Fig. 2.

T-DM1-injected embryos lacking all BLECs at 8 dpf fail to recover this cell population at later stages while showing no overt effects on intracranial lymphatics development. (A) Schematic representations of the dorsal head region of a zebrafish at 2 wpf (left) and 4 wpf (right), showing BLECs (green) and intracranial lymphatics (red), and indicating the zoomed-in areas shown in B-D and E-G. (B-G′) Maximum confocal projections of either 2, 3 or 4-week-old larvae, with mrc1a:mCitrine- and lyve1:dsRed-positive lymphatic endothelial cells [dorsal (B-G) and lateral (B′-G′) views]. Embryos were injected with either IgG-Alexa-647 (control) or T-DM1 at 3 dpf. In control-injected larvae (B-D′), BLECs are arranged as single cells in symmetric loops dorsal to the TeO (white arrowheads). Intracranial lymphatics (red arrowheads) emerge from a branch of the otolithic lymphatic vessel. BLECs cover the whole TeO after 3 weeks of age. In larvae injected with 1 ng T-DM1 (E-G′) at 3 dpf, BLECs are ablated (white asterisks). Intracranial lymphatics (red arrowheads) are unaffected. Different larvae were used for each stage. (H) Schematic representation of an extracted zebrafish brain (dorsal view). During dissection of the brain, the intracranial/dural lymphatics are removed. The dotted line indicates the region of the dissected brains shown in I-T. (I-T) Confocal images showing maximum projections of 8 dpf, 3 wpf, and 4 wpf larvae with mrc1a:mCitrine-positive lymphatic endothelial cells and kdrl: mCherry-positive blood vessels. T-DM1-injected embryos were grouped according to the number of BLECs at 8 dpf (I,L,O,R). Ventrally located BLECs are not visible in these images. Dorsally located remaining BLECs are highlighted (white arrow). Fish with 0 BLECs (L-N) or with 1-4 ventral BLECs (O-Q) did not regenerate BLECs, whereas several fish with 1-4 remaining dorsal BLECs (S) fully regenerated the BLEC population. For each stage, different larvae were used. FB, forebrain; HB, hind brain; TeO, tectum opticum; dpf, days post-fertilization; wpf, weeks post-fertilization.