XB-ART-53123
Dev Neurobiol
2017 Sep 01;778:1007-1020. doi: 10.1002/dneu.22490.
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Zebrafish transgenic constructs label specific neurons in Xenopus laevis spinal cord and identify frog V0v spinal neurons.
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A correctly functioning spinal cord is crucial for locomotion and communication between body and brain but there are fundamental gaps in our knowledge of how spinal neuronal circuitry is established and functions. To understand the genetic program that regulates specification and functions of this circuitry, we need to connect neuronal molecular phenotypes with physiological analyses. Studies using Xenopus laevis tadpoles have increased our understanding of spinal cord neuronal physiology and function, particularly in locomotor circuitry. However, the X. laevis tetraploid genome and long generation time make it difficult to investigate how neurons are specified. The opacity of X. laevis embryos also makes it hard to connect functional classes of neurons and the genes that they express. We demonstrate here that Tol2 transgenic constructs using zebrafish enhancers that drive expression in specific zebrafish spinal neurons label equivalent neurons in X. laevis and that the incorporation of a Gal4:UAS amplification cassette enables cells to be observed in live X. laevis tadpoles. This technique should enable the molecular phenotypes, morphologies and physiologies of distinct X. laevis spinal neurons to be examined together in vivo. We have used an islet1 enhancer to label Rohon-Beard sensory neurons and evx enhancers to identify V0v neurons, for the first time, in X. laevis spinal cord. Our work demonstrates the homology of spinal cord circuitry in zebrafish and X. laevis, suggesting that future work could combine their relative strengths to elucidate a more complete picture of how vertebrate spinal cord neurons are specified, and function to generate behavior. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1007-1020, 2017.
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Species referenced: Xenopus laevis
Genes referenced: elavl3 evx1 evx2 isl1 lgals4.2 mhc2-dab slc17a7
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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. |
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Zebrafish islet1 enhancer predominantly labels Rohon Beard neurons and evx1 enhancer labels V0v neurons in Stage 41 Xenopus laevis spinal cord. (A–C) DAB immunohistochemistry for EGFP in transient Tg(islet1:cfos:Gal4VP16;UAS:EGFP) transgenic spinal cord. (A) Dorsal view of spinal cord, rostral left showing labeled RB neuron soma (*) and longitudinal axons in the dorsal tract (black arrows). The smaller, more weakly labeled cells are probably not RB neurons. (B and C) lateral views, rostral left, dorsal top. (B) RB neuron with characteristic large round soma in dorsal spinal cord (*) and ascending and descending longitudinal axons (black arrows), + indicates a pigment cell located ventral and lateral to the RB neuron. (C) shows a slightly more ventrally located neuron in the same embryo as (B) with an oval soma and ventral axon (arrows). (D) Lateral view with rostral left and dorsal top showing DAB immunohistochemistry for EGFP in transient Tg(evx1:cfos:Gal4VP16;UAS;EGFP) transgenic spinal cord. Labeled cells have pear-shaped soma, axons that extend ventrally and then become commissural and are located in the dorsal 48–68% of the spinal cord. White crosses indicate dorsal pigment cells. (E) Fluorescent immunohistochemistry for EGFP in transient Tg(evx1:cfos:Gal4VP16;UAS:EGFP) spinal cord; cross section (dorsal up) showing a single labeled neuron soma and ventral commissural axon. White dotted lines show edge of spinal cord. (F–H and J–L) Double labels (in situ hybridization with BM purple plus fluorescent immunohistochemistry for EGFP) of Tg(evx1:cfos:Gal4VP16;UAS:EGFP) spinal cord cross sections. Spinal cord margins are delinated with dotted lines. (F) X. laevis evx1 RNA expression (purple). (G) EGFP expression (green) in same cross section as (F). (H) merged image of F and G. Two EGFP-labeled cells (X) co-express evx1 RNA. (J) X. laevis slc17a7 RNA expression (purple). (K) EGFP expression (green) in same cross section as (J). (L) merged image of J and K. Two EGFP-labeled cells (X) coexpress slc17a7 RNA. (I) schematics showing approximate region of spinal cord shown in dorsal view in (A) and lateral views in (B–D). (B and C) show only part of this region. (A–L) are all Stage 41. Scale bar in A = 20 μm (panels A–H and J–L) and scale bar in I = 1 mm. |
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