Fig. 6

(A) Birthdate-related segregation of the neuropil of V2a neurons in the hindbrain and the spinal cord. (i) Experimental procedure and region displayed (gray patch). Converted Kaede is shown in magenta and unconverted Kaede is shown in green. (ii-vi) Segregation of neuropil from V2a neurons born before and after 24 hpf. (ii) Dorsal view of the hindbrain and the spinal cord. Dotted rectangles indicate the locations of the images in the following panels. (iii-vi) Close-up views of the early-born neuropil (magenta arrowheads) and the late-born neuropil (green arrowheads) in the hindbrain and spinal cord. Dotted lines indicate the mediolateral extent of neuropil. (iii) Dorsal view of the caudal hindbrain. (iv) Dorsal view of the rostral spinal cord. (v) Dorsal view of the middle spinal cord. (vi) Dorsal view of the caudal spinal cord. r, rostral; c, caudal. Scale bars, 15 um. (vii-xi) Neuropil segregation between V2a neurons born before and after 36 hpf. Panels are organized as in ii-vi. (xii) Intensity profile of photoconverted Kaede in the neuropil. Normalized intensity of photoconverted Kaede is plotted as a function of normalized mediolateral position (dashed white lines in iii-vi, viii-xi) in the neuropil. Lines indicate the average of intensity distributions across fish, while shaded areas around the line represent the standard error (n = 6). Blue, photoconversion at 24 hpf; orange, photoconversion at 36 hpf. (B) Segregation of descending projections from hindbrain V2a neurons. (i) Procedure of the experiments and location of the images in panel ii and vi. (magenta, photoconverted Kaede; green, unconverted Kaede). (ii-v) Hindbrain V2a neurons are photoconverted at 42 hpf to highlight the spinal projections of the early-born and intermediate-aged hindbrain V2a neurons. Magenta arrowheads indicate the neuropil region occupied by the early-born hindbrain V2a neurons. Green arrows indicate spinal neuropil regions that have fewer projections of the early-born hindbrain V2a neurons. Dotted lines indicate the mediolateral extent of neuropil. (ii) Dorsal view of hindbrain and spinal cord. Dotted rectangles indicate the location of the images in panel iii-v. (iii) Dorsal view of the rostral spinal cord. (iv) Dorsal view of the middle spinal cord. (v) Dorsal view of the caudal spinal cord. (vi-ix) Hindbrain V2a neurons are photoconverted at 96 hpf to highlight the spinal projections of all hindbrain V2a descending neurons. Magenta arrowheads indicate the spinal neuropil filled with the hindbrain V2a descending neurons. Dotted lines indicate the mediolateral extent of neuropil. Panels vi to ix are organized as in panels ii-v. (x) Intensity profile of photoconverted Kaede in the neuropil. Normalized intensity of photoconverted Kaede is plotted against normalized mediolateral position in the neuropil (dashed white lines in iii-v, vii-ix). Lines indicate the average of intensity distributions across fish, while shaded areas around the line represent the standard error (n = 8). Blue, hindbrain photoconversion at 42 hpf; orange, hindbrain photoconversion at 96 hpf. (xi) Coronal views of spinal motoneurons in the rostral spinal cord. One side of the spinal cord is shown. Backfilled motoneurons are shown in cyan. Closed cyan arrowheads indicate primary motoneurons (PMN). Open arrowheads indicate secondary motoneurons (SMN). (xii) Coronal views of spinal interneurons in the rostral spinal cord. One side of the spinal cord is shown. Backfilled interneurons are shown in cyan. Closed cyan arrowheads indicate the late-born spinal interneuron, multipolar commissural neuron (MCoD).r, rostral; c, caudal; d, dorsal; v, ventral. Scale bars, 15 μm.