161level of BMP signaling for subsequent IM marker gene expression following in vitro
culture of the presomitic mesenchyme and in vivo ectopic activation of BMP signal
within the PAM (Mauch et al. 2000 ; James and Schultheiss 2003 ; James and
Schultheiss 2005 ). Another report has identified a putative role for Vg1/Nodal sig-
nal in the specification of IM (Fleming et al. 2013 ). These results indicate that fac-
tors from the neighboring PAM and LPM presumably signal to give the IM identity
by establishing the dorsoventral signal gradients following migration from the prim-
itive streak.
9.5.3 A-P Patterning Within the Intermediate Mesoderm
Because the distinct three types of kidney primordia form within the IM, it is essen-
tial to understand the A-P axis specification mechanism within the IM.
The initial A-P patterning within the IM becomes apparent at approximately
E9.5 (Fig. 9.4a). Although the representative IM markers Osr1 and Wt1 are uni-
formly expressed in the IM at this stage, there exist distinct differences between the
anterior and posterior regions of the body trunk. The anteriorly located IM at this
stage (just beneath the forelimb level) exhibits a condensed structure—the nephro-
genic cord—which is reminiscent of the capping mesenchyme in the metanephros
(Fig. 9.4b). Indeed, they share a molecular signature with mature metanephric neph-
ron progenitors: expression of Pax2, Six2, and Gdnf and the cell surface pattern
Itga8+/Pdgfra− (Taguchi et al. 2014 ). Contrastingly, the posterior IM (PIM; located
at the hind limb level) at this stage is negative for these markers, indicating that they
are a newly formed immature population. Meanwhile, the PIM begins to express the
posterior Hox genes including the Hox10, Hox11, and Hox12 families, which are
essential for metanephros development (Wellik et al. 2002 ; Mugford et al. 2008a).
These findings suggest that the anteriorly located IM at this stage is the formerly
developed progenitor for anterior kidney rudiments (mesonephros) and the PIM is
the later-developing precursor for the posterior kidney anlage (metanephros).
Lineage tracing experiments using Six2-CreER mice identified that the Six2-
positive anteriorly located IM at E9.5 contributes predominantly to the mesonephric
nephrons but not to the metanephros. Accordingly, sorted Osr1- or Wt1-positive
PIM cells at E9.5 could be induced to differentiate into metanephric nephron pro-
genitors in vitro (Fig. 9.5). Here, the addition of retinoic acid, activin, and Bmp4 in
the presence of a high concentration of Wnt agonist inhibited induction of nephron
progenitors. Meanwhile, addition of Fgf9 and a low concentration of Wnt agonist
synergistically promoted metanephric nephron progenitor induction from the
Wt1+ PIM.
Taken together, the above findings suggest that anteriorly located IM at E9.5,
which exhibits a mature signature pattern of nephron progenitors and predominantly
contributes to the mesonephros, may not migrate to give rise to the metanephric
nephron progenitors. Meanwhile, the PIM—which expresses posterior Hox genes—
matures later to develop into the metanephric nephron progenitors (Figs. 9.5 and 9.6).
9 Early Kidney Specification and Its Recapitulation by Pluripotent Stem Cells