Identification of a conserved 125 base-pair Hb9 enhancer that specifies gene expression to spinal motor neurons.
The homeobox gene Hb9 is expressed selectively by motor neurons (MNs) in the developing CNS. Previous studies have identified a 9-kb 5' fragment of the mouse Hb9 gene that is sufficient to direct gene expression to spinal MNs in vivo. Here, we sought to identify more discrete MN-specifying elements, using homology searches between genomic sequences of evolutionarily distant species. Based on homology screening of the mouse and human Hb9 promoters, we identified a 3.6-kb Hb9 enhancer that proved sufficient to drive MN-specific lacZ expression. We then compared mouse, human, and pufferfish (Fugu rubripes) genomic sequences, and identified a conserved 438-bp sequence, consisting of noncontiguous 313-bp and 125-bp fragments, residing within the 3.6-kb Hb9 enhancer. The zebrafish (Danio rerio) Hb9 genomic region was then found to have two identical copies of the 125-bp sequence, but no counterpart for the 313-bp sequence. Transgenic analysis showed that the 125-bp alone was both necessary and sufficient to direct spinal MN-specific lacZ expression, whereas the 313-bp sequence had no such enhancer activity. Moreover, the 125-bp Hb9 enhancer was found to harbor two Hox/Pbx consensus-binding sequences, mutations of which completely disrupted thoracolumbar Hb9 expression. These data suggest that Hox/Pbx plays a critical role in the segmental specification of spinal MNs. Together, these results indicate that the molecular pathways regulating Hb9 expression are evolutionarily conserved, and that MN-specific gene expression may be directed and achieved using a small 125-bp 5' enhancer.
Nakano
Takahiro
T
Department of Neurology and Neuroscience, Cornell University Medical Center, New York, NY 10021, USA.
Windrem
Martha
M
Zappavigna
Vincenzo
V
Goldman
Steven A
SA
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004742:Enhancer Elements, Genetic; D018507:Gene Expression Regulation, Developmental; D005801:Genes, Homeobox; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009046:Motor Neurons / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013116:Spinal Cord / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D023741:Takifugu; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish
D018398:Homeodomain Proteins; D014157:Transcription Factors; C084059:Hb9 protein, mouse