Cell News | Issue 04, 2019 - page 25

Cell News 04/2019 25
development. Strikingly, lymphatic vessels of Cxcr5-/- mice were
filled with intralymphatic CD4+ clusters (Fig. 3G). Such clusters
were biggest at the presumptive iLN site, but smaller clusters
were also disseminated throughout the lymphatic vascular
network (Fig. 3G). Furthermore, the number of isolated CD4+ cell
in the skin interstitium was increased (Fig. 3H). Our results thus
show that extravascular LTi cell accumulation is a pre-requisite
for the induction of lymphatic vessel remodeling and LN capsule
formation.
Next, we asked whether disruption of collecting vessel special-
ization in late embryogenesis affects LN development. In Foxc2-/-
mice primary lymphatic capillary plexus fails to remodel into
collecting vessels (Norrmén et al., 2009; Petrova et al., 2004).
We analyzed LN development in Foxc2f/f; Prox1-CreERT2 (Fox-
c2lecKO) mice with LEC-specific loss of FOXC2. As described pre-
viously (Sabine et al., 2015), Foxc2lecKO embryos had a complete
arrest of collecting vessel development, as determined by the
absence of valves and expression of capillary markers LYVE1 and
CCL21 in all LECs (Fig. 4A). In parallel with the failed collecting
vessel maturation, formation of the LN capsule was severely
disrupted: instead of a continuous lymphatic “cup”, the Foxc2lecKO
iLN anlage was surrounded by a mesh-like vasculature, only
partially enclosing the LN (Fig. 4B). In the adult LN, SCS contains
distinct populations of LECs, “ceiling” (cLECs) and “floor” (fLEC)
LECs (Ulvmar et al., 2014). Only cLECs produce the chemokine
receptor CCRL1 while fLECs express LYVE1, ITGA2B and Mad-
CAM1 (Cohen et al., 2010; Cordeiro et al., 2016; Ulvmar et al.,
2014). In embryonic LNs, we identified the same expression
pattern, suggesting that specialization of SCS LECs is established
prenatally (Fig. 4, C-E). Impaired lymphatic vessel maturation,
however, led to a loss of capsule organization. LN LECs in Fox-
c2lecKO mice failed to express SCS LEC markers ITGA2B or CCRL1,
whereas LYVE1 and MadCAM1 were uniformly high (Fig. 4, C-E).
Moreover, mislocalized SMCs encircled the LN lymphatic vessels
(Fig. 4, F and F’). Finally, in adult LNs, the CD169+ macrophages
inserted into the SCS fLEC layer prevent dissemination of patho-
gens and deliver antigens to adjacent B cells (Carrasco and Ba-
tista, 2007; Junt et al., 2007; Phan et al., 2007). Surprisingly, we
observed CD169+ macrophages at the bottom of wildtype E18.5
iLN anlagen, close to the lymphatic capsule (Fig. 4G). However,
in Foxc2 lecKO mice, the number of macrophages was reduced and
they were dispersed throughout the LN (Fig. 4G). These results
demonstrate that FOXC2 ensures both collecting vessel matura-
tion and pre-natal LN LEC capsule specialization.
From E18.5 onwards the Foxc2lecKO iLNs were significantly smaller
than controls (Fig. 5A). Organization of axillary and mesenteric
LNs was also perturbed, but the formation of non-encapsulated
Peyer’s patches was not affected (Fig. 5, B-F). We next investi-
gated the reason for the reduced Foxc2 lecKO LN size. We observed
accumulation of intra- and extra- vascular CD4+ cells and large
intralymphatic clusters both in the vicinity of LN anlagen and
throughout the dermal lymphatic vessels of Foxc2lecKO mice (Fig
5, G-K). These results demonstrate that collecting vessel matu-
ration is essential for LN capsule formation, SCS specialization
and further LN expansion. We then compared the expression
of chemokines (Cxcl13, Ccl21 and Ccl19), LTi (Lta and Ltb) and
PRIZE WINNERS 2019
Figure 5. Impaired LN expansion and trafficking of LTi cells in
Foxc2lecKO mice.
(A) iLN size in WT and Foxc2 lecKO embryos. E16.5, n = 4 and 3;
E18.5-E19.0, n = 9 and 12; E20.0 n = 3 and 3 for WT and Foxc2lecKO
genotypes, respectively. ns, not significant; *P < 0.05. (B) Impaired
axillary LN development in E20.5 Foxc2lecKO embryos. Staining for LYVE1
(green), PROX1 (red) and CD4 (white). Asterisk, swollen LN lymphatics
in Foxc2 lecKO mice. n = 2 per genotype. Scale bar, 100 μm. (C) Impaired
mesenteric LN development in E18.5 Foxc2lecKO embryos. Staining for
CD4 (green) and PROX1 (white), 10-μm transverse sections. n = 4
per genotype. Scale bar, 200 μm. (D) Normal development of Peyer’s
patches (PPs) in E19.0 Foxc2lecKO embryos. Whole-mount staining for
CD4 (green) and LYVE1 (red). Arrowheads, sprouting LECs. WT n = 4;
Foxc2lecKO n = 3. Scale bar, 50 μm. (E) Quantification of E18.5 PP size.
WT n = 6; Foxc2lecKO n = 4; ns, not significant. (F) Quantification of
E18.5 PP number. WT n = 9; Foxc2 lecKO n = 5; ns, not significant. (G)
Increased isolated CD4+ LTi cells in Foxc2lecKO dermis and lymphatic
vessels. Whole-mount E18.5 skin; CD4 (green) and PROX1 (red). Pink
arrowheads, intralymphatic CD4 cells; white arrowheads, extravascular
CD4 cells. WT n = 3; Foxc2lecKO n = 4. Scale bar, 50 μm. (H) Quantifica-
tion of isolated extra- and intra-lymphatic CD4+ cells in E18.5-E19.0
WT and Foxc2lecKO embryos. WT n = 6 and Foxc2lecKO n = 7. *P < 0.05. (I)
Intralymphatic CD4+ clusters in skin of Foxc2 lecKO embryo. Whole-mount
staining for CD4 (green) and PROX1 (red). E19.0 WT n = 3; Foxc2lecKO
n = 4. Scale bar, 50 μm. (J) Quantification of intralymphatic CD4+
clusters in skin of E18.5-19.0 WT (n = 9) and Foxc2lecKO ( n = 10) mice.
*P < 0.05. (K) CD4+ intralymphatic cluster size in E18.5-E19.0 skin of
WT (n = 9) or Foxc2lecKO (n = 10) embryos. *P < 0.05. All quantifications,
2-tailed unpaired Student’s t test.