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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.
