Fig 2: Depletion of Neurog2 in trigeminal placode cells does not cause increased cell death. Representative transverse section through the forming trigeminal ganglion (E3–3.5 (HH19), n = 5) after Neurog2 MO unilateral electroporation (D–F, red) followed by Tubb3 immunohistochemistry to detect placode cell-derived neurons (A,C,D,F, purple) and TUNEL staining to identify apoptotic cells (B,C,E,F, green). Scale bar in (A) is 100 µm and applies to all images. Abbreviations: e, ectoderm and NT, neural tube.

Fig 3: Depletion of Neurog2 in trigeminal placode cells impairs trigeminal ganglion development. Lateral view of the trigeminal ganglion in a chick head (E3 (HH18), n = 4, (A–F) and E3–3.5 (HH20), n = 6, (H–M)). Representative images are maximum intensity projections of confocal Z-stacks through the contralateral control (A,D,H,K) and Neurog2 MO-electroporated (B,E,I,L) sides after processing for whole-mount Tubb3 immunohistochemistry to detect placode-derived neurons (A,B,D,E,H,I,K,L, arrowheads) and tissue clearing. Bottom rows (D–F,K–M) show higher magnification images of their corresponding top rows (A–C,H–J). (C,F,J,M) MO-positive cells (arrows). Brackets indicate width of ophthalmic branch. (G,N) Quantification of the width of the ophthalmic branch on the control (blue) and Neurog2 MO-treated (orange) sides. Plots represent the median (center line), 75th percentile (top of box), and 25th percentile (bottom of box), with whiskers connecting the largest and smallest values. A paired sample t-test revealed p values of 0.05 (G) and 0.02 (N). Scale bar in (A,D) is 1 mm and applies to (B,C) and (E,F), respectively. Scale bar in (H,K) is 200 µm and applies to (I,J) and (L,M), respectively. Abbreviations: mmV, maxillomandibular lobe; opV, ophthalmic lobe; and TG, trigeminal ganglion.

Fig 4: The Neurog2 MO reduces Neurog2 protein levels. At ~E1.5 (HH9+ to 10), placode cells were unilaterally electroporated either with a Neurog2 or control (Ctrl) MO. After re-incubation to E2.5–3 (HH16–18), the forming trigeminal ganglion on the electroporated side was dissected and pooled from multiple embryos. Lysates were prepared, and equivalent amounts of protein per sample were separated on a 10% SDS-PAGE gel. Immunoblotting for Neurog2 and Beta actin (control) was then performed, and band intensity was calculated from unmodified immunoblot images using Image Lab software (Bio-Rad). Relative protein levels were ascertained by normalizing Neurog2 volumes to Beta actin volumes. Knockdown amount was determined by comparing normalized ratios between Ctrl MO and Neurog2 MO samples, with the Ctrl MO sample set as one.

Fig 5: Analysis of apoptosis and neurogenesis in ischemic brain: Western blot analysis of (A) Caspase 3, (B) Bax, (C) Bcl-2, (D) Tbr 2, (E) Ngn 2, (F) Pax 6. For these analyses, n = 5 animals for each group were employed. Data were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant: * p < 0.05 versus sham; ** p < 0.01 versus sham; *** p < 0.001 versus sham.