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including separation of the trachea and esophagus, branching morphogenesis, and
differentiation of lung epithelium (Kimura et al. 1996 ; Minoo et al. 1999 ).
The coordinated expression of RA, FGF, Wnt, and BMP signaling pathways is
important for the efficient induction of NKX2–1+ progenitors. Work featuring a
mouse foregut culture system has found that Fgf10 signals from the mesoderm help
to induce Nkx2–1 expression and lung bud outgrowth (Chen et al. 2007 ; Desai et al.
2004 ). Wnt2/Wnt2b expression from the mesoderm surrounding the ventral foregut
endoderm is essential for efficient Nkx2–1 induction as their combined deletion or
that of β-catenin in epithelial cells blocks lung formation and allows minimal, tran-
sient Nkx2–1 expression (Goss et al. 2009 ; Harris-Johnson et al. 2009 ). The local
mesenchymal expression of BMP ligands is likewise essential for robust Nkx2–1
induction, possibly in part through repression of Sox2, with deletion of Bmpr1a and
Bmpr1b resulting in tracheal agenesis (Domyan et al. 2011 ). BMP-induced repres-
sion of Sox2 results in derepression of Nkx2–1, potentially facilitating the induction
capacity of canonical Wnt signaling. Recently published data in Xenopus suggests
two temporally distinct roles for RA in this process (Rankin et al. 2016 ). Through
mechanisms that remain to be defined, RA promotes the WNT2/2B–mediated spec-
ification of Nkx2–1+ lung progenitors via induction of endodermal hedgehog (Hh)
signaling at developmental stage NF15–NF25. Inhibition of RA signaling during
this stage resulted in loss of Wnt2b, Fgf10, Nkx2–1, and Sftpc expression. This pro-
cess is independent of FGF10, as evidenced by the failure of recombinant FGF10 to
rescue the phenotype. Slightly later in Xenopus development (NF25–NF38), how-
ever, recombinant FGF10 was able to restore expression of Wnt2b and Nkx2–1
together with subsequent expression of Sftpc, consistent with stage-specific mecha-
nistic differences in regulation. While additional work by the same group has con-
firmed that RA acts upstream of Hh/Wnt to specify Nkx2–1 lung progenitors in
mouse explant cultures, the stage-specific relationship between RA and FGF10 has
yet to be confirmed in mammals.
At approximately the same time as Nkx2–1 specification, a gradient of BMP and
Wnt signaling along the D/V axis of the developing foregut tube promotes its sepa-
ration into two separate tracheal and esophageal tubes. BMP, FGF, and Wnt ligands
are expressed from the ventral mesoderm, while the BMP antagonist Noggin is
expressed dorsally (Rodewald 2008 ). Together, these signals pattern the foregut
resulting in high levels of Sox2 expression dorsally in the nascent esophagus and
Nkx2–1 ventrally in the nascent trachea. Hh signaling likewise plays a key role in
this process, with Shh ligand from the foregut and early respiratory epithelium sig-
naling to the surrounding mesenchyme where they support mesenchymal prolifera-
tion and stimulate the expression of Wnt and BMP family members (Pepicelli et al.
1998 ). This is clearly demonstrated in Shh−/− (Litingtung et al. 1998 ) or Gli2−/−/
Gli3+/− (Motoyama et al. 1998 ) mutant mice that are characterized by profound
foregut defects, including esophageal atresia and tracheoesophageal fistula.
To successfully derive Nkx2–1 expressing respiratory progenitor cells, in vitro
directed differentiation protocols seek to recapitulate temporally coordinated
expression of these same signaling pathways (Green et al. 2011 ; Longmire et al.
2012 ; Mou et al. 2012 ). Following the generation of C-KIT+/CXCR4+/EPCAM+
A. Wilson and L. Ikonomou