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The evolutionary origin of the Runx/CBFbeta transcription factors--studies of the most basal metazoans.

Sullivan JC, Sher D, Eisenstein M, Shigesada K, Reitzel AM, Marlow H, Levanon D, Groner Y, Finnerty JR, Gat U - BMC Evol. Biol. (2008)

Bottom Line: Comparative structural modeling indicates that the Runx-CBFbeta-DNA complex from most cnidarians and sponges is highly similar to that found in humans, with changes in the residues involved in Runx-CBFbeta dimerization in either of the proteins mirrored by compensatory changes in the binding partner.These results reveal that Runx and CBFbeta likely functioned together to regulate transcription in the common ancestor of all metazoans, and the structure of the Runx-CBFbeta-DNA complex has remained extremely conserved since the human-sponge divergence.The expression data suggest a hypothesis that these genes may have played a role in nerve cell differentiation or maintenance in the common ancestor of cnidarians and bilaterians.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Boston University, 5 Cummington St, Boston, MA 02215, USA. jamescsullivan@gmail.com

ABSTRACT

Background: Members of the Runx family of transcriptional regulators, which bind DNA as heterodimers with CBFbeta, are known to play critical roles in embryonic development in many triploblastic animals such as mammals and insects. They are known to regulate basic developmental processes such as cell fate determination and cellular potency in multiple stem-cell types, including the sensory nerve cell progenitors of ganglia in mammals.

Results: In this study, we detect and characterize the hitherto unexplored Runx/CBFbeta genes of cnidarians and sponges, two basal animal lineages that are well known for their extensive regenerative capacity. Comparative structural modeling indicates that the Runx-CBFbeta-DNA complex from most cnidarians and sponges is highly similar to that found in humans, with changes in the residues involved in Runx-CBFbeta dimerization in either of the proteins mirrored by compensatory changes in the binding partner. In situ hybridization studies reveal that Nematostella Runx and CBFbeta are expressed predominantly in small isolated foci at the base of the ectoderm of the tentacles in adult animals, possibly representing neurons or their progenitors.

Conclusion: These results reveal that Runx and CBFbeta likely functioned together to regulate transcription in the common ancestor of all metazoans, and the structure of the Runx-CBFbeta-DNA complex has remained extremely conserved since the human-sponge divergence. The expression data suggest a hypothesis that these genes may have played a role in nerve cell differentiation or maintenance in the common ancestor of cnidarians and bilaterians.

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Expression pattern of Nv-Runx in adult Nematostella. Digoxigenin-labeled sense-strand and antisense-strand riboprobes corresponding to nucleotides 100–539 of the ORF (exons 1–3, which contain the Runt domain) were used to characterize the spatial expression of Runx in adult Nematostella. Similar results were obtained using a longer probe corresponding to the entire Nv-Runx transcript (not shown). A) Labeled anti-sense probes detect Nv-Runx expression in the oral region of the anemones, particularly in the ectoderm of the tentacle tips (animal on left). No specific staining was observed using sense-strand probes (animal on right). B-C) While expression was always limited to the tentacle and head region there was some background staining that varied between individual animals. Note that the dark color in the mouth of the animal depicted in panel B (arrowhead) does not represent Runx expression, since it is not detected in sections of this region. The arrowhead in Panel C reveals the strong expression at the tentacle tips. D-H) These panels show ectodermal expression of Nv-Runx in the tentacles as seen in cryostat sections of anemones after whole mount in-situ hybridization. D) Low magnification micrograph of a section through the head and tentacles, revealing general architecture as well as the location of the enlarged micrographs in E and F. Bar = 100 μm E-G) Expression of Nv-Runx in the ectoderm of the tentacles, Bars in E and F = 50 μm, in G = 20 μm, H) A thin section of a tentacle from Nematostella, stained with Methylene Blue. Numerous spirocysts (Sp) and several nematocysts (N) can be observed, as can darkly and heterogeneously stained gland cells (G) found towards the apical part of the ectoderm. The elongated cells (S) are probably sensory cells [61]. The mesoglea (Mes) is schematically marked by a dashed line. Bar = 10 μm. I) Expression of Nv-Runx in scattered cells in the ectoderm of the body wall (arrowhead). Bar = 20 μm.
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Figure 7: Expression pattern of Nv-Runx in adult Nematostella. Digoxigenin-labeled sense-strand and antisense-strand riboprobes corresponding to nucleotides 100–539 of the ORF (exons 1–3, which contain the Runt domain) were used to characterize the spatial expression of Runx in adult Nematostella. Similar results were obtained using a longer probe corresponding to the entire Nv-Runx transcript (not shown). A) Labeled anti-sense probes detect Nv-Runx expression in the oral region of the anemones, particularly in the ectoderm of the tentacle tips (animal on left). No specific staining was observed using sense-strand probes (animal on right). B-C) While expression was always limited to the tentacle and head region there was some background staining that varied between individual animals. Note that the dark color in the mouth of the animal depicted in panel B (arrowhead) does not represent Runx expression, since it is not detected in sections of this region. The arrowhead in Panel C reveals the strong expression at the tentacle tips. D-H) These panels show ectodermal expression of Nv-Runx in the tentacles as seen in cryostat sections of anemones after whole mount in-situ hybridization. D) Low magnification micrograph of a section through the head and tentacles, revealing general architecture as well as the location of the enlarged micrographs in E and F. Bar = 100 μm E-G) Expression of Nv-Runx in the ectoderm of the tentacles, Bars in E and F = 50 μm, in G = 20 μm, H) A thin section of a tentacle from Nematostella, stained with Methylene Blue. Numerous spirocysts (Sp) and several nematocysts (N) can be observed, as can darkly and heterogeneously stained gland cells (G) found towards the apical part of the ectoderm. The elongated cells (S) are probably sensory cells [61]. The mesoglea (Mes) is schematically marked by a dashed line. Bar = 10 μm. I) Expression of Nv-Runx in scattered cells in the ectoderm of the body wall (arrowhead). Bar = 20 μm.

Mentions: We next used whole mount in-situ hybridization, followed by cryosectioning, to characterize the spatial expression of Runx and CBFβ. In agreement with the RT-PCR results, Nv-Runx was expressed mainly in the tentacles, with the strongest expression seen in the tentacle tips (Figure 7A). We found that the expression is limited to small foci found exclusively in the ectoderm, often in tight association with nematocytes (Figure 7G). Sporadic Runx-expressing cells were also seen in the outer body wall in the pharyngeal region (Figure 7I). The same general pattern of Runx expression was observed in ten different anemones using two different anti-sense RNA probes – a 450 nucleotide probe corresponding to exons 1–3 of the gene (containing the Runt domain; Figure 2A) as well as a 1.5 kb probe corresponding to the entire ORF (data not shown). No staining was observed with the complementary negative control sense-strand RNA probes (Figure 7A). While the general pattern of expression was similar in all of the individual anemones, possible differences in the relative level of expression were observed between different animals within the same experiment (compare Figure 7, panels B and C).


The evolutionary origin of the Runx/CBFbeta transcription factors--studies of the most basal metazoans.

Sullivan JC, Sher D, Eisenstein M, Shigesada K, Reitzel AM, Marlow H, Levanon D, Groner Y, Finnerty JR, Gat U - BMC Evol. Biol. (2008)

Expression pattern of Nv-Runx in adult Nematostella. Digoxigenin-labeled sense-strand and antisense-strand riboprobes corresponding to nucleotides 100–539 of the ORF (exons 1–3, which contain the Runt domain) were used to characterize the spatial expression of Runx in adult Nematostella. Similar results were obtained using a longer probe corresponding to the entire Nv-Runx transcript (not shown). A) Labeled anti-sense probes detect Nv-Runx expression in the oral region of the anemones, particularly in the ectoderm of the tentacle tips (animal on left). No specific staining was observed using sense-strand probes (animal on right). B-C) While expression was always limited to the tentacle and head region there was some background staining that varied between individual animals. Note that the dark color in the mouth of the animal depicted in panel B (arrowhead) does not represent Runx expression, since it is not detected in sections of this region. The arrowhead in Panel C reveals the strong expression at the tentacle tips. D-H) These panels show ectodermal expression of Nv-Runx in the tentacles as seen in cryostat sections of anemones after whole mount in-situ hybridization. D) Low magnification micrograph of a section through the head and tentacles, revealing general architecture as well as the location of the enlarged micrographs in E and F. Bar = 100 μm E-G) Expression of Nv-Runx in the ectoderm of the tentacles, Bars in E and F = 50 μm, in G = 20 μm, H) A thin section of a tentacle from Nematostella, stained with Methylene Blue. Numerous spirocysts (Sp) and several nematocysts (N) can be observed, as can darkly and heterogeneously stained gland cells (G) found towards the apical part of the ectoderm. The elongated cells (S) are probably sensory cells [61]. The mesoglea (Mes) is schematically marked by a dashed line. Bar = 10 μm. I) Expression of Nv-Runx in scattered cells in the ectoderm of the body wall (arrowhead). Bar = 20 μm.
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Related In: Results  -  Collection

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Figure 7: Expression pattern of Nv-Runx in adult Nematostella. Digoxigenin-labeled sense-strand and antisense-strand riboprobes corresponding to nucleotides 100–539 of the ORF (exons 1–3, which contain the Runt domain) were used to characterize the spatial expression of Runx in adult Nematostella. Similar results were obtained using a longer probe corresponding to the entire Nv-Runx transcript (not shown). A) Labeled anti-sense probes detect Nv-Runx expression in the oral region of the anemones, particularly in the ectoderm of the tentacle tips (animal on left). No specific staining was observed using sense-strand probes (animal on right). B-C) While expression was always limited to the tentacle and head region there was some background staining that varied between individual animals. Note that the dark color in the mouth of the animal depicted in panel B (arrowhead) does not represent Runx expression, since it is not detected in sections of this region. The arrowhead in Panel C reveals the strong expression at the tentacle tips. D-H) These panels show ectodermal expression of Nv-Runx in the tentacles as seen in cryostat sections of anemones after whole mount in-situ hybridization. D) Low magnification micrograph of a section through the head and tentacles, revealing general architecture as well as the location of the enlarged micrographs in E and F. Bar = 100 μm E-G) Expression of Nv-Runx in the ectoderm of the tentacles, Bars in E and F = 50 μm, in G = 20 μm, H) A thin section of a tentacle from Nematostella, stained with Methylene Blue. Numerous spirocysts (Sp) and several nematocysts (N) can be observed, as can darkly and heterogeneously stained gland cells (G) found towards the apical part of the ectoderm. The elongated cells (S) are probably sensory cells [61]. The mesoglea (Mes) is schematically marked by a dashed line. Bar = 10 μm. I) Expression of Nv-Runx in scattered cells in the ectoderm of the body wall (arrowhead). Bar = 20 μm.
Mentions: We next used whole mount in-situ hybridization, followed by cryosectioning, to characterize the spatial expression of Runx and CBFβ. In agreement with the RT-PCR results, Nv-Runx was expressed mainly in the tentacles, with the strongest expression seen in the tentacle tips (Figure 7A). We found that the expression is limited to small foci found exclusively in the ectoderm, often in tight association with nematocytes (Figure 7G). Sporadic Runx-expressing cells were also seen in the outer body wall in the pharyngeal region (Figure 7I). The same general pattern of Runx expression was observed in ten different anemones using two different anti-sense RNA probes – a 450 nucleotide probe corresponding to exons 1–3 of the gene (containing the Runt domain; Figure 2A) as well as a 1.5 kb probe corresponding to the entire ORF (data not shown). No staining was observed with the complementary negative control sense-strand RNA probes (Figure 7A). While the general pattern of expression was similar in all of the individual anemones, possible differences in the relative level of expression were observed between different animals within the same experiment (compare Figure 7, panels B and C).

Bottom Line: Comparative structural modeling indicates that the Runx-CBFbeta-DNA complex from most cnidarians and sponges is highly similar to that found in humans, with changes in the residues involved in Runx-CBFbeta dimerization in either of the proteins mirrored by compensatory changes in the binding partner.These results reveal that Runx and CBFbeta likely functioned together to regulate transcription in the common ancestor of all metazoans, and the structure of the Runx-CBFbeta-DNA complex has remained extremely conserved since the human-sponge divergence.The expression data suggest a hypothesis that these genes may have played a role in nerve cell differentiation or maintenance in the common ancestor of cnidarians and bilaterians.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Boston University, 5 Cummington St, Boston, MA 02215, USA. jamescsullivan@gmail.com

ABSTRACT

Background: Members of the Runx family of transcriptional regulators, which bind DNA as heterodimers with CBFbeta, are known to play critical roles in embryonic development in many triploblastic animals such as mammals and insects. They are known to regulate basic developmental processes such as cell fate determination and cellular potency in multiple stem-cell types, including the sensory nerve cell progenitors of ganglia in mammals.

Results: In this study, we detect and characterize the hitherto unexplored Runx/CBFbeta genes of cnidarians and sponges, two basal animal lineages that are well known for their extensive regenerative capacity. Comparative structural modeling indicates that the Runx-CBFbeta-DNA complex from most cnidarians and sponges is highly similar to that found in humans, with changes in the residues involved in Runx-CBFbeta dimerization in either of the proteins mirrored by compensatory changes in the binding partner. In situ hybridization studies reveal that Nematostella Runx and CBFbeta are expressed predominantly in small isolated foci at the base of the ectoderm of the tentacles in adult animals, possibly representing neurons or their progenitors.

Conclusion: These results reveal that Runx and CBFbeta likely functioned together to regulate transcription in the common ancestor of all metazoans, and the structure of the Runx-CBFbeta-DNA complex has remained extremely conserved since the human-sponge divergence. The expression data suggest a hypothesis that these genes may have played a role in nerve cell differentiation or maintenance in the common ancestor of cnidarians and bilaterians.

Show MeSH
Related in: MedlinePlus