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Fig. 6. Both high-affinity Pax2-binding sites are essential for the initiation of
mid-hindbrain-specific Brn1 expression. (A) Expression of the
6.2wt-lacZ transgene at E9.5. A 6.2 kb AflII-NotI
fragment from the 5' region of Brn1 (–6267/–59
relative to the start codon) directs expression of a lacZ reporter
gene in the posterior forebrain (fb), mid-hindbrain boundary (arrowhead)
region and spinal cord (sc), which correspond to a subset of the endogenous
Brn1 expression domains at E9.5 (see
Fig. 2F). (B,C) Inactivation of
the Pax2-binding site Dd or Pc prevents mid-hindbrain-specific expression of
the 6.2Ddm-lacZ or 6.2Pcm-lacZ transgene, respectively,
while leaving the forebrain and spinal cord expression unaffected. The
expression of all transgenes was analyzed by X-gal staining of injected
founder (G0) embryos at E9.5. Each transgenic construct gave rise
to three lacZ-expressing embryos with a similar ß-galactosidase
staining pattern. The embryo shown in B revealed ectopic ß-galactosidase
expression in the epidermis (ep) from the forebrain to the hindbrain (hb),
which was not seen with other embryos carrying the same 6.2Ddm-lacZ
gene. Arrowheads in B,C indicate the midbrain-hindbrain boundary (mhb). (D)
Brn1 promoter sequence. The mouse (m) DNA sequence of promoter
element P is shown together with the transcription initiation sites, two
conserved CCAAT boxes and the functional Pax2/5/8-binding site Pc. Only the
divergent nucleotides of the corresponding human (h) Brn1 sequence
are indicated.
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