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Meteorin regulates mesendoderm development by enhancing nodal expression.

Kim YY, Moon JS, Kwon MC, Shin J, Im SK, Kim HA, Han JK, Kong YY - PLoS ONE (2014)

Bottom Line: Embryoid body culture using Meteorin- embryonic stem (ES) cells showed reduced Nodal expression and concomitant impairment of mesendoderm specification.Meteorin- embryos displayed reduced levels of Nodal transcripts before the gastrulation stage, and impaired expression of Goosecoid, a definitive endoderm marker, during gastrulation, while the proximo-distal and anterior-posterior axes and primitive streak formation were preserved.Our results show that Meteorin is a novel regulator of Nodal transcription and is required to maintain sufficient Nodal levels for endoderm formation, thereby providing new insights in the regulation of mesendoderm allocation.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, South Korea ; Division of Molecular and Life Science, Department of Life Sciences, POSTECH, Pohang, South Korea.

ABSTRACT
During gastrulation, distinct lineage specification into three germ layers, the mesoderm, endoderm and ectoderm, occurs through an elaborate harmony between signaling molecules along the embryonic proximo-distal and anterior-posterior axes, and Nodal signaling plays a key role in the early embryonic development governing embryonic axis formation, mesoderm and endoderm specification, and left-right asymmetry determination. However, the mechanism by which Nodal expression is regulated is largely unknown. Here, we show that Meteorin regulates Nodal expression and is required for mesendoderm development. It is highly expressed in the inner cell mass of blastocysts and further in the epiblast and extra-embryonic ectoderm during gastrulation. Genetic ablation of the Meteorin gene resulted in early embryonic lethality, presumably due to impaired lineage allocation and subsequent cell accumulation. Embryoid body culture using Meteorin- embryonic stem (ES) cells showed reduced Nodal expression and concomitant impairment of mesendoderm specification. Meteorin- embryos displayed reduced levels of Nodal transcripts before the gastrulation stage, and impaired expression of Goosecoid, a definitive endoderm marker, during gastrulation, while the proximo-distal and anterior-posterior axes and primitive streak formation were preserved. Our results show that Meteorin is a novel regulator of Nodal transcription and is required to maintain sufficient Nodal levels for endoderm formation, thereby providing new insights in the regulation of mesendoderm allocation.

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Restoration of Nodal signaling in Meteorin+/Δ ES cells by Meteorin expression and reduced expression of Nodal in Meteorin−/− embryos.(A) At 48-mMeteorin plasmid into Meteorin−/Δ ES cells, Meteorin and Nodal expression was higher than that of pCAGGS mock-transfected Meteorin−/Δ ES cells. (B) When conditioned medium obtained from Meteorin+/+ ES cells (WT CM) was applied to Meteorin−/Δ ES cells, the expression levels of Nodal and Lefty1 were higher than those seen when conditioned medium from Meteorin−/Δ (KO CM) was applied. (C)Nodal and Lefty1 expressions were analyzed by qRT-PCR 48 hrs after the addition of recombinant mouse Meteorin (rmMeteorin) to Meteorin−/Δ ES cells. Both gene expressions were induced by Meteorin protein addition. Error bars indicate s.e.m. **p<0.01. (D) Expression of Lefty1 transcript was assessed by qRT-PCR after the addition of SB431542, an inhibitor of type I TGF-beta receptors, to Meteorin+/+ or Meteorin−/Δ ES cell lines. The residual activity of TGF-beta/Nodal signaling in Meteorin−/Δ ES cells was further inhibited by SB431542 treatment. Error bars indicate s.e.m. **p<0.01. (E–F) Expression of Nodal transcripts was analyzed by in situ hybridization in Meteorin+/+ and Meteorin−/− embryos at each developmental stage. At E6.5, the expression level of Nodal was significantly lower in Meteorin−/− embryos than in Meteorin+/+ embryos, especially in the proximal epiblast. All scale bars: 100 µm.
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pone-0088811-g005: Restoration of Nodal signaling in Meteorin+/Δ ES cells by Meteorin expression and reduced expression of Nodal in Meteorin−/− embryos.(A) At 48-mMeteorin plasmid into Meteorin−/Δ ES cells, Meteorin and Nodal expression was higher than that of pCAGGS mock-transfected Meteorin−/Δ ES cells. (B) When conditioned medium obtained from Meteorin+/+ ES cells (WT CM) was applied to Meteorin−/Δ ES cells, the expression levels of Nodal and Lefty1 were higher than those seen when conditioned medium from Meteorin−/Δ (KO CM) was applied. (C)Nodal and Lefty1 expressions were analyzed by qRT-PCR 48 hrs after the addition of recombinant mouse Meteorin (rmMeteorin) to Meteorin−/Δ ES cells. Both gene expressions were induced by Meteorin protein addition. Error bars indicate s.e.m. **p<0.01. (D) Expression of Lefty1 transcript was assessed by qRT-PCR after the addition of SB431542, an inhibitor of type I TGF-beta receptors, to Meteorin+/+ or Meteorin−/Δ ES cell lines. The residual activity of TGF-beta/Nodal signaling in Meteorin−/Δ ES cells was further inhibited by SB431542 treatment. Error bars indicate s.e.m. **p<0.01. (E–F) Expression of Nodal transcripts was analyzed by in situ hybridization in Meteorin+/+ and Meteorin−/− embryos at each developmental stage. At E6.5, the expression level of Nodal was significantly lower in Meteorin−/− embryos than in Meteorin+/+ embryos, especially in the proximal epiblast. All scale bars: 100 µm.

Mentions: We next examined whether reduced Nodal expression in the Meteorin−/Δ ES cells can be rescued by introduction of Meteorin. When Meteorin−/Δ ES cells were transfected with a Meteorin-expressing vector, the expression of Nodal increased (Fig. 5A). Since Meteorin is a secreted protein, we applied the conditioned medium (CM) obtained from control ES cells to Meteorin−/Δ ES cells. As expected, the addition of control CM to Meteorin−/Δ ES cells led to increase in Nodal and Lefty1 expression (Fig. 5B). To rule out the possibility that this result was caused by Nodal protein in the conditioned medium, not by Meteorin protein, we directly applied recombinant mouse Meteorin (rmMeteorin) protein to Meteorin−/Δ ES cells. The addition of rmMeteorin to Meteorin-deficient ES cells also rescued the expression of Nodal and Lefty1, directly suggesting that Meteorin is required to produce sufficient amounts of Nodal signaling for normal development (Fig. 5C). On the other hand, residual Lefty1 expression in the Meteorin−/Δ ES cells was further decreased by SB431542, an inhibitor of type I TGF-beta receptors, (Fig. 5D), indicating that TGF-beta/Nodal signaling in the Meteorin−/Δ ES cells, albeit decreased, is functional. Indeed, in Meteorin−/− embryos, Nodal expresses, but the level was relatively lower than that in Meteorin+/+ embryos, especially in the proximal region of embryos (Fig. 5E–F).


Meteorin regulates mesendoderm development by enhancing nodal expression.

Kim YY, Moon JS, Kwon MC, Shin J, Im SK, Kim HA, Han JK, Kong YY - PLoS ONE (2014)

Restoration of Nodal signaling in Meteorin+/Δ ES cells by Meteorin expression and reduced expression of Nodal in Meteorin−/− embryos.(A) At 48-mMeteorin plasmid into Meteorin−/Δ ES cells, Meteorin and Nodal expression was higher than that of pCAGGS mock-transfected Meteorin−/Δ ES cells. (B) When conditioned medium obtained from Meteorin+/+ ES cells (WT CM) was applied to Meteorin−/Δ ES cells, the expression levels of Nodal and Lefty1 were higher than those seen when conditioned medium from Meteorin−/Δ (KO CM) was applied. (C)Nodal and Lefty1 expressions were analyzed by qRT-PCR 48 hrs after the addition of recombinant mouse Meteorin (rmMeteorin) to Meteorin−/Δ ES cells. Both gene expressions were induced by Meteorin protein addition. Error bars indicate s.e.m. **p<0.01. (D) Expression of Lefty1 transcript was assessed by qRT-PCR after the addition of SB431542, an inhibitor of type I TGF-beta receptors, to Meteorin+/+ or Meteorin−/Δ ES cell lines. The residual activity of TGF-beta/Nodal signaling in Meteorin−/Δ ES cells was further inhibited by SB431542 treatment. Error bars indicate s.e.m. **p<0.01. (E–F) Expression of Nodal transcripts was analyzed by in situ hybridization in Meteorin+/+ and Meteorin−/− embryos at each developmental stage. At E6.5, the expression level of Nodal was significantly lower in Meteorin−/− embryos than in Meteorin+/+ embryos, especially in the proximal epiblast. All scale bars: 100 µm.
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Related In: Results  -  Collection

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pone-0088811-g005: Restoration of Nodal signaling in Meteorin+/Δ ES cells by Meteorin expression and reduced expression of Nodal in Meteorin−/− embryos.(A) At 48-mMeteorin plasmid into Meteorin−/Δ ES cells, Meteorin and Nodal expression was higher than that of pCAGGS mock-transfected Meteorin−/Δ ES cells. (B) When conditioned medium obtained from Meteorin+/+ ES cells (WT CM) was applied to Meteorin−/Δ ES cells, the expression levels of Nodal and Lefty1 were higher than those seen when conditioned medium from Meteorin−/Δ (KO CM) was applied. (C)Nodal and Lefty1 expressions were analyzed by qRT-PCR 48 hrs after the addition of recombinant mouse Meteorin (rmMeteorin) to Meteorin−/Δ ES cells. Both gene expressions were induced by Meteorin protein addition. Error bars indicate s.e.m. **p<0.01. (D) Expression of Lefty1 transcript was assessed by qRT-PCR after the addition of SB431542, an inhibitor of type I TGF-beta receptors, to Meteorin+/+ or Meteorin−/Δ ES cell lines. The residual activity of TGF-beta/Nodal signaling in Meteorin−/Δ ES cells was further inhibited by SB431542 treatment. Error bars indicate s.e.m. **p<0.01. (E–F) Expression of Nodal transcripts was analyzed by in situ hybridization in Meteorin+/+ and Meteorin−/− embryos at each developmental stage. At E6.5, the expression level of Nodal was significantly lower in Meteorin−/− embryos than in Meteorin+/+ embryos, especially in the proximal epiblast. All scale bars: 100 µm.
Mentions: We next examined whether reduced Nodal expression in the Meteorin−/Δ ES cells can be rescued by introduction of Meteorin. When Meteorin−/Δ ES cells were transfected with a Meteorin-expressing vector, the expression of Nodal increased (Fig. 5A). Since Meteorin is a secreted protein, we applied the conditioned medium (CM) obtained from control ES cells to Meteorin−/Δ ES cells. As expected, the addition of control CM to Meteorin−/Δ ES cells led to increase in Nodal and Lefty1 expression (Fig. 5B). To rule out the possibility that this result was caused by Nodal protein in the conditioned medium, not by Meteorin protein, we directly applied recombinant mouse Meteorin (rmMeteorin) protein to Meteorin−/Δ ES cells. The addition of rmMeteorin to Meteorin-deficient ES cells also rescued the expression of Nodal and Lefty1, directly suggesting that Meteorin is required to produce sufficient amounts of Nodal signaling for normal development (Fig. 5C). On the other hand, residual Lefty1 expression in the Meteorin−/Δ ES cells was further decreased by SB431542, an inhibitor of type I TGF-beta receptors, (Fig. 5D), indicating that TGF-beta/Nodal signaling in the Meteorin−/Δ ES cells, albeit decreased, is functional. Indeed, in Meteorin−/− embryos, Nodal expresses, but the level was relatively lower than that in Meteorin+/+ embryos, especially in the proximal region of embryos (Fig. 5E–F).

Bottom Line: Embryoid body culture using Meteorin- embryonic stem (ES) cells showed reduced Nodal expression and concomitant impairment of mesendoderm specification.Meteorin- embryos displayed reduced levels of Nodal transcripts before the gastrulation stage, and impaired expression of Goosecoid, a definitive endoderm marker, during gastrulation, while the proximo-distal and anterior-posterior axes and primitive streak formation were preserved.Our results show that Meteorin is a novel regulator of Nodal transcription and is required to maintain sufficient Nodal levels for endoderm formation, thereby providing new insights in the regulation of mesendoderm allocation.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, South Korea ; Division of Molecular and Life Science, Department of Life Sciences, POSTECH, Pohang, South Korea.

ABSTRACT
During gastrulation, distinct lineage specification into three germ layers, the mesoderm, endoderm and ectoderm, occurs through an elaborate harmony between signaling molecules along the embryonic proximo-distal and anterior-posterior axes, and Nodal signaling plays a key role in the early embryonic development governing embryonic axis formation, mesoderm and endoderm specification, and left-right asymmetry determination. However, the mechanism by which Nodal expression is regulated is largely unknown. Here, we show that Meteorin regulates Nodal expression and is required for mesendoderm development. It is highly expressed in the inner cell mass of blastocysts and further in the epiblast and extra-embryonic ectoderm during gastrulation. Genetic ablation of the Meteorin gene resulted in early embryonic lethality, presumably due to impaired lineage allocation and subsequent cell accumulation. Embryoid body culture using Meteorin- embryonic stem (ES) cells showed reduced Nodal expression and concomitant impairment of mesendoderm specification. Meteorin- embryos displayed reduced levels of Nodal transcripts before the gastrulation stage, and impaired expression of Goosecoid, a definitive endoderm marker, during gastrulation, while the proximo-distal and anterior-posterior axes and primitive streak formation were preserved. Our results show that Meteorin is a novel regulator of Nodal transcription and is required to maintain sufficient Nodal levels for endoderm formation, thereby providing new insights in the regulation of mesendoderm allocation.

Show MeSH
Related in: MedlinePlus