Increasing apoptosis leads to deposition of fewer proneuromast (A,B) PH3 positive cells are labeled green. (D,E,G,I,K,M) Primary lateral line morphology revealed by klf4 in situ hybridization. Proneuromasts are numbered and the terminal proneuromasts are labeled with a ‘T’. Presence of terminal proneuromasts demonstrates completed primordium migration. (A,C) Primordia treated with DMSO from 26 to 32 hpf proliferate normally. (D) DMSO treated primordia migrate and deposit a normal complement of proneuromasts along the trunk. (B,C) Treatment with 75 μM aphidicolin + 7.5 mM hydroxyurea (HUA) from 26 to 32 hpf causes a reduction in the proportion of mitotic cells expressing PH3. (E) HUA treatment from 26 to 54 hpf leads to a significant reduction in the number of deposited proneuromasts without preventing primordium migration. (F,H,J,L) Whole mount TUNEL assay at 32 hpf. Nuclei are labeled with DAPI and red spots represent TUNEL signal. Dying cells are rapidly extruded and form a cloud surrounding and trailing behind the migrating primordium. (F) No appreciable apoptosis is detectable in a wt primordium. (G) A typical 48 hpf wt primary lateral line. (H) bap28 homozygous mutants show a dramatic increase in apoptosis. (I) bap28 mutants possess fewer primary proneuromasts but still produce terminal proneuromasts. (J) Injection of toxic tcf7 ATG MO leads to apoptosis in the primordium. (K) tcf7 ATG MO also causes deposition of fewer proneuromasts, though to a lesser extent than bap28 mutants. (L) Maternal/zygotic tcf7 null mutants show no apoptosis in the primordium. (M) tcf7 mutants have a normal number of primary proneuromasts. (N) Quantification of proneuromast number. Manipulations that decrease the rate of cell proliferation or increase apoptosis lead to deposition of fewer proneuromasts. This effect of apoptosis can be rescued by co-injection of p53 MO. Error bars equal standard deviation. Scale bars equal 50 μm.
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