Fig. 1.

Heterogeneity of myeloid cell populations in X. laevis larvae. (A) Schematic representation of a double transgenic Xenopus larva expressing Green Fluorescent Protein (GFP) under the control of the Xenopus lurp1 gene promoter and the mCherry Fluorescent Protein (mCherry) under the control of zebrafish mpeg1 gene promoter. Myeloid cells are differentially labeled according to GFP or mCherry expression. (B) Dual channel confocal projections merged with the best in focus transmitted light image. Granulocyte-like cells, labeled in green are located all along the larval body. Macrophage-like cells appeared labeled in yellow or red due the mCherry expression driven from the mpeg1 promoter. The labeled myeloid cells are located in scattered positions along the tail and in close proximity to the major blood vessels, such as the dorsal aorta (DA, colored red mask), the posterior cardinal vein (PCV, colored blue mask) and the dorsal longitudinal anatomizing vessel (DLAV, colored blue mask). (C) Myeloid cell subpopulations density was calculated by counting each cell labeled group by mm² in different larvae (n=5). (D) and (E) Transmitted light images of the ventral fin of Xenopus larvae. lurp+/mpeg− (Granulocyte-like) and lurp+/mpeg+ macrophage-like cells are denoted by green and orange arrowheads, respectively. Cellular projections are denoted with arrowheads and dashed lines. See Video 3, Video 4, Video 5, associated with panels D and E. (F) Cell length was calculated between the most separated end points of each cell and cell area was calculated by creating masks of the fluorescent expression of each cell (ImageJ software). (G) Dual channel confocal projection merged with the best in focus transmitted light image. A small wound was inflicted in the ventral fin to activate myeloid cell migration (white overshadow at the right hand site). See supplementary video 6, associated with this panel. (H) Time of activation was calculated from the moment the wound was made and when the cells started to migrate (continuous changes in position of more than one cell body size) for the given subpopulation in 5 different larvae. lurp+/mpeg+ cells (macrophage-like) are denoted in orange and lurp+/mpeg− cells (granulocyte-like) are denoted in green. Mann–Whitney U-test, * p<0.05. (I) Speed of migration was calculated by tracking individual cells during a 1-h time block as total path length by time. (J) Normalized Lamellipodia Area (NLA) was calculated as the ratio between the whole cell area and the cell body area (area mask generated in ImageJ software). Mann–Whitney U-test, n=8, **p<0.01.