microscope; 0.1× MMR was used as imaging medium. (D) Total magnification 300× objective (Olympus LCPlan 20×/0.4 NA). (E) Total magnification 40× objective (Olympus UPlanFL 4×/0.13 NA). Photoshop CS2 (Adobe Systems, San Jose, CA) or ImageJ 1.38 (National Institutes of Health, Bethesda, MD) were used for image or time-lapse video processing.
Mentions: To analyze the initial steps of primitive myeloid cell migration, we performed homotypic transplantations of aVBIs from microruby-injected embryos into stage-14 and -16 host embryos. This allowed us to observe the migration of primitive myeloid cells away from the aVBI, using time-lapse fluorescence video microscopy (Figure 2). By stages 19 and 20, migration of microruby-positive cells was observed as single individual cells leaving the transplant (Figure 2D,E; Videos S1,S2, available on the Blood website; see the Supplemental Materials link at the top of the online article). The overall morphology and cell population behavior throughout the colonization process was very dynamic. It started with a “blebbing” cell behavior with low motility as cells left the aVBI, changing to a behavior with higher protrusive activity and low migratory speeds (Videos S1,S2). As the distance from the aVBI increased the cells changed to a highly motile bipolar behavior, which allowed the cells to colonize the whole embryo within 4 to 8 hours (Figure 2E; Video S3). Once that process was completed, a third phase ensued as isolated primitive myeloid cells began to patrol the embryo, coincident with less protrusive activity in the cells. Throughout these migratory behaviors, primitive myeloid cells were capable of undergoing cell divisions and some were phagocytic (data not shown). In summary, the behavior of these cells is consistent with the previously described Xenopus primitive macrophages or zebrafish primitive macrophages and neutrophils.27,31–34

Bottom Line: Furthermore, we isolated spib, an ETS transcription factor, specifically expressed in primitive myeloid precursors.Using spib antisense morpholino knockdown experiments, we show that spib is required for myeloid specification, and, in its absence, primitive myeloid cells retain hemangioblast-like characteristics and fail to migrate.Thus, we conclude that spib sits at the top of the known genetic hierarchy that leads to the specification of primitive myeloid cells in amphibians.

View Article: PubMed Central - PubMed

Affiliation: The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom. ricardo.costa@manchester.ac.uk

ABSTRACT
Vertebrate blood formation occurs in 2 spatially and temporally distinct waves, so-called primitive and definitive hematopoiesis. Although definitive hematopoiesis has been extensively studied, the development of primitive myeloid blood has received far less attention. In Xenopus, primitive myeloid cells originate in the anterior ventral blood islands, the equivalent of the mammalian yolk sac, and migrate out to colonize the embryo. Using fluorescence time-lapse video microscopy, we recorded the migratory behavior of primitive myeloid cells from their birth. We show that these cells are the first blood cells to differentiate in the embryo and that they are efficiently recruited to embryonic wounds, well before the establishment of a functional vasculature. Furthermore, we isolated spib, an ETS transcription factor, specifically expressed in primitive myeloid precursors. Using spib antisense morpholino knockdown experiments, we show that spib is required for myeloid specification, and, in its absence, primitive myeloid cells retain hemangioblast-like characteristics and fail to migrate. Thus, we conclude that spib sits at the top of the known genetic hierarchy that leads to the specification of primitive myeloid cells in amphibians.

Show MeSH
Related in: MedlinePlus