Tbx3 promotes liver bud expansion during mouse development by suppression of cholangiocyte differentiation†

In Tbx3-/- embryos at E9.0, the ventral foregut endoderm expresses Afp indistinguishably from wild-type.

“In Tbx3 Ϫ/Ϫ embryos of E9.0 (16-somite stage), the ventral foregut endoderm expressed Afp indistinguishably from the wild-type”

Tbx3-deficient livers at E14.5 show a dramatic reduction of Afp and albumin expression.

“Tbx3-deficient livers were characterized by a dramatic reduction of the hepatoblast marker gene alpha fetoprotein (Afp) and the hepatocyte marker gene albumin (Fig. 1C)”

In Tbx3-/- embryos on an NMRI outbred background, homozygous mutant embryos are viable until E14.5.

“we maintained a new Tbx3 null allele (Tbx3 cre ) 7 on an NMRI outbred background that supported viability of homozygous mutant embryos until E14.5.”

At E12.5, dramatic reduction of Afp-positive cells confirmed the complete failure to expand the hepatic cell population in the mutant.

“Dramatic re-duction of Afp-positive cells at E12.5 confirmed the complete failure to expand this hepatic cell population in the mutant.”

At E10.5, few Afp-positive cells were found in the underlying mesenchyme of the mutant compared to the densely populated wild-type.

“At E10.5 the mesenchyme adjacent to the liver bud was densely populated with hepatoblasts in the wild-type, whereas few Afp-positive cells were found in the underlying mesenchyme of the mutant.”

Tbx3-deficient livers at E14.5 show increased Ck18 expression detected in 10% to 20% of cells.

“whereas expression of Ck18, an antigen confined to cholangiocytes, was increased and detected in 10% to 20% of cells in the mutant liver (Fig. 1D).”

qRT-PCR confirmed that expression levels of p19Arf, p21, p27, Cdk1, and CyclinD1 are unchanged in Tbx3-/- liver buds.

“Quantitative PCR on reverse-transcribed mRNA isolated from E9.5 liver buds (qRT-PCR) independently confirmed that expression levels of the cell cycle regulators p19 Arf , p21 Cip1 , p27 Kip1 , Cdk1, and CyclinD1 are unchanged in Tbx3 Ϫ/Ϫ liver buds”

At E10.5, Tbx3 is markedly down-regulated and confined to hepatoblasts populating the hepatic lobes.

“At E10.5, Tbx3 was markedly down-regulated and confined to hepatoblasts populating the hepatic lobes.”

In Tbx3-/- embryos, delamination of hepatoblasts fails.

“In Tbx3 Ϫ/Ϫ embryos, delamination of hepatoblasts failed”

• Also stated in: § Proliferation and Migration Defects in the Tbx3-Deficient Liver Bud Are Secondary to Cell Fate Changes. · plain

  “Afp-expression shows that hepatic fate is specified in the Tbx3deficient foregut endoderm, but delamination of hepatoblasts does not occur.”

From E12.5, Tbx3 expression is barely detectable in the liver by in situ hybridization.

“From E12.5, Tbx3 expression was barely detectable in the liver by in situ hybridization analysis.”

Expression of Hlx, Gata4, and Gata6 was unaltered in the mesenchyme of the septum transversum in Tbx3 mutant.

“Similarly, expression of the genes encoding the H2.0-like homeodomain protein (Hlx) and the Zn-finger transcription factors GATA-binding proteins 4 and 6 (Gata4 and Gata6) 14,15 was unaltered in the mesenchyme of the septum transversum region”

Up-regulation of Onecut1 expression directly correlates with the expression profile of Tbx3 and the temporal onset of defects in the Tbx3-deficient embryo.

“First, up-regulation of Onecut1 expression directly correlates with the expression profile of Tbx3 and the temporal onset of defects in the Tbx3-deficient embryo.”

Loss and gain of Tbx3 expression does not result in immediate changes of cell-cycle regulators, including p19 Arf and p21 Cip1.

“Our molecular analyses of Tbx3 loss-and gain-of-function scenarios both in vivo and in vitro, however, clearly show that loss and gain of Tbx3 expression does not result in immediate changes of cell-cycle regulators, including p19 Arf and p21 Cip1 .”

p57 was expressed in the hepatic endoderm and underlying mesenchyme in wildtype and indistinguishably in mutant embryos.

“p57 Kip2 was expressed in the hepatic endoderm and in the underlying mesenchyme in wildtype and indistinguishably in mutant embryos.”

In Tbx3-/- embryos, cells of the hepatic epithelium retain strong expression of E-cadherin.

“cells of the hepatic epithelium retained strong expression of E-cadherin.”

Expression of Pdx1 was unchanged in the Tbx3-/- embryo.

“Expression of Pdx1, a marker for pancreatic fate, 23 was unchanged in the Tbx3 Ϫ/Ϫ embryo (Supporting Fig. 2)”

Overexpression of Tbx3 in 9A1 hepatoblasts led to increased levels of Hnf4a and Cebpa.

“Overexpression of Tbx3 in 9A1 hepatoblasts led to increased levels of Hnf4a and Cebpa”

TUNEL staining showed that apoptosis was unaffected in the mutant liver at E9.5.

“Terminal deoxynucleotidyl transferase-mediated nick-end labeling staining showed that apoptosis was unaffected in mutant liver at this stage (Fig. 4B).”

Expression of Shh remained excluded from the hepatic endoderm in the Tbx3-mutant embryo.

“Expression of the gene encoding the signaling molecule Sonic hedgehog (Shh) remained excluded from the hepatic endoderm in the Tbx3-mutant embryo (Supporting Fig. 2)”

At E9.0 (18-somite), BrdU labeling indices of epithelial cells of wild-type and mutant hepatic endoderm were similar.

“At the 18-somite stage (E9.0), when Tbx3 is only weakly expressed, labeling indices of epithelial cells of wild-type and mutant hepatic endoderm were similar.”

Expression of p15, p16, p18, p19INK4d, p19Arf, and p27 was detected in the hepatic endoderm of neither wild-type nor mutant embryos at E9.5.

“Expression of p15 INK4b , p16 INK4a , p18 INK4c , p19 INK4d , p19 Arf , and p27 Kip1 was detected in the hepatic endoderm of neither wild-type nor mutant embryos”

Tbx3 mRNA is first detected at low levels in the hepatic endoderm at the 18-somite stage (E9.0).

“Tbx3 messenger RNA (mRNA) was first detected at low levels in the hepatic endoderm at the 18-somite stage (E9.0).”

Expression of p21 was found throughout the hepatic and foregut endoderm in either genotype.

“Expression of p21 Cip1 was found throughout the hepatic and foregut endoderm in either genotype.”

Ectopic expression of Onecut1 in hepatoblasts and hepatocytes represses transcription of Hnf4a and Cebpa.

“Second, ectopic expression of One-cut1 in hepatoblasts and hepatocytes represses transcription of Hnf4a and Cebpa”

In Tbx3-/- embryos, the laminin-rich membrane around the liver bud remains intact.

“the laminin-rich membrane around the liver bud remained intact”

Ectopic expression of Onecut1 enhances the effect of Tbx3 inhibition on cholangiocyte differentiation.

“and enhances the effect of Tbx3 inhibition on cholangiocyte differentiation.”

Expression of Hex was unchanged in the Tbx3-deficient liver bud at E9.5.

“Expression of hematopoietically expressed homeobox that regulates the transition from a columnar to a pseudostratified epithelium 13 was unchanged in the Tbx3deficient liver bud at E9.5 (Fig. 5B).”

Overexpression of Tbx2-VP16 in 9A1 hepatoblasts resulted in downregulation of Hnf4a, Cebpa, and albumin.

“whereas expression of Tbx2-VP16 resulted in downregulation of Hnf4a, Cebpa and albumin (Fig. 7A).”

Proliferation of hepatic endoderm is severely reduced in Tbx3-/- embryos.

“Proliferation of hepatic endoderm is severely reduced in Tbx3Ϫ/Ϫ embryos.”

High Tbx3 expression tightly correlates with the onset of morphological and molecular changes in Tbx3-mutant livers at E9.5.

“and tightly correlates with the onset of morphological and molecular changes in Tbx3-mutant livers at E9.5.”

Levels of p19 Arf are unaffected by changes of Tbx3 and Onecut1 expression.

“Levels of p19 Arf were unaffected by changes of Tbx3 and Onecut1 expression”

E-cadherin and laminin expression remains high in the mutant liver bud.

“Expression of both antigens remains high in the mutant liver bud.”

High Tbx3 expression is confined to a short time window in hepatic development.

“Our in situ hybridization analysis has shown that high Tbx3 expression is confined to a short time window in hepatic development”

Onecut1 expression is activated by Tbx3 in all cell lines tested.

“Onecut1 expression was surprisingly activated by Tbx3 in all cell lines tested (Fig. 7).”

Tbx3-deficient mice exhibit severe liver hypoplasia at E14.5.

“Tbx3-deficient mice exhibit severe liver hypoplasia at E14.5.”

Apoptosis in the hepatic endoderm at E9.5 does not differ between wild-type and Tbx3-mutant embryos.

“Terminal deoxynucleotidyl transferase-mediated nick-end labeling staining for apoptosis in the hepatic endoderm (white outline) at E9.5 (23s) does not reveal differences between wild-type and Tbx3-mutant embryos.”

Hepatic fate is specified in the Tbx3-deficient foregut endoderm.

“Afp-expression shows that hepatic fate is specified in the Tbx3deficient foregut endoderm”

In Tbx3-deficient livers, blood cells and Ck18+ cholangiocytes have replaced Alb-expressing hepatocytes.

“Blood cells and Ck18 ϩ -cholangiocytes have replaced Alb-expressing hepatocytes.”

Hepatic specification occurred normally in Tbx3-/- embryos.

“In summary, hepatic specification occurred normally in Tbx3 Ϫ/Ϫ embryos.”

Failure of migration of hepatoblasts into the surrounding mesenchyme may be caused by the inability to maintain Prox1 expression.

“Thus, failure of migration of hepatoblasts into the surrounding mesenchyme may be caused by the inability to maintain Prox1 expression.”

Tbx3 controls hepatobiliary fate decision by antagonistically regulating expression of key transcriptional mediators of the hepatocyte and cholangiocyte pathways.

“that are fully compatible with the notion that Tbx3 controls hepatobiliary fate decision by antagonistically regulating expression of key transcriptional mediators of the hepatocyte and cholangiocyte pathways.”

Absence of albumin-expressing hepatocytes but presence of Ck18-positive cholangiocytes at E14.5 indicated that the hepatoblast lineage decision was affected in Tbx3-deficient livers.

“Absence of albumin-expressing hepatocytes but presence of Ck18-positive cholangiocytes at E14.5 (Fig. 1C,D) indicated that the hepatoblast lineage decision was affected in Tbx3-deficient livers.”

Tbx3 controls morphogenesis of the liver bud by coordinately regulating proliferation, migration, and differentiation of hepatoblasts.

“We correlate the hepatic expression of Tbx3 with onset of liver defects in Tbx3deficient embryos and show that Tbx3 controls morphogenesis of the liver bud by coordinately regulating proliferation, migration, and differentiation of hepatoblasts.”

Expression of Pdx1 unchanged argues against an expansion of pancreatic fates into the liver region.

“Expression of Pdx1, a marker for pancreatic fate, 23 was unchanged in the Tbx3 Ϫ/Ϫ embryo (Supporting Fig. 2), arguing against an expansion of pancreatic fates into the liver region.”

A recent study has provided strong evidence for a primary requirement of Tbx3 in hepatogenesis.

“However, a recent study has provided strong evidence for a primary requirement of Tbx3 in hepatogenesis.6,7”

• cites [6]Not matched

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• cites [7]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Hepatoblasts started to differentiate into hepatocytes in E9.5 wildtype embryos.

“arguing that hepatoblasts started to differentiate into hepatocytes.”

De-repression of p19Arf and proliferation defects, and impairment of hepatoblast migration, is a consequence rather than a cause of aberrant cholangiocyte differentiation.

“Based on molecular phenotyping and overexpression experiments in vitro, we propose that de-repression of p19 Arf and proliferation defects, and impairment of hepatoblast migration is a consequence rather than a cause of aberrant cholangiocyte differentiation.”

Severe reduction of cell proliferation in the hepatic epithelium at E9.5 is likely to cause the failure of hepatoblast liver bud expansion in Tbx3-/- mice.

“Hence, severe reduction of cell proliferation in the hepatic epithelium at E9.5 is likely to cause the failure of hepatoblast liver bud expansion in Tbx3 Ϫ/Ϫ mice.”

There is a Tbx3-repressed transcriptional repressor of Hnf4a and Cebpa transcription.

“our cell culture experiments with a dominant-negative form of Tbx3, Tbx2-VP16, argue for the presence of a Tbx3-repressed transcriptional repressor of Hnf4a and Cebpa transcription.”

Proliferation and migration defects in the Tbx3-deficient liver bud are secondary/late consequences of cell fate changes, not their cause.

“Proliferation and migration defects in the Tbx3-deficient liver bud are a consequence rather than a cause of aberrant cell differentiation.”

• Also stated in: § Proliferation and Migration Defects in the Tbx3-Deficient Liver Bud Are Secondary to Cell Fate Changes. · hedged

  “Hence, proliferation and migration defects and up-regulation of p19 Arf are likely to be secondary and late consequences of the cell fate changes in the liver bud.”

Tbx3 functions indirectly by repressing a transcriptional repressor of Hnf4 and Cebpa.

“downregulation of Hnf4 and Cebpa is compatible with the notion that Tbx3 functions indirectly by repressing a transcriptional repressor of these genes.”

• AssumptionRests on an assumption: Tbx2-VP16 competes with Tbx3 for the same conserved DNA binding sites and activates transcription, thus acting as a dominant negative version of the transcriptional repressor Tbx3.

Hepatoblast differentiation became redirected to cholangiocytes in Tbx3-/- hepatic epithelium.

“suggest that hepatoblast differentiation became redirected to cholangiocytes.”

Tbx3 expression in multipotent hepatoblasts supports proliferation and hepatic differentiation of these progenitor cells by repression of p19 Arf (Cdkn2a).

“The authors showed that Tbx3 expression in multipotent hepatoblasts supports proliferation and hepatic differentiation of these progenitor cells by repression of the tumor suppressor gene p19 Arf (Cdkn2a).”

• cites [6]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [7]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The profile of Tbx3 expression is compatible with a primary role of Tbx3 in hepatoblast expansion, migration, or differentiation during liver bud development.

“Hence, the profile of Tbx3 expression is compatible with a primary role of Tbx3 in hepatoblast expansion, migration, or differentiation during liver bud development.”

Expression of Shh remained excluded indicates that deregulation of Sonic hedgehog does not underlie this hepatic phenotype.

“Expression of the gene encoding the signaling molecule Sonic hedgehog (Shh) remained excluded from the hepatic endoderm in the Tbx3-mutant embryo (Supporting Fig. 2), indicating that deregulation of Sonic hedgehog does not underlie this hepatic phenotype”

Tbx3 is a transcriptional regulator of hepatobiliary lineage decision in the liver bud.

“Here, we have identified Tbx3 as a transcriptional regulator of hepatobiliary lineage decision in the liver bud.”

Expansion and differentiation of hepatic progenitors is severely disturbed in Tbx3-deficient hepatic tissue.

“This suggests that expansion and differentiation of hepatic progenitors is severely disturbed in Tbx3-deficient hepatic tissue.”

Tbx3 is required for the transition from a hepatic diverticulum with a pseudo-stratified epithelium to a cell-emergent liver bud in the mouse.

“Here, we show that in the mouse the transcriptional repressor Tbx3, a member of the T-box protein family, is required for the transition from a hepatic diverticulum with a pseudo-stratified epithelium to a cell-emergent liver bud.”

• Also stated in: § Tbx3 and Onecut1 Antagonistically Regulate Hepatobiliary Fate Decision. · plain

  “Tbx3 promotes the progression from the pseudo-stratified epithelium (E9.0) to a cell-emergent liver bud (E9.5).”

Inhibition of Tbx3 and activation of Onecut1 synergize to promote cholangiocyte differentiation in Hepa1-6 cells.

“Cotransfection of Onecut1 in these cells further repressed Hnf4a expression and activated the cholangiocyte differentiation marker Ck7, robustly suggesting that inhibition of Tbx3 and activation of Onecut1 synergize in (trans-) differentiation into cholangiocytes (Fig. 7B).”

Low-level expression of Tbx3 in mature hepatocytes is required to maintain their fate and prevent trans-differentiation into cholangiocytes.

“our overexpression experiments in hepatoma cells argue that low-level expression of Tbx3 in mature hepatocytes is required to maintain the fate of these cells and prevent trans-differentiation into cholangiocytes.”

In Hepa1-6 cells, continued low-level Tbx3 expression is required to maintain hepatocyte phenotype.

“Hepa1-6 also resulted in the repression of hepatocyte marker genes, arguing that continued albeit low-level expression of Tbx3 is required to maintain the phenotype of hepatocytes (Fig. 7B).”

Onecut1 and Oc2 are unlikely to be causally involved in the proliferation and migration defects in Tbx3-deficient liver buds.

“Up-regulation of Onecut1 in the Tbx3-decifient liver buds makes it unlikely that the two factors are causally involved in the proliferation and migration defects.”

Endogenous Onecut1 is unlikely to mediate Tbx3 function on hepatocyte marker genes in these cellular systems.

“However, it is unlikely that endogenous Onecut1 mediates Tbx3 function on hepatocyte marker genes in these cellular systems, because Onecut1 expression was surprisingly activated by Tbx3 in all cell lines tested (Fig. 7).”

Down-regulation of Prox1 may cause the proliferation/migration phenotype in Tbx3-deficient liver buds.

“We favor instead that down-regulation of Prox1 may cause this phenotype.”

Tbx3 maintains hepatocyte differentiation by controlling an early transcription factor network.

“In summary, our in vitro experiments further support the role of Tbx3 in the maintenance of hepatocyte differentiation by controlling an early transcription factor network.”

Hepatocyte fate is lost at the expense of cholangiocyte fate in E9.5 Tbx3-/- liver bud.

“indicating that the hepatocyte fate is lost at the expense of the cholangiocyte fate.”

p19 Arf is not involved in the hepatobiliary lineage decision.

“Levels of p19 Arf were unaffected by changes of Tbx3 and Onecut1 expression, providing additional evidence that p19 Arf is not involved in the hepatobiliary lineage decision.”

Onecut1 is an inducer of cholangiocyte differentiation.

“They indicate an important role of Onecut1 as an inducer of cholangiocyte differentiation.”

Tbx3 promotes the progression from the pseudo-stratified epithelium (E9.0) to a cell-emergent liver bud (E9.5).

“Tbx3 promotes the progression from the pseudo-stratified epithelium (E9.0) to a cell-emergent liver bud (E9.5).”

Down-regulation of Prox1 in the Tbx3 mutant likely reflects the hepatoblast fate switch at E9.5.

“down-regulation of Prox1 in the mutant is likely to reflect the hepatoblast fate switch at E9.5.”

• cites [31]Not matched

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Tbx3 is a bona fide transcriptional repressor.

“because Tbx3 is a bona fide transcriptional repressor.29”

• cites [29]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.