Cell News | Issue 04, 2019 - page 26

Cell News 04/2019 26
LTo (LtbR and Vcam1) cell markers in Foxc2lecKO or wildtype iLNs.
Only Cxcl13 mRNA was significantly decreased in the absence of
FOXC2 (Fig. 6, A-B). On the protein level CXCL13 but not VCAM1
was reduced in Foxc2lecKO iLNs (Fig. 6, C-F). These results show
that lymphatic vessel specialization is important for stromal
CXCL13 expression and LN expansion.
Mechanistically, we then reasoned that LTβR signaling, supplied
by LTαβ+ LTi cells (Vondenhoff et al., 2009a), may be necessary
but not sufficient to fully activate Cxcl13 expression in LTo cells.
By absorbing plasma leaking from blood capillaries, lymphatic
vessels generate interstitial fluid flow (IFF), which has import-
ant morphogenetic functions (Starling, 1896; Wiig and Swartz,
2012). We reasoned that engulfment of LN anlagen by growing
LN capsule creates high IFF, from the moment when embryonic
maturing lymphatics initiate directional lymph transport. To an-
alyze whether IFF affects Cxcl13 expression in vitro, we cultured
mouse embryonic fibroblasts (MEFs) in 3D in the presence or ab-
sence of LTβR-activating antibody in static or IFF conditions (Fig.
7A). As expected from previous work (Chambliss et al., 2013),
IFF induced formation of actin stress fibers indicating cytoskel-
eton reorganization (Fig. 7B). Most importantly, IFF potentiated
the production of Cxcl13 mRNA in response to LTβR activation
(Fig. 7C). To study IFF in vivo, we microinjected FITC-dextran in
the forelimb interstitium of E18.5 control or Foxc2lecKO em-
bryos. Wildtype lymphatic vessels readily drained the dye but
transport into lymphatic vessels of Foxc2lecKO mice was strongly
reduced (Fig. 7, D and E). These results suggest that in addition
to their role in LTi cell transport, perinodal lymphatics promote
IFF, which cooperates with LTβR signaling to induce CXCL13 in
fibroblastic LTo cells and thereby enhances LTi cell retention in
the LN anlage (Fig. 8).
In this study, we propose that LTi cells initially transmigrate from
veins at LN development sites using gaps in venous mural cell
coverage. This process is independent of lymphatic vasculature,
but lymphatic vessels are indispensable for the transport of LTi
cells that egress from blood capillaries and serve as an essential
LN expansion reservoir. At later stages, lymphatic collecting
vessels ensure efficient LTi cell transport and formation of the
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Figure 7. Interstitial fluid flow potentiates Cxcl13 induction in response
to LTß signaling.
(A) MEFs were cultured in the left central channel of microfluidic
device. Transfer of medium from one to the other side of the device
creates a hydrostatic pressure gradient and interstitial fluid flow (IFF).
(B) IFF induces actin stress fibers in MEFs. DAPI (blue) and F-actin
(green). n = 2. Scale bar, 100 μm. (C) IFF potentiates Cxcl13 expression
in response to LTß signaling. RT-qPCR analysis of the indicated genes
after 48 hours of IFF. Cxcl13 n = 6; LtbR, Vcam1, Rankl n = 4; 1-way
ANOVA test with Tukey post-hoc test; *P < 0.05. (D) Reduced interstitial
FITC-dextran transport in E18.5 Foxc2 lecKO embryos. Arrowheads, FITC-
dextran-filled lymphatics; asterisks, injection site. Scale bar, 2.5 mm. (E)
FITC-dextran lymphatic uptake in WT and Foxc2lecKO mice. E18.5 n = 14
per genotype. 2-tailed unpaired Student’s t test; *P < 0.05.
Figure 6. Reduced Cxcl13 in iLN of Foxc2lecKO mice.
(A-B) Confirmation of Foxc2 deletion at E18.5 in Foxc2lecKO model
(A) and Cxcl13 mRNA selective reduction in E18.5 Foxc2lecKO iLNs
(B). RT-qPCR for the indicated genes in iLNs and skins. WT n = 3-6,
Foxc2lecKO n = 2-5; *P < 0.05. (C) Comparable VCAM1 levels in E18.5
WT and Foxc2lecKO embryos. Whole-mount iLN (5-μm) for CD4 (green),
VCAM1 (red) and LYVE1 (white). E18.5 WT n = 3, Foxc2lecKO n = 4. Scale
bar, 50 μm. (D) Quantification of VCAM1 intensity. E18.5 WT n = 3,
Foxc2lecKO n = 4; ns, not significant. (E) Reduced CXCL13 levels in Fox-
c2lecKO iLNs. Whole-mount iLN (1-μm); CD4 (green), CXCL13 (red) and
LYVE1 (white). E18.5 WT n = 3 and Foxc2lecKO n = 4. Scale bar, 50 μm.
(F) Quantification of CXCL13 intensity. E18.5 WT n = 3 and Foxc2lecKO n
= 4; *P < 0.05. All quantifications, 2-tailed unpaired Student’s t test.