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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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Xenopus spib identification. (A) Conserved protein motifs in spib and spi1; the PEST degradation domain and ETS DNA-binding domain. (B) ClustalW tree alignment on vertebrate spib and spi1 protein sequences and percentage identity over their entirety and DNA-binding domains. (C) Genome organization around the known vertebrate spib and spi1 genes, showing synteny between various vertebrate genomes. Genes shown in the same color represent putative orthologous genes. Expression pattern of Xenopus tropicalis spib and (D-G) Xenopus laevis spib paralogs (SpiBa and SpiBb, H-J) is indistinguishable and marks a population of primitive myeloid progenitors in the anterior ventral blood islands (aVBIs). spib expression is transient and first detected at stage 17. (F) Cleared embryo shows only mesodermal expression, and (J) spib expression is eventually down-regulated after spib-expressing cells associate with vitelline veins (arrowheads). Anterior is to the left, and dorsal to the top in all lateral views; panels D, G, and I,J represent ventral views. The following database sequences were used: Hs spib Q01892, Mm spib O35906, Xl spiba BC046671, Xl spibb BC130210, Xt spib IMAGE 7023083, Hs spi1 P17947, Mm spi1 P17433, Gg spi1 NP_990354, Xt spi1 BC098077, and Dr spi1 CAD58735 (outgroup NP_001008139 and P14921).
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Figure 1: Xenopus spib identification. (A) Conserved protein motifs in spib and spi1; the PEST degradation domain and ETS DNA-binding domain. (B) ClustalW tree alignment on vertebrate spib and spi1 protein sequences and percentage identity over their entirety and DNA-binding domains. (C) Genome organization around the known vertebrate spib and spi1 genes, showing synteny between various vertebrate genomes. Genes shown in the same color represent putative orthologous genes. Expression pattern of Xenopus tropicalis spib and (D-G) Xenopus laevis spib paralogs (SpiBa and SpiBb, H-J) is indistinguishable and marks a population of primitive myeloid progenitors in the anterior ventral blood islands (aVBIs). spib expression is transient and first detected at stage 17. (F) Cleared embryo shows only mesodermal expression, and (J) spib expression is eventually down-regulated after spib-expressing cells associate with vitelline veins (arrowheads). Anterior is to the left, and dorsal to the top in all lateral views; panels D, G, and I,J represent ventral views. The following database sequences were used: Hs spib Q01892, Mm spib O35906, Xl spiba BC046671, Xl spibb BC130210, Xt spib IMAGE 7023083, Hs spi1 P17947, Mm spi1 P17433, Gg spi1 NP_990354, Xt spi1 BC098077, and Dr spi1 CAD58735 (outgroup NP_001008139 and P14921).

Mentions: In an unrelated screen,29 we identified a novel transcript expressed in the anterior ventral blood islands at neurula stage (Figure 1). In tailbud embryos, expression of this gene was observed in a punctate pattern reminiscent of primitive myeloid markers such as XLURP-1, XPOX2, and MMP7.27,30,31 Sequence analysis showed that this gene belonged to the spi subfamily of ETS transcription factors, with a C-terminal ETS domain and a PEST domain (Figure 1A,B). This architecture is conserved in all members of the spi subfamily, of which spi1 (also known as pu.1 and Sfpi1) is the most well-characterized member. To determine to which member of the spi subfamily our gene belonged, we used genome and synteny information to conclude that our gene is the Xenopus tropicalis ortholog of spib, given that it is located between the pold1 and mybpc2 genes (Figure 1C). We also isolated the Xenopus tropicalis ortholog of spi1, which shows remarkable synteny with the human, mouse, and chick genomes (Figure 1C). Interestingly, however, we noted that spib sequences appear to be absent from the chicken and fish genomes.


spib is required for primitive myeloid development in Xenopus.

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

Xenopus spib identification. (A) Conserved protein motifs in spib and spi1; the PEST degradation domain and ETS DNA-binding domain. (B) ClustalW tree alignment on vertebrate spib and spi1 protein sequences and percentage identity over their entirety and DNA-binding domains. (C) Genome organization around the known vertebrate spib and spi1 genes, showing synteny between various vertebrate genomes. Genes shown in the same color represent putative orthologous genes. Expression pattern of Xenopus tropicalis spib and (D-G) Xenopus laevis spib paralogs (SpiBa and SpiBb, H-J) is indistinguishable and marks a population of primitive myeloid progenitors in the anterior ventral blood islands (aVBIs). spib expression is transient and first detected at stage 17. (F) Cleared embryo shows only mesodermal expression, and (J) spib expression is eventually down-regulated after spib-expressing cells associate with vitelline veins (arrowheads). Anterior is to the left, and dorsal to the top in all lateral views; panels D, G, and I,J represent ventral views. The following database sequences were used: Hs spib Q01892, Mm spib O35906, Xl spiba BC046671, Xl spibb BC130210, Xt spib IMAGE 7023083, Hs spi1 P17947, Mm spi1 P17433, Gg spi1 NP_990354, Xt spi1 BC098077, and Dr spi1 CAD58735 (outgroup NP_001008139 and P14921).
© Copyright Policy - creativecommons
Related In: Results  -  Collection

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

Figure 1: Xenopus spib identification. (A) Conserved protein motifs in spib and spi1; the PEST degradation domain and ETS DNA-binding domain. (B) ClustalW tree alignment on vertebrate spib and spi1 protein sequences and percentage identity over their entirety and DNA-binding domains. (C) Genome organization around the known vertebrate spib and spi1 genes, showing synteny between various vertebrate genomes. Genes shown in the same color represent putative orthologous genes. Expression pattern of Xenopus tropicalis spib and (D-G) Xenopus laevis spib paralogs (SpiBa and SpiBb, H-J) is indistinguishable and marks a population of primitive myeloid progenitors in the anterior ventral blood islands (aVBIs). spib expression is transient and first detected at stage 17. (F) Cleared embryo shows only mesodermal expression, and (J) spib expression is eventually down-regulated after spib-expressing cells associate with vitelline veins (arrowheads). Anterior is to the left, and dorsal to the top in all lateral views; panels D, G, and I,J represent ventral views. The following database sequences were used: Hs spib Q01892, Mm spib O35906, Xl spiba BC046671, Xl spibb BC130210, Xt spib IMAGE 7023083, Hs spi1 P17947, Mm spi1 P17433, Gg spi1 NP_990354, Xt spi1 BC098077, and Dr spi1 CAD58735 (outgroup NP_001008139 and P14921).
Mentions: In an unrelated screen,29 we identified a novel transcript expressed in the anterior ventral blood islands at neurula stage (Figure 1). In tailbud embryos, expression of this gene was observed in a punctate pattern reminiscent of primitive myeloid markers such as XLURP-1, XPOX2, and MMP7.27,30,31 Sequence analysis showed that this gene belonged to the spi subfamily of ETS transcription factors, with a C-terminal ETS domain and a PEST domain (Figure 1A,B). This architecture is conserved in all members of the spi subfamily, of which spi1 (also known as pu.1 and Sfpi1) is the most well-characterized member. To determine to which member of the spi subfamily our gene belonged, we used genome and synteny information to conclude that our gene is the Xenopus tropicalis ortholog of spib, given that it is located between the pold1 and mybpc2 genes (Figure 1C). We also isolated the Xenopus tropicalis ortholog of spi1, which shows remarkable synteny with the human, mouse, and chick genomes (Figure 1C). Interestingly, however, we noted that spib sequences appear to be absent from the chicken and fish genomes.

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