DEV015297 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. A cmyb:eGFP transgene marks cells in the AGM, along the pronephric ducts and in the thymic lobes. (A) Overview of regions shown in high-magnification fluorescent images. Purple region denotes left thymic lobe, blue region the left pronephric duct and red region the AGM (space between axial vessels). (B-E) cmyb is expressed in the first thymic immigrants beginning at ∼48 hpf. (F-I) GFP⁺ cells appear along the pronephric tubules beginning at ∼32 hpf, and increase in number over time. Broken lines indicate the boundaries of the duct. (J-N) Within the embryo, GFP⁺ cells are first observed in the AGM region at ∼27 hpf. After 48 hpf, the AGM region greatly expands as the aorta and vein move apart. The upper broken line indicates the ventral wall of the dorsal aorta, the lower line indicates the dorsal wall of the cardinal vein. GFP⁺ ductal cells appear ventrolateral to cells within the demarcated AGM region. Images are merged fluorescence and Nomarski photographs. Embryos are positioned anterior towards the left and dorsal side upwards.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. HSCs and neurons can be segregated by light scatter characteristics from cmyb:eGFP embryos. (A) Lateral view of a cmyb:eGFP embryo at 42 hpf. The spinal cord shows small round GFP⁺ cells, whereas larger and brighter cells are observed in the AGM region (dorsal side upwards, anterior towards the left). (B) cmyb:EGFP⁺ cells were analyzed by forward (FSC) and side (SSC) scatter characteristics. Cells were sorted by size (red gate, larger; green gate, smaller). (C) QPCR analysis shows that FSC^lo cells are highly enriched for NCAM-expressing cells, whereas FSC^hi cells are enriched for CD45-expressing cells.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. A CD45:DsRed transgene marks a subset of AGM HSCs and differentiated leukocytes. (A) Deconvolved image stacks through the 76 hpf AGM shows a subset of CD45:DsRed⁺ cells (upper left panel) also express the CD41:eGFP transgene (upper left panel). Lower panels show merged channels, with Nomarski overlay on the right. Arrowheads mark double positive cells, brackets mark the boundaries of the dorsal aorta (DA) and cardinal vein (CV). (B) Double transgenic animals show GFP⁺ cells along the pronephric ducts and DsRed⁺ cells within the anterior pronephros. All animals oriented dorsal side upwards, anterior towards the left. OV, otic vesicle.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Circulation is not required for translocation of hematopoietic precursors to the pronephric tubules or anterior pronephros. (A,B) silent heart morphants did not develop circulation, as shown by erythrocyte pooling (black brackets). (D,E) cmyb:eGFP⁺ cells translocated normally to the pronephric tubules (majority of ductal cells flanked by white brackets), and migrated to the anterior pronephri (arrowheads). (C,F) cmyb:eGFP⁺ cells (asterisk) only rarely appeared (3/18 morphants) within the CHT. All animals photographed at 72 hpf with dorsal side upwards, anterior towards the left.

• Movie 1 -

  Movie 1. Translocation of CD41:eGFP⁺ cells from the AGM to the duct. High-magnification view of the AGM region shows CD41⁺ cells to exhibit dynamic behavior. GFP⁺ cells translocate from between the axial vessels (the majority of GFP⁺ cells are spindle-shaped cells along the ventral wall of the dorsal aorta) to the pronephric ducts (ventral to the AGM). Timelapse between 54 and 61 hpf, one frame captured every 6 minutes. Embryo is oriented anterior towards the left, dorsal side upwards.

• Movie 2 -

  Movie 2. Colonization of the thymus by CD41:eGFP⁺ cells. Timelapse shows the initial colonization of the left thymic lobe (just ventral to the otic vesicle, which is found posterior to the eye). GFP⁺ cells migrate into the thymic region, where they begin to cluster and divide. Timelapse between 54 and 61 hpf, one frame captured every 6 minutes. Embryo is oriented anterior towards the left, dorsal side upwards.

• Movie 3 -

  Movie 3. Lateral view of directed ductal migration of CD41:eGFP⁺ cells. Between 2 and 3 dpf, CD41⁺ cells are observed to migrate slowly from the posterior regions of the pronephric ducts towards the anterior pronephros. Ductal GFP⁺ cells appear just dorsal to the yolk ball. GFP⁺ streaks are due to CD41⁺ thrombocytes in circulation. Timelapse between 55 and 70 hpf, one frame captured every 10 minutes. Embryo is oriented anterior towards the right, dorsal side upwards.

• Movie 4 -

  Movie 4. Dorsal view of directed ductal migration of CD41:eGFP⁺ cells. CD41⁺ cells move in bilateral stripes as they migrate anteriorly along the pronephric ducts. GFP⁺ streaks are due to CD41⁺ thrombocytes in circulation. Timelapse between 65 and 75 hpf, one frame captured every 10 minutes. Embryo is oriented anterior towards the bottom right, dorsal side towards the viewer.

• Movie 5 -

  Movie 5. Dorsal view of directed ductal migration of cmyb:eGFP⁺ cells. Timelapse imaging shows that cmyb⁺ cells migrate slowly along each pronephric tubule in an anterior direction. Timelapse between 65 and 75 hpf, one frame captured every 10 minutes. Embryo is positioned anterior side upwards, dorsal side towards the viewer.