61et al. 2005 ; Pieper et al. 2012 ; Saint-Jeannet and Moody 2014 ). The cranial placodes
are arranged anteroposteriorly, and the development of each placode is dependent
upon the expression of various Pax proteins. Pax proteins are transcription factors
that modulate organogenesis through regulation of cell proliferation, apoptosis,
migration, and maintenance of stem cell pluripotency (Chi and Epstein 2002 ; Lang
et al. 2007 ). In vertebrates, Six1 is necessary for Pax protein expression and placode
development (Christophorou et al. 2009 ). Pax2 and Pax8 are central to inner ear
development and are markers of the otic epibranchial placode domain (OEPD)
(Baker and Bronner-Fraser 2001 ; Bouchard et al. 2004 ; Ohyama et al. 2006 ;
Schlosser 2006 ; Pieper et al. 2012 ). Induction of the OEPD and subsequent Pax2/
Pax8 expression is mediated by FGF signaling. Despite the lack of uniformity of
FGF signaling among species, Fgf3/8/10/19 mediates induction of OEPD in mam-
mals. (Wright and Mansour 2003 ; Schimmang 2007 ; Ladher et al. 2010 ; Urness
et al. 2010 ).
4.2.3 Formation of the Otic Placode
After induction of the OEPD, the functional segregation of the otic and epibranchial
placodes is mediated by Wnt signaling and further upregulation of Pax2 within the
otic placode ( Groves and Bronner-Fraser 2000 ; Wright and Mansour 2003 ). In
mice, specification of the otic placode is mediated by expression of Wnt ligands
including Wnt8a from the caudal hindbrain ( Ladher et al. 2000 ; Freter et al. 2008 ;
Vendrell et al. 2013 ). In addition, the role of Wnt signaling in otic specification is
further supported by mouse studies in which deletion of β-catenin reduces the size
of the otic placode (Wright and Mansour 2003 ). It has been postulated that Pax2
contributes to the thickened pseudostratified morphology of the otic placode as
compared to the surrounding epithelium (Christophorou et al. 2010 ). This thicken-
ing may enable actions on the basal and apical aspects of placodal cells, such as
expression of cellular adhesion molecules, facilitating subsequent placode invagina-
tion, and formation of the otic vesicle (Christophorou et al. 2010 ; Sai and Ladher
2015 ).
From its superficial position in the embryo, the otic placode undergoes morpho-
genetic changes to attain the relatively insulated position of the fully developed
inner ear. Invagination into the head mesenchyme may be mediated by expansion of
the basal region of the placodal epithelium followed by apical constriction (Sai et al.
2014 ). This biphasic process promotes depression of the otic placode to form the
otic pit, followed by complete encapsulation as a hollow sphere known as the otic
vesicle or otocyst wherein differentiation of sensory cells and neuronal components
takes place (Freter et al. 2008 ).
4 Inner Ear Organoids: Recapitulating Inner Ear Development in 3D Culture