Identification of a conserved 125 base-pair Hb9 enhancer that specifies gene expression to spinal motor neurons

Abstract

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.

Acetyl l-carnitine protects motor neurons and Rohon-Beard sensory neurons against ketamine-induced neurotoxicity in zebrafish embryos

“…The transcription factor hb9 is found in developing motor neurons of both mammals (William et al, 2003) and zebrafish (Cheesman et al, 2004;Park et al, 2004). In the hb9-GFP transgenic fish, motor neurons are labeled with strong neuron-specific expression of GFP under the control of the regulatory elements of the zebrafish hb9 gene (Flanagan-Steet et al, 2005;Nakano et al, 2005). Post-treatment with ketamine, images of the hb9-GFP transgenic embryos were acquired using an Olympus SZX 16 binocular microscope and a DP72 camera.…”

“…Importantly, in another study we also showed that acetyl L-carnitine counteracts ketamine's negative effects on heart rate and ERK (MAPK) activity in zebrafish embryos (Kanungo et al, 2012). In order to quantitate the effect of ketamine on motor neurons in vivo, hb9:green fluorescent protein (hb9:GFP) transgenic zebrafish embryos were used, in which the promoter of the transcription factor hb9 that is found in developing motor neurons of both mammals (William et al, 2003) and zebrafish (Cheesman et al, 2004;Park et al, 2004), drives GFP expression specifically in motor neurons (Flanagan-Steet et al, 2005;Nakano et al, 2005).…”

Spectrum ofHLXB9gene mutations in Currarino syndrome and genotype-phenotype correlation

“…However, genetic studies in mouse, chicken, and frog embryos have recently suggested that Hlxb9 is expressed selectively by postmitotic MNs in the developing vertebrate spinal cord, important for the consolidation and maintenance of MN identity [Saha et al, 1997;Arber et al, 1999;Lee and Pfaff, 2003;Lee et al, 2004;Nakano et al, 2005]. We will focus on transcription factor pathways involved in MN specification to identify possible candidate genes.…”

“…We will focus on transcription factor pathways involved in MN specification to identify possible candidate genes. Nonspecific general activator proteins such as E2F and Sp1 located throughout the neural tube interact with the proximal region of HLXB9-gene promoter and nonselectively stimulate HB9 transcription [Nakano et al, 2005]. High level of Hlxb9 expression in postmitotic MNs is activated and maintained by specific transcription factors binding two recently identified, nonoverlapping MN-specific enhancer-complexes, namely a 125-base pair (bp) region (from -8557 to -8412 bp upstream of the HLXB9 gene starting codon) and a 246-bp region (from -7621 to -7409 bp) [Arber et al, 1999;Lee and Pfaff, 2003;Lee et al, 2004;Nakano et al, 2005].…”

“…Outside the HD, the HB9 sequence differs from that of other known HD-containing proteins with the exception of MNR2 [Treisman et al, 1989;Draganescu et al, 1995;Wilson et al, 1996;Narlikar and Hartemink, 2006]. HB9 target genes are not known, but several regulators of Hlxb9 gene expression have been identified during MN development in other vertebrates, namely proneuronal basic helix-loop-helix (bHLH) proteins such as Ngn2, NeuroM, and LIM domain proteins such as Isl1, Lhx3, and Lhx4 [Lee and Pfaff, 2003;Lee et al, 2004;Nakano et al, 2005]. As with other HD-containing proteins, HB9 has been shown to interact with DNA sequences containing a TAAT motif, and is able to regulate its own expression when binding to the HLXB9 gene promoter TAAT motif, located in the 5 0 UTR region of the gene [Lee et al, 2004].…”