Identifying Transcription Factors Expressed by Ventral Spinal Cord Interneurons

Summary: We isolated and expression profiled V0v, V1, V2a, V2b and Kolmer Agduhr (KA) spinal interneurons, as well as all spinal neurons and all trunk cells using Fluorescence Activated Cell Sorting (FACS) of different transgenic zebrafish lines in which these cells express fluorescent proteins at 27 hours post fertilization (hpf). We then used several different bioinformatic comparisons to identify mainly transcription factor genes that either were candidates for being expressed by specific spinal cord cell types or very little was known about. Templates for in situ hybridization probes were either obtained from existing sources or PCR amplified from first-strand cDNA. Digoxigenin-labeled antisense RNA probes were then used to analyze the expression pattern of the corresponding gene from 24-36 hpf. The expression of some of these genes was also analyzed in mindbomb1 mutants at 24 hpf, since this can improve detection of expression in certain spinal interneurons. These data are deposited in the ZFIN database and details for probe preparation are provided below and in the figure legends. These results provide novel descriptions of the expression of several transcription factors, not only in the spinal cord but also in other tissues during early zebrafish development.

METHODS: Embryos from AB, TL or AB/TL wild-type fish were collected, allowed to develop at 28.5 °C until the appropriate stage, manually dechorionated and then fixed by incubating over night in 4% paraformaldehyde (PFA) at 4 °C. Embryos older than 24 hpf were often incubated in 0.003% 1-phenyl-2-thiourea (PTU) to prevent pigment formation. After fixation, embryos were washed with PBT (PBS+ 0.1% tween) and then dehydrated with 100% methanol. Embryos were then stored in 100% methanol at -20 °C. in situ hybridization (ISH) was performed as previously described (Concordet, J.P., Lewis, K.E., Moore, J.W., Goodrich, L.V., Johnson, R.L., Scott, M.P., Ingham, P.W., 1996. Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning. Development 122(9), 2835–2846). Once staining was developed, embryos were mounted in 70% glycerol and photographs were taken using a Zeiss Axio Imager M1 microscope and processed using Adobe Photoshop CS. Templates for digoxigenin-labeled antisense RNA probes were obtained either from published sources or by PCR amplification of a specific region of the gene in question. PCRs were performed with the following protocol, except where noted in the figure legends. Total RNA was extracted from 27 hpf wild-type embryos using TRIzol Reagent (Ambion, 15596-026). RNA integrity and quality was assessed using agarose gel electrophoresis. First-strand cDNA was synthesized with iScript cDNA Synthesis Kit (Bio-Rad, 170-8891). 50 µl PCR reactions were performed using 5 µl first-strand cDNA template, Phusion High-Fidelity DNA Polymerase (NEB, M0530L) and forward and reverse primers (primer sequences are provided in the figure legends). The 5’' terminus of each reverse primer contains the T3 RNA Polymerase Promoter sequence, so that anti-sense ISH probes can be synthesized. PCR conditions were: Initiation: 94 °C for 3 minutes. 35 cycles: Denaturation: 94 °C for 30 seconds, Annealing: 56.5 °C for 30 seconds and Extension: 72 °C for 1.5 minutes. Final extension: 72 °C for 10 minutes. PCR products were purified by phenol:chloroform extraction. in situ hybridization probes were made using 1 µg purified PCR product, T3 RNA Polymerase (Roche, 11031171001) and DIG RNA Labeling Mix (Roche, 11277073910).

Acknowledging this work: All clones, information, and images used from this work should cite this summary and first description of this work posted in ZFIN: (England, S., Hilinski, W., de Jager, S., Andrzejczuk, L., Campbell, P., Chowdhury, T., Demby, C., Fancher, W., Gong, Y., Lin, C., Machikas, A., Rodriguez-Larrain, G., Roman Rivera, V. and Lewis, K. E. Identifying Transcription Factors Expressed by Ventral Spinal Cord Interneurons. ZFIN on-line publication, 2014). This research was supported by Grant Number R21NS073979 from the National Institute Of Neurological Disorders And Stroke, NIH. Please acknowledge this grant number in all publications using these materials. The content of this data submission is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the National Institutes of Health. If you have any questions or comments on this project, please contact K. E. Lewis.