19911.2.1 Induction of the Primitive Streak During Development
and In Vitro
The generation of both endodermal and mesodermal tissues commences with gas-
trulation, the process by which cells of the epiblast go through an epithelial-to-
mesenchymal (EMT) transition to form the primitive streak (PS) (Fig. 11.2). In the
mouse at 6.5 days post coitum, the anterior visceral endoderm forms on the distal
epiblast and then migrates to the anterior side where it produced inhibitors of nodal,
BMP and Wnt signalling. This results in the formation of the PS on the opposite side
of the epiblast in response to activity of these same pathways (Perea-Gomez et al.
2002 ). Expression of BMP4 by the extraembryonic ectoderm (EE) adjacent to the
posterior PS activates Wnt3 expression (Ben-Haim et al. 2006 ), resulting in canoni-
cal β-catenin signalling within PS cells that in turn results in the expression of
Nodal, Mixl1, Eomes and Brachyury (T) (Funa et al. 2015 ). Conversely, nodal sig-
nalling is essential for PS formation via upregulation of Bmp4 in the EE. Both
mesoderm and endoderm arise from the PS with fate determined by the position of
cells along the streak. While Mixl1 is expressed along the PS, expression is higher
in the anterior PS which will form endoderm, this region also expressing Sox17,
Foxa2 and Eomes. Conversely, the posterior PS is marked by T, Meox1 and KDR.
This fate is patterned by the Bmp4/nodal gradient along the axis of the PS which is
established via the presence of the BMP antagonists, chordin and noggin, in the
anterior PS and the node counteracting the BMP4 secreted from the EE near the
posterior PS (Bachiller et al. 2000 ). Activation of nodal/activin receptors upregu-
lates the transcription of Mixl1 (Hart et al. 2002 ; Pereira et al. 2012 ) determining
endodermal fate.
Protocols for the induction of PS have been key for the generation of a variety of
both endodermal and mesodermal tissues from hPSCs. Based on this understanding
of early patterning, most employ combinations of nodal signalling (usually mim-
icked by activin A), BMP signalling (usually via the addition of recombinant BMP4)
and canonical Wnt signalling (either via the addition of recombinant Wnt3a or
inhibitors of GSK3β such as CHIR99021 or BIO) (Davis et al. 2008 ; Sumi et al.
2008 ; Burridge et al. 2012 ). Anterior PS has been induced via inhibition of BMP
signalling using noggin, while posterior PS patterning is favoured via inhibition of
nodal signalling. Mesodermal tissues such as cardiomyocyte are often initiated
using a combination of activin A and BMP4, sometimes with FGF2. We have shown
that varying the relative concentrations of BMP4 and activin A can alter specifica-
tion from anterior (low BMP4, high activin A) to posterior (high BMP4, low Activin
A) PS based on relative changes in Sox17 versus T expression (Takasato et al. 2014 ).
Indeed, induction of posterior PS from human iPSC was shown to be as effective
using canonical Wnt activation alone (CHIR99021) (Takasato et al. 2014 ). In the
mouse system, based on evidence of a later role for activin A signalling (Jackson
et al. 2010 ), Taguchi et al. ( 2014 ) induced a T-positive ‘nascent mesoderm’ state
using BMP4 and high Wnt signalling.
11 Recapitulating Development to Generate Kidney Organoid Cultures