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CNE_22004_sm_supplfig1.tif31992KSupplemental Figure 1. Trigeminal placode extends into caudal forebrain level. A. At the forebrain/eye level, an HH10 (9ss) embryo (collected at HH15 (25ss)) shows two post-mitotic cells (arrows) that migrated to position of future opV nerve. B. Higher power of A. C. DiI label at HH11 (12ss) (collected at HH14 (21ss)) shows delaminate placode cells at forebrain level (small arrow). D. Embryo treated at HH9 (8ss) (collected at HH15 (26;ss)) (same level as A,B) has two NFM+/BrdU- cells. E. Higher power of D. F. DAPI of cells in E. G-I. Pax3, a marker for specified trigeminal placode cells, is expressed at forebrain levels at HH9, 10, 11 (8ss, 11ss, 14ss). A-F. Large arrows indicate post-mitotic cells. G-I. Large arrow indicates Pax3+ cell in ectoderm. FB=forebrain, cFB=caudal forebrain, HB=hindbrain. Scale bars are approximately 10 μm. For triple merge with DAPI of D-E, see Figure2.
CNE_22004_sm_supplfig2.tif13479KSupplemental Figure 2. Post-mitotic opV placode cells increase in number over time and become localized in condensing opV ganglion and nerve. A-B. Between mid-eye level and opV ganglion region, the number of post-mitotic cells within the forming opV nerve (B) by this stage are increasing to 2–5 per section in an embryo treated at HH10 (9ss) and collected at HH15 (25ss). C–D. At a similar axial level in an older embryo treated at HH14 (22ss) and collected at HH16 (28ss), additional post-mitotic cells can be found within the condensing opV nerve approaching the ganglion region. E–G. As a comparison to C–D, this embryo was treated at the same time as C–D at HH14 (23ss) but allowed to develop until HH18 (36ss). In a longitudinal section at the level of the condensed trigeminal ganglion (contains opV and mmV cells at this level), post-mitotic cells are seen in the opV nerve (E) both rostral towards the eye (F) and caudal at the ganglion edge (G). H. Hu+ neurons (white arrows) are shown in the forming opV at the level of forebrain caudal to the eye at HH17 (29ss). I. Expression of NF at level of opV nerve at HH24. J. Adjacent section of I shows NF expression within opV nerve. K. Nerve cell bodies are Hu+ within the maturing opV nerve. A–G. Black arrows indicate post-mitotic cells. ECTO= ectoderm, TG= trigeminal ganglion, FB= forebrain, opVN= ophthalmic trigeminal nerve. Scale bars are approximately 10 μm.
CNE_22004_sm_supplfig3.tif46900KSupplemental Figure 3. opV placode cells that express Pax3 begin exiting cell cycle at HH8 (3–5ss). A–C. All ectoderm cells that give rise to future Pax3+ opV placode cells are actively dividing at HH7 (2ss) (collected at HH10 (11ss)). Pax3 stains migrating neural crest in the mesenchyme at HH10. D–F. Pax3+/BrdU- cells detected in the mesenchyme first treated at HH8 (5ss) (collected at HH13 (19ss)) similar to embryo treated with 3H-Thy. G-I. More Pax3+/BrdU- cells become detectable in a spur of delaminating cells when treated at HH9 (8ss) (collected at HH16 (28ss)). J-L. Additional placode cells exiting the cell cycle by HH10 (11ss) (collected at HH16 (28ss)). All sections at level of middle midbrain. BrdU (magenta;A,D,G,J); Pax3 (green;B,E,H,K); Merge of BrdU, Pax3 (C,F,I,L). Arrows indicate post-mitotic cells and arrowheads indicate cells undergoing DNA synthesis at time of initial BrdU application. Scale bars are approximately 10 μm. For triple merge with DAPI of C, F, I, L, see Figure4.
CNE_22004_sm_supplfig4.tif7976KSupplemental Figure 4. opV placode cells that express NFM, begin exiting cell cycle at HH8 (3–5ss). A–C. A few NFM+/BrdU- cells detected in embryos treated at HH8 (5ss) (collected at HH13 (18ss)). C'. Higher power view of C showing NFM+/BrdU+ cell. C''. Higher power view of C showing NFM+/BrdU- cell. D–F. More NFM+/BrdU- cells in embryos treated at HH9 (8ss) (collected at HH15 (26ss)). F'. Higher power view of F showing NFM+/BrdU- cell. F''. Higher power view of F showing NFM+/BrdU+ cell. G–I. Additional placode cells have exited the cell cycle by HH10 (9ss) and express NFM (collected at HH15 (25ss)). I'. Higher power view of I showing NFM+/BrdU- cell. I''. Higher power view of I showing NFM+/BrdU+ cell. Arrows indicate post-mitotic cells and arrowheads indicate cells undergoing DNA synthesis at time of initial BrdU application. BrdU (magenta;A,D,G); NFM (green;B,E,H); Merge of BrdU, NFM (blue;C,F,I). All sections at level of middle midbrain. Scale bars are approximately 10 μm. For triple merge with DAPI of C-C'', F-F'', I-I'', see Figure5.
CNE_22004_sm_supplfig5.tif37964KSupplemental Figure 5. Pulse of BrdU shows opV placode cells that express neuronal markers have exited the cell cycle. Embryos were exposed to a 30 minute pulse of BrdU immediately before fixation. A–C. However, Pax3 expression does not necessarily correlate with cell cycle exit. At HH10 (10ss), opV placode cells in ectoderm express Pax3 (green) with (arrowhead) and without (arrow) BrdU. D–F. At HH11 (13ss), NFM+ (green)/BrdU- cells are detected in ectoderm and mesenchyme. G–I. By HH18 (33ss), opV placode cells condensing in opV ganglion are Hu+ (green)/Brdu-(arrows). A–F at level of mid midbrain, G–I at level of caudal midbrain. Scale bars are approximately 10 μm. For triple merge with DAPI of C, F, I see Figure6.

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