Fig. S2

(related to Figure 2). Determination of cxcr4a expression pattern of using recombineered Cxcr4a BAC constructs.

(A) Construction of a targeted BAC construct driving TagRFP-T expression from cxcr4a promoter-enhancer sequences. (B-E) Confocal images of Tg(fli1a:EGFP)^y1 embryo (green) at 35 hpf (B and C) and 48 hpf (D and E) injected with a recombineered BAC clone with TagRFP-T driven by the cxcr4a promoter (red). TagRFP-T is detected in ISV, DA, and a subset of cells in PCV from which lymphatic progenitor emerges (arrowhead in B and C). (B and D) Merged image. (C and E) Red channel only. (F-H) Confocal images of 2 dpf Tg(fli1a:EGFP)^y1 embryo (green) injected with a recombineered BAC clone with TagRFP-T driven by the cxcr4a promoter (red). TagRFP-T is detected in lymphatic progentor sprouting from PCV and PC. (F) Green channel only. (G) Merged image. (H) Red channel only. (I) Quantification of TagRFP-T expression domains in modified Cxcr4a BAC-injected Tg(fli1a:EGFP)^y1 embryo at 2.5 dpf. Numbers above the bars show the number of TagRFP-T expressing embryos counted. (J-N) WISH images showing cxcr4b expression in various stages of Zebrafish. Cxcr4b is detected in a subset of PCV cells at 32 hpf (black arrows; J) and at 36 hpf (black arrows; K), in a sprouting lymphatic progenitor (white arrow in K), in dorsoventrally migrating lymphatic cells at 65 hpf (black arrows; L), and in lymphatic cells forming the TD at 3 dpf (black arrows; M) and 4 dpf (black arrows; N).