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signaling—this was recapitulated in vitro in the experiments to induce anterior pitu-
itary gland tissue from floating ESC aggregates (Suga 2011 ). Consistent with these
findings, we observed that addition of human BMP4 to the aggregates on day 3
upregulated the NNE marker Dlx3 and the mesendoderm marker brachyury with
concurrent downregulation of the neuroectoderm marker Sox1. Brachyury expres-
sion indicated unwanted formation of mesodermal and endodermal cell types. To
more effectively induce the NNE, we supplemented the BMP4 treatment with the
small molecule SB431542 (SB) which inhibits the transforming growth factor beta
(TGF-β) signaling pathway and mediates suppression of brachyury (Chambers et al.
2009 ). Day 5 analysis of aggregates treated with BMP4/SB demonstrated expres-
sion of the NNE marker Tfap2a mainly localized to the outer epithelium (E-cadherin+)
of the aggregates along with markedly reduced brachyury expression (Fig. 4.3e–f).
Within the outer epithelium, we also identified a layer that was positive for Sox1
and N-cadherin (Ncad), consistent with neuroectoderm formation. The core of each
aggregate was positive for Nanog, a factor necessary for maintaining pluripotency
of ESCs (Misui et al. 2003 ). Additionally, qPCR analysis of day 5 aggregates treated
with BMP4/SB demonstrated upregulation of the NNE marker Dlx3 alongside
downregulation of the neuroectodermal marker Sox1 as compared to aggregates
treated with vehicle controls. It was therefore determined that day 5 aggregates
treated with BMP4/SB represent an outer epithelium of NNE surrounding a layer of
mesendodermal and neuroectodermal components and an inner core of still-
pluripotent stem cells. Morphologically, the outer epithelium of aggregates treated
with BMP4/SB was noticeably thicker and relatively undisrupted when compared to
aggregates treated with BMP4 or SB alone. As a confirmation of the NNE identity
of this outer epithelium, long-term culture of these aggregates for 20 days in basal
medium resulted in expression of Krt5 and p63, which is consistent with epidermis
formation from NNE. The apical aspect of this epidermis is oriented toward the
aggregate interior.
4.3.2 Formation of the Preplacodal Region (PPR)
The next step toward otic differentiation is formation of the PPR, which later gives
rise to the cranial placodes, including the otic placode. Based on studies of in vivo
PPR formation, it was determined that the BMP signaling necessary for induction
of the NNE must be attenuated for PPR induction, which is also dependent on acti-
vation of the FGF signaling pathway. Based upon these observations from in vivo
organogenesis, the aggregates were treated with a combination of the BMP inhibitor
LDN-193189 (LDN) and human recombinant FGF2. We observed that BMP inhibi-
tion on day 4.5 did not counteract specification of the NNE given that aggregates
treated with LDN at that time point continued to express Dlx3. Support for a mor-
phology consistent with the PPR was found in the presence of thickened epithelial
patches in aggregates treated with BMP4/SB/LDN which were not present in aggre-
gates in the BMP4/SB treatment group. Consistent with in vivo data, this thickening
A.N. Elghouche et al.