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Fig. S5. Utilizing Xenopus system to understand the genetic interaction between Tbx5 and Shh signaling.
A. To study the interaction of Tbx5 and Hedgehog signaling, we utilized: (1) a dexamethasone (DEX) inducible glucocorticoid receptor-Tbx5 (GR-Tbx5) fusion protein, and (2) pharmacological agonists (purmorphamine) and antagonists (cyclopamine) of SMO to activate or
repress Hh signaling similar to Fig. 4 (top). Experimental details for testing Hedgehog response in anterior mesendoderm (AME, red, Shh+) and posterior mesendoderm (PME, blue, Shh-) tissues measured by gli1 expression (bottom). Dissected AME or PME tissue was cultured for 6 hours in combinations of dexamethasone (DEX, translocates GR-Tbx5 fusion protein to the nucleus),
purmorphamine (Hh agonist), and cyclopamine (Hh antagonist). B. Expression of gli1 examined
by qRT-PCR in culture conditions as indicated by the checked boxes. All conditions were normalized to the AME controls (far left). C. Experimental plan for investigating markers of lung development in embryos injected with mistmatched morpholinos (MM-MOs) or Tbx5-MOs. Embryos were cultured in DMSO (grey) or Hh agonist (purmorphamine, blue) for 48 hours. D. qRT-PCR of shh, dhh, gli1, and nkx2-1 from Tbx5-morphant or control embryonic explants treated with DMSO or Hh agonist. |
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Display additional annotations [+]
Gene |
Clone |
Species |
Stages |
Anatomy |
nkx2-1
|
|
laevis
|
NF stage 35 and 36
|
forebrain
,
thyroid primordium
,
lung primordium
|
tbx5
|
|
laevis
|
NF stage 35 and 36
|
otic vesicle
,
retina
,
cardiac mesoderm
,
lateral plate mesoderm
|
wnt2b
|
|
laevis
|
NF stage 35 and 36
|
otic vesicle
,
lens
,
midbrain
,
midbrain-hindbrain boundary
,
ventral blood island
,
[+]
|
|
|
Fig. S1. Xenopus whole mount in situ hybridization. RNA in situ hybridization was performed in NF35 Xenopus embryos for tbx5, wnt2b, nkx2-1, and shh (whole mount side-view on top, transverse cross-section on bottom). lpm = lateral plate mesoderm, thy = thyroid, nc = notochord,
phe = pharyngeal endoderm, fge = foregut endoderm, gb = gall bladder, fp = floor plate. |
|
|
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shh (sonic hedgehog) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35 & 36, lateral view, anterior left, dorsal up (top image) and transverse section (below).
Key: nc = notochord, phe = pharyngeal endoderm, fge = foregut endoderm, gb = gall bladder, fp = floor plate. |