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Fig. 6. Cdx4 overexpression in the zebrafish hindbrain disrupts rhombomere
formation and promotes spinal cord development. Wild-type embryos
(A,C,E,G,I,K,M,O,Q)
and their siblings carrying one copy of a phsp70:cdx4 transgene
(B,D,F,H,J,L,N,P,R)
were heat shocked for 1 hour at 37°C at the three-somite stage and then
grown at 28°C for a total of 16 (14 somites, A,B), 19 (20 somites,
C-H,M-R) or 50 (I-L) hours. (A,B) Confocal images of the hindbrain region of
14-somite stage embryos injected with membrane-anchored RFP
(gap43-RFP) mRNA to reveal rhombomere furrow formation (arrowhead).
In all 12 embryos examined, cdx4 overexpression impaired furrow
formation. (C,D) Distinctive rhombomere bulges (arrowheads in C) fail to
appear in cdx4-overexpressing embryos (D) at the 20-somite stage
(n=15). (E,F) Embryos overexpressing cdx4 fail to express
the rhombomere boundary marker rfng (purple, arrowheads).
krx20 expression was used to visualize r3 and r5 (red). (G,H) Loss of
epha4a expression in r1 and reduction in r3 and r5 (brackets) in
embryos overexpressing cdx4. (I,J) cdx4 overexpression
results in the rostral expansion of vagal (nX) motor neurons and the loss of
facial (nVII) and trigeminal (nV, not shown) motor neurons in
isl1-GFP embryos. (K,L) In wild-type embryos, the RMO44-positive MiD3
reticulospinal neurons are found forming a cluster in r6 (brackets, K). This
cluster is replaced by individual r7/8-like, T reticular interneurons in
cdx4-overexpressing embryos (arrowheads, L). On a few occasions, MiD2
(r5) and Mauthner (r2) neurons (Ma) were also lost in
cdx4-overexpressing embryos (data not shown). (M,N) Expression of the
r4 marker hoxb1a is not affected in embryos overexpressing
cdx4. (O,P) hoxb3a, a gene that is normally transcribed in
rhombomeres posterior to r4 (O, arrowhead), is expressed throughout the
rostral hindbrain in cdx4-overexpressing embryos (bracket in P).
(Q,R) cdx4 overexpression induces ectopic hindbrain expression of the
spinal motor neuron and oligodendrocyte marker olig2 (purple, arrows
in R). For the 20-somite stage embryos (C-H,M-R), a minimum of 36 embryos from
at least three independent experiments were analyzed. All embryos mounted
anterior to the left, lateral views except for A,B,I-L, which are dorsal. o,
otic vesicle; r, rhombomere; s, somite. Scale bars: 100 µm.
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