Xenbase Image ID: 155842

Figure 1. Zebrafish enhancers label appropriate neurons in Xenopus laevis spinal cord. (A) Schematic of shuffle-LAGAN analysis of evx2 genomic region, with zebrafish evx2 used as the baseline and compared to orthologous genomic regions in mouse and human. Conserved coding sequences are indicated in purple and conserved UTR regions are indicated in light blue. CNEs are indicated in pink. Percentage of sequence conservation is indicated by peak heights (scale is provided on RHS), gray arrow indicates 5′-3′ gene orientation. Red dotted box indicates region amplified to create evx2 enhancer transgenic constructs. (B) Schematic of Stage 41 X. laevis tadpole. Red box indicates the approximate spinal cord region shown in subsequent lateral views. (D–F and I) show only a small part of this region. (C–I) Lateral views of one side of Stage 41 X. laevis spinal cord, dorsal top, rostral left. (C–F, H, and I) show dissected spinal cords. The tissue shown is the full dorsal-ventral extent of the spinal cord and no other tissue is included except for a few pigment cells. (C) DAB immunohistochemistry (dark brown staining) for EGFP in transient transgenic Tg(elavl3:EGFP) spinal cord showing several different labeled post-mitotic neurons. For example, we have indicated a couple of RB neurons (*), a group of three motoneurons in the ventral spinal cord on the RHS of the panel (†), and some commissural cells (x). The dorsal black cells are pigment cells (+). (D–F) DAB immunohistochemistry for EGFP in transient transgenic Tg(evx2:βcarp:EGFP) spinal cords. (D) shows a region of rostral spinal cord with several labeled cells. The cells have pear shaped somata approximately 4.7 µm wide along the rostral-caudal axis and 6 µm tall in the dorsal-ventral axis, they are located in the dorsal 48–68% of the spinal cord, and they all have axons that project to the ventral spinal cord and then cross the midline to become commissural. (E and F) show two different focal planes of the same spinal cord in a region with just one labeled cell, black arrows indicate axon trajectory, black cells in F and dorsally in E are pigment cells. The cell soma is visible in (E) and its axon is visible on the contralateral side of the spinal cord in (F). (E) is slightly more rostral than (F). (G–I) Live expression of EGFP in transient transgenic Tg(elavl3:Gal4VP16;UAS:EGFP) spinal cords. (G) shows expression on one side of the mid-trunk spinal cord of an intact tadpole. The white dotted lines show the dorsal and ventral limits of the spinal cord. The expression appears weaker/more diffuse because we are looking through the skin and muscle overlying the spinal cord. (H and I) show expression in dissected live spinal cords where these other tissues have been removed. (H) shows an example where many cells are labeled. (I) shows an example of more sparse labeling where individual cells and their axons can be observed and identified. Scale bar in C = 20 μm (panels C–F) and 30 μm (panels G–I). Scale bar in B = 1 mm. Image published in: Juárez-Morales JL et al. (2017) Copyright © 2017. Image reproduced with permission of the Publisher, John Wiley & Sons. Larger Image Printer Friendly View

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