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Fig. 1. Characterization of a motoneuron enhancer. (A) Schematic representation of
mouse genomic sequences of Hb9. Gray boxes represent areas of high
homology between mouse and human. Percent nucleotide conservation is shown and
percent gapping in the alignment is listed for each area of homology. Mouse
promoter sequences (black line) were fused to the reporter GFP gene to
determine the activity of each DNA fragment. The presence (+) or absence
(–) of high level GFP expression in motoneurons from HH stage 24 chicks
electroporated with each reporter construct is listed on the right (MN Expr.).
Each result is representative of 
20 embryos. (B) Fragments of the 2.5 kb
distal fragment –8129 to –5575 were subcloned into a
NheI:GFP or TATA:GFP vector as indicated, electroporated into
chick embryos and GFP expression was analyzed at HH stage 24. Gray rectangles
correspond to evolutionarily conserved sequences 5'-CD (5'
conserved domain), M50 (50 nucleotide segment from enhancer), M100
(100 nucleotide segment from enhancer) and 3'-CD (3'
conserved domain). A 231 nucleotide segment (M250, –7121 to –6890)
contains an enhancer active in embryonic motoneurons. (C-G) GFP expression in
HH stage 24 chick embryos following electroporation of Hb9:GFP
reporter constructs listed in A and B. (C) The upstream 9.2 kb region of
Hb9 directs motoneuron-specific expression of GFP. (D) The proximal
NheI fragment of Hb9 drives weak GFP expression along
the entire dorsoventral axis of the neural tube. (E) The 2.5 kb distal element
produces a strong GFP signal (green) in Hb9+ motoneurons (red).
Medial red cells lacking GFP are probably Mnr2+ motoneuron
progenitors that label with the anti-Hb9 antibody. (F) A 231 nucleotide
fragment (M250), drove high levels of GFP in motoneurons (MN), but some
ectopic expression of the reporter is also detected in the dorsal neural tube
(non-MN). The M250+NheI construct corresponds to
MNE::GFP in Lee and Pfaff (Lee
and Pfaff, 2003). (G) M250 linked to synthetic TATA box is active
in motoneurons without labeling ectopic cells. (H) E11.5 transgenic mouse
embryos with M250+NheI:GFP reporter stained with anti-GFP
antibody. (I,J) Hb9::d4GFP (destabilized GFP) reporter
activity in HH stage 20 chick embryos. The 9.2 kb promoter of Hb9 and
M250 enhancer (–7121 to –6890) are both active in `mature'
(laterally-located) motoneurons (MN, bracket). (K) The enhancer of
Hb9 (MNE) mediates activation (blue factors) and
maintenance (yellow factors) of transcription in developing motoneurons.
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