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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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Impaired mesendodermal development in EB culture of Meteorin−/Δ cells.(A) Schematic view of EB culture. After 2 passages maintained in feeder-free embryonic stem cells, cells were dissociated and cultured using hanging drops for 2 days. Then, they were collected and cultured on bacterial-grade dishes. At each day of culture, indicated as arrowheads in the schematic view, cells were harvested for subsequent qRT-PCR experiments. (B–H) The expression of early developmental lineage markers on each day of culture was analyzed by qRT-PCR. (B) Successful gene deletion in Meteorin−/Δ ES cells was confirmed by observing the transcript level of Meteorin. (C) Expression of Oct3/4, a pluripotency marker, was similarly maintained in Meteorin−/Δ EB culture compared to Meteorin+/+ EB culture. Expression levels of Nestin, an early neuroectoderm marker (D), and AFP, a visceral endoderm marker (E), were higher in Meteorin−/Δ EB culture than in Meteorin+/+ EB culture. The expression levels of Brachyury, an early mesendoderm marker (F), Gsc, an endoderm marker (G), and Nodal, a posterior epiblast marker (H), significantly decreased in Meteorin−/Δ EB culture throughout the culture period. The expression of Lefty1(I) and Cerl(J), downstream molecules of Nodal signaling, was also lower in Meteorin−/Δ EB culture than in the control. Error bars indicate standard error of the mean (s.e.m.). All experiments were conducted more than 3 times and the representative graphs are shown.
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pone-0088811-g003: Impaired mesendodermal development in EB culture of Meteorin−/Δ cells.(A) Schematic view of EB culture. After 2 passages maintained in feeder-free embryonic stem cells, cells were dissociated and cultured using hanging drops for 2 days. Then, they were collected and cultured on bacterial-grade dishes. At each day of culture, indicated as arrowheads in the schematic view, cells were harvested for subsequent qRT-PCR experiments. (B–H) The expression of early developmental lineage markers on each day of culture was analyzed by qRT-PCR. (B) Successful gene deletion in Meteorin−/Δ ES cells was confirmed by observing the transcript level of Meteorin. (C) Expression of Oct3/4, a pluripotency marker, was similarly maintained in Meteorin−/Δ EB culture compared to Meteorin+/+ EB culture. Expression levels of Nestin, an early neuroectoderm marker (D), and AFP, a visceral endoderm marker (E), were higher in Meteorin−/Δ EB culture than in Meteorin+/+ EB culture. The expression levels of Brachyury, an early mesendoderm marker (F), Gsc, an endoderm marker (G), and Nodal, a posterior epiblast marker (H), significantly decreased in Meteorin−/Δ EB culture throughout the culture period. The expression of Lefty1(I) and Cerl(J), downstream molecules of Nodal signaling, was also lower in Meteorin−/Δ EB culture than in the control. Error bars indicate standard error of the mean (s.e.m.). All experiments were conducted more than 3 times and the representative graphs are shown.

Mentions: To investigate the nature of Meteorin−/− embryo abnormalities, we adopted the in vitro EB culture system, which recapitulates the differentiation process during gastrulation. To first generate Meteorin- ES cells, we sequentially targeted both alleles of the Meteorin gene by homologous recombination (Fig. S3A and B). After the forced expression of Cre recombinase, Meteorin transcripts were no longer expressed in Meteorin−/Δ ES cells (Fig. S3D and Fig. 3B). There was no apparent difference in morphology or proliferation rate between Meteorin−/Δ and control ES cells (data not shown).


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)

Impaired mesendodermal development in EB culture of Meteorin−/Δ cells.(A) Schematic view of EB culture. After 2 passages maintained in feeder-free embryonic stem cells, cells were dissociated and cultured using hanging drops for 2 days. Then, they were collected and cultured on bacterial-grade dishes. At each day of culture, indicated as arrowheads in the schematic view, cells were harvested for subsequent qRT-PCR experiments. (B–H) The expression of early developmental lineage markers on each day of culture was analyzed by qRT-PCR. (B) Successful gene deletion in Meteorin−/Δ ES cells was confirmed by observing the transcript level of Meteorin. (C) Expression of Oct3/4, a pluripotency marker, was similarly maintained in Meteorin−/Δ EB culture compared to Meteorin+/+ EB culture. Expression levels of Nestin, an early neuroectoderm marker (D), and AFP, a visceral endoderm marker (E), were higher in Meteorin−/Δ EB culture than in Meteorin+/+ EB culture. The expression levels of Brachyury, an early mesendoderm marker (F), Gsc, an endoderm marker (G), and Nodal, a posterior epiblast marker (H), significantly decreased in Meteorin−/Δ EB culture throughout the culture period. The expression of Lefty1(I) and Cerl(J), downstream molecules of Nodal signaling, was also lower in Meteorin−/Δ EB culture than in the control. Error bars indicate standard error of the mean (s.e.m.). All experiments were conducted more than 3 times and the representative graphs are shown.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928293&req=5

pone-0088811-g003: Impaired mesendodermal development in EB culture of Meteorin−/Δ cells.(A) Schematic view of EB culture. After 2 passages maintained in feeder-free embryonic stem cells, cells were dissociated and cultured using hanging drops for 2 days. Then, they were collected and cultured on bacterial-grade dishes. At each day of culture, indicated as arrowheads in the schematic view, cells were harvested for subsequent qRT-PCR experiments. (B–H) The expression of early developmental lineage markers on each day of culture was analyzed by qRT-PCR. (B) Successful gene deletion in Meteorin−/Δ ES cells was confirmed by observing the transcript level of Meteorin. (C) Expression of Oct3/4, a pluripotency marker, was similarly maintained in Meteorin−/Δ EB culture compared to Meteorin+/+ EB culture. Expression levels of Nestin, an early neuroectoderm marker (D), and AFP, a visceral endoderm marker (E), were higher in Meteorin−/Δ EB culture than in Meteorin+/+ EB culture. The expression levels of Brachyury, an early mesendoderm marker (F), Gsc, an endoderm marker (G), and Nodal, a posterior epiblast marker (H), significantly decreased in Meteorin−/Δ EB culture throughout the culture period. The expression of Lefty1(I) and Cerl(J), downstream molecules of Nodal signaling, was also lower in Meteorin−/Δ EB culture than in the control. Error bars indicate standard error of the mean (s.e.m.). All experiments were conducted more than 3 times and the representative graphs are shown.
Mentions: To investigate the nature of Meteorin−/− embryo abnormalities, we adopted the in vitro EB culture system, which recapitulates the differentiation process during gastrulation. To first generate Meteorin- ES cells, we sequentially targeted both alleles of the Meteorin gene by homologous recombination (Fig. S3A and B). After the forced expression of Cre recombinase, Meteorin transcripts were no longer expressed in Meteorin−/Δ ES cells (Fig. S3D and Fig. 3B). There was no apparent difference in morphology or proliferation rate between Meteorin−/Δ and control ES cells (data not shown).

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