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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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Effect of spib knockdown in primitive myeloid progenitors. (A-I) Primitive macrophages defined by the expression of spi1 and mmp7 are absent from spib-depleted embryos. (J-L,O-Q,T-V) WMISH analysis of aVBI in spib morphants; at the earliest point we can identify a pool of primitive myeloid progenitors by the coexpression of spib, mpo, and cebpa. (M,R,W) Quantification of the number of embryos that express spib, mpo, and cebpa and (N,S,X) size of primitive myeloid progenitor pool, by the area of tissue-expressing progenitor markers. Error bars represent the SD of the number of embryos analyzed (n). (O) Amount of tissue was calculated using area ratios of a and a′. Similar results were obtained in either ATG or e1i1 morphants and are in contrast with the uninjected and ATG mismatch control group (CTL). Student t test P values lower than .001 are marked as * and considered statistically significant; P values higher than .05 are not statistically significant and marked as ***.
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Figure 6: Effect of spib knockdown in primitive myeloid progenitors. (A-I) Primitive macrophages defined by the expression of spi1 and mmp7 are absent from spib-depleted embryos. (J-L,O-Q,T-V) WMISH analysis of aVBI in spib morphants; at the earliest point we can identify a pool of primitive myeloid progenitors by the coexpression of spib, mpo, and cebpa. (M,R,W) Quantification of the number of embryos that express spib, mpo, and cebpa and (N,S,X) size of primitive myeloid progenitor pool, by the area of tissue-expressing progenitor markers. Error bars represent the SD of the number of embryos analyzed (n). (O) Amount of tissue was calculated using area ratios of a and a′. Similar results were obtained in either ATG or e1i1 morphants and are in contrast with the uninjected and ATG mismatch control group (CTL). Student t test P values lower than .001 are marked as * and considered statistically significant; P values higher than .05 are not statistically significant and marked as ***.

Mentions: Of all the markers we analyzed, only cebpa expression was relatively unaffected by spib loss of function, although cebpa-expressing cells were found to cluster around the vitelline veins (Figure 5M-O red arrowheads). Together with the fact that cebpa is expressed prior to spib, this result suggests that cebpa may act upstream of spib in the hierarchy of myeloid development. However, the markers expressed well after spib, such as spi1 and mmp7, are particularly sensitive to spib depletion (Figure 5G-L,P-Q; Figure 6D-I). This demonstrates that spib is necessary for spi1 and mmp7 expression, suggesting that spib functions upstream of spi1 and mmp7. Furthermore, loss of primitive myeloid cell differentiation was sustained until tailbud stages as observed by the reduction in the number of neutrophils at stage 42 (Figure S2B).


spib is required for primitive myeloid development in Xenopus.

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

Effect of spib knockdown in primitive myeloid progenitors. (A-I) Primitive macrophages defined by the expression of spi1 and mmp7 are absent from spib-depleted embryos. (J-L,O-Q,T-V) WMISH analysis of aVBI in spib morphants; at the earliest point we can identify a pool of primitive myeloid progenitors by the coexpression of spib, mpo, and cebpa. (M,R,W) Quantification of the number of embryos that express spib, mpo, and cebpa and (N,S,X) size of primitive myeloid progenitor pool, by the area of tissue-expressing progenitor markers. Error bars represent the SD of the number of embryos analyzed (n). (O) Amount of tissue was calculated using area ratios of a and a′. Similar results were obtained in either ATG or e1i1 morphants and are in contrast with the uninjected and ATG mismatch control group (CTL). Student t test P values lower than .001 are marked as * and considered statistically significant; P values higher than .05 are not statistically significant and marked as ***.
© Copyright Policy - creativecommons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577559&req=5

Figure 6: Effect of spib knockdown in primitive myeloid progenitors. (A-I) Primitive macrophages defined by the expression of spi1 and mmp7 are absent from spib-depleted embryos. (J-L,O-Q,T-V) WMISH analysis of aVBI in spib morphants; at the earliest point we can identify a pool of primitive myeloid progenitors by the coexpression of spib, mpo, and cebpa. (M,R,W) Quantification of the number of embryos that express spib, mpo, and cebpa and (N,S,X) size of primitive myeloid progenitor pool, by the area of tissue-expressing progenitor markers. Error bars represent the SD of the number of embryos analyzed (n). (O) Amount of tissue was calculated using area ratios of a and a′. Similar results were obtained in either ATG or e1i1 morphants and are in contrast with the uninjected and ATG mismatch control group (CTL). Student t test P values lower than .001 are marked as * and considered statistically significant; P values higher than .05 are not statistically significant and marked as ***.
Mentions: Of all the markers we analyzed, only cebpa expression was relatively unaffected by spib loss of function, although cebpa-expressing cells were found to cluster around the vitelline veins (Figure 5M-O red arrowheads). Together with the fact that cebpa is expressed prior to spib, this result suggests that cebpa may act upstream of spib in the hierarchy of myeloid development. However, the markers expressed well after spib, such as spi1 and mmp7, are particularly sensitive to spib depletion (Figure 5G-L,P-Q; Figure 6D-I). This demonstrates that spib is necessary for spi1 and mmp7 expression, suggesting that spib functions upstream of spi1 and mmp7. Furthermore, loss of primitive myeloid cell differentiation was sustained until tailbud stages as observed by the reduction in the number of neutrophils at stage 42 (Figure S2B).

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