Distant regulatory elements in a Sox10-beta GEO BAC transgene are required for expression of Sox10 in the enteric nervous system and other neural crest-derived tissues.
Sox10 is an essential transcription factor required for development of neural crest-derived melanocytes, peripheral glia, and enteric ganglia. Multiple transcriptional targets regulated by Sox10 have been identified; however, little is known regarding regulation of Sox10. High sequence conservation surrounding 5' exons 1 through 3 suggests these regions might contain functional regulatory elements. However, we observed that these Sox10 genomic sequences do not confer appropriate cell-specific transcription in vitro when linked to a heterologous reporter. To identify elements required for expression of Sox10 in vivo, we modified bacterial artificial chromosomes (BACs) to generate a Sox10betaGeoBAC transgene. Our approach leaves endogenous Sox10 loci unaltered, circumventing haploinsufficiency issues that arise from gene targeting. Sox10betaGeoBAC expression closely approximates Sox10 expression in vivo, resulting in expression in anterior dorsal neural tube at embryonic day (E) 8.5 and in cranial ganglia, otic vesicle, and developing dorsal root ganglia at E10.5. Characterization of Sox10betaGeoBAC expression confirms the presence of essential regulatory regions and additionally identifies previously unreported expression in thyroid parafollicular cells, thymus, salivary, adrenal, and lacrimal glands. Fortuitous deletions in independent Sox10betaGeoBAC lines result in loss of transgene expression in peripheral nervous system lineages and coincide with evolutionarily conserved regions. Our analysis indicates that Sox10 expression requires the presence of distant cis-acting regulatory elements. The Sox10betaGeoBAC transgene offers one avenue for specifically testing the role of individual conserved regions in regulation of Sox10 and makes possible analysis of Sox10+ derivatives in the context of normal neural crest development.
Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0275, USA.
Karen K
KK
Deal
V Ashley
VA
Cantrell
Ronald L
RL
Chandler
Thomas L
TL
Saunders
Douglas P
DP
Mortlock
E Michelle
EM
Southard-Smith
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D022202:Chromosomes, Artificial, Bacterial; D017615:Enteric Nervous System / Q000196:embryology* / Q000378:metabolism*; D005260:Female; D006609:High Mobility Group Proteins / Q000096:biosynthesis* / Q000235:genetics*; D008297:Male; D008546:Melanoma, Experimental; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009432:Neural Crest / Q000196:embryology / Q000378:metabolism*; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D055757:SOXE Transcription Factors; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics*; D019076:Transgenes
D006609:High Mobility Group Proteins; D055757:SOXE Transcription Factors; C527924:Sox10 protein, mouse; C527925:Sox10 protein, rat; D014157:Transcription Factors