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Figure 3. Whole-mount in situ hybridization. A: Oocytes contain flotillin1 transcripts throughout the cytoplasm at stages I and II. By stage IV, transcripts are only detected in the animal hemisphere (an) by in situ hybridization. v, vegetal pole. B: In 64-cell embryos, flotillin1 transcripts are abundant in the animal hemisphere (asterisk denotes animal pole). C: At gastrula stage 12.5, flotillin1 transcripts are detected throughout the embryonic ectoderm, but not in the vegetal yolk plug (asterisk). There is a slight enrichment on the dorsal (d) side. v, ventral side. D,D prime : Stage 14 dorsal view, with anterior to the top; flotillin1 expression is enhanced in the dorsal, presumptive neural ectoderm (D) compared with the ventral, presumptive epidermal ectoderm of same embryo (D prime ). Arrow points to dorsal midline. E,E prime : At stage 15, flotillin1 expression is enhanced in the thickened neural plate (E, np) compared with the ventral epidermis (E prime , e) in the same tissue section (same magnification). F: At stage 16/17, flotillin1 expression is enhanced in the neural plate (np) compared with epidermis (e). Arrow points to dorsal midline. G: At stage 19/20, flotillin1 is detected throughout the length of the neural tube (nt) and in the paraxial mesoderm (pm; dorsal view). H: At stage 21/22, there is additional expression in the branchial arches (arrows; side view). I: At stage 25/26, staining is detected in lens (l), neural tube (nt), olfactory placode (op), otocyst (ot), retina (r), and branchial arches 1-4. J: Tissue section of stage 25 just posterior to otocyst demonstrates expression in ectoderm overlying branchial arch 4, including pigmented superficial layer (arrow) and thickened sensorial layer (asterisk) of the placode. K: Stage 31 side view using flotillin1A probe. l, lens; nt, neural tube, op, olfactory placode, ot, otocyst; r, retina; branchial arches, 1-4; and cranial ganglia (V, VII). L: Stage 31 side view using flotillin1B probe. M: Stage 31 side view using n-tubulin probe to confirm neural identity of olfactory pit (op) and cranial ganglia (V, VII). N: Stage 32/33 (sagittal section), showing flotillin1 staining in olfactory pit (op) and underlying branchial arch 1 mesenchyme (bam). r, central retina is unstained. O: Stage 32/33 (transverse section) flotillin1 staining is in the ciliary margin (cm) but not central region of the retina, lens (l), and dorsal (d) but not ventral (v) forebrain (fb). P: Stage 32/33 ectoderm overlying branchial arch 4. Staining is reduced in the pigmented superficial layer (arrow) but intense in the thickened sensorial layer (asterisk) of the placode. Q: Stage 32/33 dorsal hindbrain (hb), otocyst (ot), and cranial ganglia (VII, IX/X) are stained. Note lack of staining in the notochord (n). Q prime : Tissue section at comparable level stained with an anti-N-Tubulin antibody confirms the identity of the two cranial ganglia. R: Stage 32/33 dorsal neural tube at the spinomedullary junction (smj) is stained for flotillin1A expression. Arrows indicate intensified staining in dorsolateral primary neurons. Note staining in the dorsal part (asterisks) of somite (so) but not in notochord (n). S: Stage 32/33 dorsal neural tube at the spinomedullary junction (smj) stained for flotillin1B. A restricted dorsolateral region is stained (arrow), but there is no staining in the somite (so). T: Midtrunk level of stage 33/34 shows flotillin1A expression in dorsal spinal cord (sc), dorsal part (asterisks) of somite (so) and dorsal fin epidermis (e). Arrow denotes enhanced staining in two dorsal primary neurons. U: At comparable level and stage, flotillin 1B expression (arrows) is detected only in dorsal spinal cord (sc). V: Stage 39/40 side view using flotillin1A probe. Staining is apparent in the olfactory pit (op), hindbrain (hb), cranial ganglia (VII, IX/X), and branchial tissues (asterisk).W: Stage 39/40 side view using flotillin1B probe. Staining is apparent in the olfactory pit (op), hindbrain (hb), cranial ganglia (VII, IX/X), dorsal primary neurons (small arrows), and only faint staining in distal branchial tissues (asterisk).X: Stage 35/36 dorsal primary spinal neurons (arrows) are prominently stained with flotillin1A probe. Y: Stage 39/40 staining of flotillin 1A in lens (l) and retina (r) is dramatically reduced, whereas branchial arch 1 mesenchyme (bam) staining persists. Z: Stage 39/40 staining of flotillin 1A in the otocyst (ot) is dramatically reduced, whereas cranial ganglion (IX/X) and branchial arch 4 mesenchyme (bam) staining (arrows) persists. |
