200
11.2.2 Inducing the Intermediate Mesoderm
During Development and In Vitro
The trunk mesoderm of the vertebrate embryo is formed by migration of cells from
the posterior PS. This mesoderm extends along the trunk of the embryo and is pat-
terned from the middle of the embryo outwards (mediolaterally) into three regions:
the paraxial, intermediate and lateral plate mesoderm. It is the intermediate meso-
derm (IM), marked by Osr1, Lhx1 and Pax2, that gives rise to the kidneys and the
indeterminate gonad (Fig. 11.2). Hence, to generate this particular mesodermal
region requires an understanding of how this mediolateral patterning occurs. Three
growth factor pathways are involved in this process: BMP, nodal and FGF signalling
(Takasato and Little 2015 ). High BMP signalling specifies lateral plate mesoderm as
this region, as well as the overlying surface ectoderm, produces BMP4 (Obara-
Ishihara et al. 1999 ; James and Schultheiss 2005 ). Inhibition of BMP signalling,
specified in the embryo via the production of the BMP inhibitor, noggin, by the
notochord, is required for paraxial mesoderm fate (Wijgerde et al. 2005 ). It is there-
fore proposed that the IM develops under an intermediate level of BMP signalling.
Similarly, inhibition of nodal signalling via the production of the inhibitor Cer1 from
the paraxial mesoderm is critical for paraxial mesoderm specification (Biben et al.
1998 ). In the absence of Cer1, there is an expansion of the IM (Fleming et al. 2013 ).
Finally, FGF9 is produced by the paraxial and intermediate mesoderm in the caudal
embryo, within this more restricted to IM in the trunk region (Colvin et al. 1999 ).
The trunk of the embryo extends from the head to the tail across time; hence
there are also distinct differences in specification within all mesodermal domains
across time and along this rostro-caudal (head-to-tail) axis. Indeed, three distinct
pairs of excretory organs form along this axis (pronephros, mesonephros and meta-
nephros) with the metanephroi representing the most caudal of these organs. A com-
mon component to all three excretory organs is the epithelium of the nephric duct.
This initially forms early, and hence from anterior IM, and then extends along the
embryo as the trunk elongates (Takasato et al. 2014 ; Taguchi et al. 2014 ; Takasato
and Little 2015 ). Conversely, the cells contributing to the MM arise later and in a
more caudal location. As a result, cells of the MM are subjected to prolonged canon-
ical Wnt signalling within the caudal embryo and are also protected from retinoic
acid signalling via the expression of Cyp26a. Clearly, to recreate a kidney, both
anterior and posterior IM derivatives are required.
Attempts to recreate IM in vitro from hPSCs initially focussed on the generation
of OSR1+ mesenchymal populations (Mae et al. 2013 ). However, after initial poste-
rior PS patterning, we have reported the spontaneous formation of OSR1+ FOXF1+
lateral plate mesoderm (Takasato et al. 2014 ), highlighting the non-specificity of
OSR1 as an IM marker. Instead, it has been shown that FGF signalling, using either
FGF2 or FGF9 with or without retinoic acid (RA), results in the induction of PAX2+
LHX1+ IM cells (Lam et al. 2014b). This suggested in human that FGF9 signalling
is sufficient to specify IM even in the absence of BMP suppression. To test the
hypothesis that rostro-caudal patterning could also be regulated in vitro, we varied
M.H. Little et al.