16
knock-out mice, the formation of hippocampal structure was severely affected (Lee
et al. 2000 ; Galceran et al. 2000 ). In contrast, the formation of ectopic cortical hem
by chimera mouse of conditional Lhx2 knock-out showed ectopic generation of hip-
pocampal structure next to additionally induced hem (Mangale et al. 2008 ). Taken
together, these in vivo results suggest the necessity and sufficiency of cortical hem
and Wnt signaling for induction of hippocampus and the necessity of BMP signal-
ing for the patterning of dorsomedial telencephalic tissues.
1.4.2 Generation of Dorsomedial Telencephalic Tissues
In Vitro
Based on in vivo developmental cues, the more dorsal regions (cortical hem, cho-
roid plexus, and medial pallium) can be generated by treatment with Wnt and BMP
signals (Eiraku et al. 2008 ; Sakaguchi et al. 2015 ). Regarding choroid plexus induc-
tion in vitro, there have been three reports using mouse and human ES cells (Eiraku
et al. 2008 ; Watanabe et al. 2012 ; Sakaguchi et al. 2015 ). In mouse ES cells, treat-
ment with BMP4 promoted the generation of transthyretin (TTR)-positive cells, and
TTR is a marker specific for choroid plexus in nervous tissues (Eiraku et al. 2008 ;
Watanabe et al. 2012 ). The induced aggregates contained a thin epithelial or vesicle-
like choroid plexus structure, with tight junctions confirmed by ZO-1 expression
and electron microscopy (Watanabe et al. 2012 ). In addition, the structure could
integrate into endogenous choroid plexus epithelium following intraventricular
transplantation. This dorsalizing strategy can also be applied to a human ES cell
system, and treatment with BMP4 alone could sufficiently induce TTR+ choroid
plexus tissues as in the mouse study (Watanabe et al. 2012 ; Sakaguchi et al. 2015 ).
Furthermore, treatment with BMP4 combined with Wnt signaling further enhanced
choroid plexus induction (Sakaguchi et al. 2015 ) (Fig. 1.6b). The treatment of
SFEBq-induced neocortical tissues with BMP4 ligand plus CHIR 99021 (GSK3
inhibitor, also known to accelerate canonical Wnt signaling), a condition designed
to mimic the in vivo dorsal midline and cortical hem signaling center, significantly
increased lmx1a, otx2, and ttr mRNA expression. Immunohistochemistry showed
that Lmx1a (Failli et al. 2002 ), water channel Aqp1 (Praetorius and Nielsen 2006 ),
and TTR signal were mainly seen apically in the thin NE portion and tight junction
marker ZO-1 was on the surface of epithelia (Fig. 1.6c, d). Interestingly, these
aggregates have a pleated NE structure as seen in human embryonic choroid plexus
tissues. This morphological character does not occur without Wnt treatment
(Sakaguchi et al. 2015 ). These characteristics in morphology and marker distribu-
tion are reminiscent of those in human fetal choroid plexus epithelia. Collectively,
treatment with CHIR + BMP signals to SFEBq-induced neocortical tissues could
dominantly induce choroid plexus tissues from human ESCs in 3D culture. This
culture system might be useful for exploring choroid plexus development with
regard to structure formation, CSF generation, barrier formation, and so on.
T. Kadoshima et al.