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MicroPubl Biol
2025 May 07;2025. doi: 10.17912/micropub.biology.001610.
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Temporal induction of the homeodomain transcription factor Nkx2-1 is sufficient to respecify foregut and hindgut endoderm to a pulmonary fate in Xenopus laevis.
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The ability of transcription factors (TFs) to regulate cell fate decisions is paramount in developmental, homeostatic, and pathogenic contexts. The homeodomain TF NKX2-1 is an essential and evolutionarily conserved master regulator of pulmonary fate in vertebrates. In this study, we tested the spatial-temporal ability of Xenopus and Human NKX2-1 to respecify foregut and hindgut endoderm in developing Xenopus laevis embryos into a pulmonary fate, as indicated by expression of pulmonary surfactant genes sftpc and sftpb . Interestingly, we find that both Human and Xenopus NKX2-1 can induce the ectopic expression of pulmonary surfactant genes in foregut and hindgut endoderm over a wide range of developmental times, as well as suppress the expression of midgut and hindgut specific genes. These results suggest a single pulmonary TF can reprogram developing endoderm and specify pulmonary fate. In addition, our work provides a comparative platform for future studies investigating how mutations in Human NKX2-1 may affect its transcriptional activity.
Figure 1. Spatial-temporal overexpression of Xenopus and Human Nkx2-1 results in ectopic pulmonary fate and suppressed midgut and hindgut fates.
(A) Experimental schematic of Nkx2-1 Gain of function (GOF) assay in Xenopus embryos. 400pg of mRNA encoding GR-fusions of Xenopus Nkx2-1 and Human NKX2-1 was co-injected with 50pg GFP mRNA into dorsal-vegetal (targeting FG) or ventral vegetal blastomeres (targeting HG) blastomeres at the 8-cell stage, and embryos were transferred into culture buffer containing Dexamethasone (Dex) at the indicated stages to induce nuclear Nkx2-1 activity. (B) GFP fluorescence images showing representative FG and HG targeting. (C-E) In situ hybridization analysis of experimental embryos. Representative staining patterns are shown for each condition with the numbers of embryos displaying that pattern listed; numbers are the sum of 4 combined injection experiments. Green arrows indicate normal endogenous expression of the gene assayed, red arrows indicate ectopic expression domains, and black arrows indicate reduced expression. (C) Analysis at NF39 for the indicated genes after Dex induction at NF13: pulmonary specific marker
sftpc, sox2 marks foregut/esophogeal and stomach (st) endoderm, ifabp
marks midgut endoderm, and cdx2 marks mid/hindgut endoderm. (D) Analysis of sftpc expression at NF29, before the normal onset of endogenous sftpc, reveals an early, precocious onset of ectopic
sftpc at NF29 in response to Xenopus Nkx2-1. (E) Nkx2-1 is sufficient to induce pulmonary fate in progressively older HG endoderm. Xenopus Nkx2-1 was injected into the HG and activity was induced via DEX treatment at NF 17, NF24, or NF29; embryos were assayed at NF39. Ectopic activation of
sftpc in the HG is observed in response to all temporal inductions. Abbreviations: lb is lung bud, st is stomach, mg is midgut, and hg is hindgut.
Figure 1.
Spatial-temporal overexpression of Xenopus and Human
Nkx2-1
results in ectopic pulmonary fate and suppressed midgut and hindgut fates
.
(A)
Experimental schematic of
Nkx2-1
Gain of function (GOF) assay in Xenopus embryos. 400pg of mRNA encoding GR-fusions of Xenopus Nkx2-1 and Human NKX2-1 was co-injected with 50pg GFP mRNA into dorsal-vegetal (targeting FG) or ventral vegetal blastomeres (targeting HG) blastomeres at the 8-cell stage, and embryos were transferred into culture buffer containing Dexamethasone (Dex) at the indicated stages to induce nuclear Nkx2-1 activity. (
B)
GFP fluorescence images showing representative FG and HG targeting. (
C-E
)
In situ
hybridization analysis of experimental embryos. Representative staining patterns are shown for each condition with the numbers of embryos displaying that pattern listed; numbers are the sum of 4 combined injection experiments. Green arrows indicate normal endogenous expression of the gene assayed, red arrows indicate ectopic expression domains, and black arrows indicate reduced expression. (
C
) Analysis at NF39 for the indicated genes after Dex induction at NF13: pulmonary specific marker
sftpc
,
sox2
marks foregut/esophogeal and stomach (st) endoderm,
ifabp
marks midgut endoderm, and
cdx2
marks mid/hindgut endoderm
.
(D)
Analysis of
sftpc
expression at NF29, before the normal onset of endogenous
sftpc
, reveals an early, precocious onset of ectopic
sftpc
at NF29 in response to Xenopus Nkx2-1.
(E)
Nkx2-1 is sufficient to induce pulmonary fate in progressively older HG endoderm. Xenopus Nkx2-1 was injected into the HG and activity was induced via DEX treatment at NF 17, NF24, or NF29; embryos were assayed at NF39. Ectopic activation of
sftpc
in the HG is observed in response to all temporal inductions. Abbreviations: lb is lung bud, st is stomach, mg is midgut, and hg is hindgut.
