Figure 2

Islet1 knock-down reduces the number of dorsal GFP⁺neurons in the Tg(-3.4neurog1:gfp)sb4 line. (A-C) Tg(-3.4neurog1:gfp)sb4 24 hours post-fertilization (hpf) Islet1 knock-down reduces the number of dorsal GFP⁺ neurons in the Tg(-3.4neurog1:gfp)sb4 line embryos express green fluorescent protein (GFP) in Rohon-Beard cells (RBs)[38] (white arrow, white outlined cell as example) and dorsal lateral ascending interneurons (DoLAs; asterisk, red circle). (A) In control embryos, central axons (red arrowhead) and peripheral processes (white arrowhead) are GFP⁺. (B) The 5-base mismatched islet1(Sp)E3 MO (CtlMO) has no effect on RB or DoLA morphology. (C) After Islet1 knock-down, central axons (yellow arrowhead) and dorsal cells with RB-like (yellow arrow) or DoLA-like (yellow asterisk) somata remain GFP⁺. However, few peripherally projecting processes are present (white arrowhead in A and B). (D) Tg(-3.4neurog1:gfp)sb4 E3 morphants have 29% less GFP⁺ dorsal neurons versus controls (*P < 0.0001, t-test). GFP⁺ RB neurons were counted in 200 μm spinal cord regions (above yolk sac-yolk sac extension boundary). The number in the bar indicates sample size. (E-G) Tg(mnx1:gfp)ml2 embryos were co-processed for GFP and HNK-1-like immunoreactivities to assess both RB (red) and primary motor neuron (PMN; green) peripheral processes. (E) RB somata (white arrows), central axons and peripheral processes (white arrowhead) are HNK-1 positive. (F) CtlMO has no effect on either motor/interneuron (green) or RB (red) morphology. (G) After Islet1 knock-down, few ventral cells express GFP versus uninjected (Uninj) (E) or control (F) embryos. Few HNK-1⁺ (red) or GFP⁺ (green) projecting peripheral processes are present. (H) The number of DoLA interneurons (twelve hemisegments) were counted in 24 hpf control and morphant Tg(-3.4neurog1:gfp)sb4 embryos. Islet1 knock-down has no effect on the DoLA interneuron number. (I) Commissural primary ascending interneurons (CoPAs) are positive for anti-neurofilament antibody 3A10 staining (asterisk). (J) In 24 hpf embryos, although several spinal neurons are positive for anti-Islet1/2 immunoreactivity (red), CoPAs are not. Scale bars = 50 μm in A (for A to C), E (for E to G) and I (for I and J).