Flow sorting and <i>Wnt</i> expression in lymphatic-associated macrophages.

<p>(A) Dot plots showing the gating used for sorting CD45+, CD11b+ and F4/80+ macrophages from embryonic dermis at E15.5. DN, population double negative for CD11b and F4/80. (B) End-point PCR showing the expression of Wnt ligand transcripts in dermal macrophages and whole embryos at E15.5. (C, D) Expression of transcripts for the <i>Frizzled</i> family receptors (C) and the co-receptors <i>Lrp5</i> and <i>Lrp6</i> (D), in dermal macrophages and whole embryos from E15.5. The observations were repeated for total of n = 3 from as many litters.</p

Myeloid <i>Wls</i> deficiency does not change dermal myeloid cell numbers.

<p>(A-D) Labeling of lymphatic capillaries and myeloid cells for LYVE1 (green), blood vessels for PECAM1 (red) and for both (merge) in dorsal skin of <i>Wls</i>^<i>fl/fl</i> and <i>Wls</i>^<i>fl/fl</i>; <i>Csf1r-icre</i> embryos of the indicated gestational age. (C, D) Lymphatic vessel boundaries are outlined with white dotted line in LYVE1 panels. (E-F) Labeling of lymphatic capillaries for PDPN (green), for myeloid cells with F4/80 (red) and for nuclei with Hoechst 33258 (blue) in the dorsal skin of E14.5 <i>Wls</i>^<i>fl/fl</i> and <i>Wls</i>^<i>fl/fl</i>; <i>Csf1r-icre</i> embryos. (G) Scatter plot representing the number of F4/80+ macrophages per field in <i>Wls</i>^<i>fl/fl</i> and <i>Wls</i>^<i>fl/fl</i>; <i>Csf1r-icre</i> embryos at E14.5. n = 4 animals for each time point per genotype, from 3 separate litters. p-value was calculated using Student’s t-test. NS, p value not significant. The charts are plotted with SEM as error bars.</p

Myeloid <i>Wls</i> deficiency results in elevated numbers and migration of lymphatic progenitors.

<p>Whole mount labeling of embryos at E10.5 (A-D) and E9.75 (F, G). LECs were labeled for PROX1 (green), blood vessels for PECAM1 (red) and nuclei with Hoechst 33258 (blue). (A), (C) and (F) show <i>Wls</i>^<i>fl/fl</i> embryos. (B), (D) and (G) show <i>Wls</i>^<i>fl/fl</i>; <i>Csf1r-icre</i> embryos. (E) Chart showing the total number of PROX1+ LECs in the region of jugular lymph sac in <i>Wls</i>^<i>fl/fl</i> and <i>Wls</i>^<i>fl/fl</i>; <i>Csf1r-icre</i> embryos at E10.5. (H and I) show the PROX1+ cells color-coded according to their distance from the cardinal vein. (J) Percentage of PROX1+ cells binned according to their distance from the cardinal vein. The chart shows cells divided into 6 equal sized bins from 0 to 500 μm. Arrows indicate PROX1+ cells in heart and lens region. For both quantifications, n = 3 animals for each time point from 2 independent litters were used. The p-value was calculated using Student’s t-test. The charts are plotted with SEM as error bars.</p

Myeloid Wnt5a gain-of-function results in elevated lymphatic vessel caliber.

<p>(A-B) Dermal tissues from <i>R26R</i>^<i>Wnt5a</i> and <i>R26R</i>^<i>Wnt5a</i>; <i>Csf1r-icre</i> animals at E15.5. Lymphatic vessels are labeled for PDPN in green. In these preparations, a wide region of dorsal skin was harvested and the lymphatic vessels visualized. The midline is marked by the dashed line. (C) Quantification of mean vessel diameter (caliber) of dermal lymphatic vessels in <i>R26R</i>^<i>Wnt5a</i> and <i>R26R</i>^<i>Wnt5a</i>; <i>Csf1r-icre</i> embryos at E15.5. (D) Quantification of branch-points per unit length of lymphatic vessels in dermal tissue from <i>R26R</i>^<i>Wnt5a</i> and <i>R26R</i>^<i>Wnt5a</i>; <i>Csf1r-icre</i> embryos at E15.5. <i>R26R</i>^<i>Wnt5a</i> (n = 3) and <i>R26R</i>^<i>Wnt5a</i>; <i>Csf1r-icre</i> (n = 4), from 3 independent litters. The p-value was calculated using Student’s t-test. NS, p value not significant. The charts are plotted with SEM as error bars.</p

Myeloid <i>Lrp5</i> deficiency results in elevated dermal lymphatic caliber.

<p>(A-F) Visualization of lymphatic capillary vessels with PDPN (green), blood vessels with PECAM1 (red) and both (merge) in dorsal skin of <i>Lrp5</i>^<i>fl/fl</i> and <i>Lrp5</i>^<i>fl/fl</i>; <i>Csf1r-icre</i> embryos of the indicated gestational age. (G) Scatter plot showing the mean lymphatic vessel caliber at different embryonic stages of development in <i>Lrp5</i>^<i>fl/fl</i> and <i>Lrp5</i>^<i>fl/fl</i>; <i>Csf1r-icre</i> embryos E14.5 (<i>Lrp5</i>^<i>fl/fl</i>; n = 4 and <i>Lrp5</i>^<i>fl/fl</i>; <i>Csf1r-icre</i>; n = 5), E16.5 (<i>Lrp5</i>^<i>fl/fl</i>; n = 4 and <i>Lrp5</i>^<i>fl/fl</i>; <i>Csf1r-icre</i>; n = 4) and E18.5 (<i>Lrp5</i>^<i>fl/fl</i>; n = 4 and <i>Lrp5</i>^<i>fl/fl</i>; <i>Csf1r-icre</i>; n = 4). (H) Scatter plot quantifying branch-points per unit length of lymphatic capillary plexus in <i>Lrp5</i>^<i>fl/fl</i> and <i>Lrp5</i>^<i>fl/fl</i>; <i>Csf1r-icre</i> embryos at E14.5 (<i>Lrp5</i>^<i>fl/fl</i>; n = 5 and <i>Lrp5</i>^<i>fl/fl</i>; <i>Csf1r-icre</i>; n = 4), E16.5 (<i>Lrp5</i>^<i>fl/fl</i>; n = 5 and <i>Lrp5</i>^<i>fl/fl</i>; <i>Csf1r-icre</i>; n = 4) and E18.5 (<i>Lrp5</i>^<i>fl/fl</i>; n = 4 and <i>Lrp5</i>^<i>fl/fl</i>; <i>Csf1r-icre</i>; n = 4), from a total of 3 independent litters for each stage. p-value was calculated using Student’s t-test. NS, p value not significant. The charts are plotted with SEM as error bars.</p