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Dev Biol
1997 Dec 15;1922:537-50. doi: 10.1006/dbio.1997.8788.
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Differential regulation of chordin expression domains in mutant zebrafish.
Miller-Bertoglio VE
,
Fisher S
,
Sánchez A
,
Mullins MC
,
Halpern ME
.
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Patterning along the dorsal-ventral (D-V) axis of Xenopus and Drosophila embryos is believed to occur through a conserved molecular mechanism, with homologous proteins Chordin and Short gastrulation (Sog) antagonizing signaling by bone morphogenetic protein 4 (BMP-4) and Decapentaplegic (Dpp), respectively. We have isolated a zebrafish gene that is highly homologous to chordin and sog within cysteine-rich domains and exhibits conserved aspects of expression and function. As in Xenopus embryos, zebrafish chordin is expressed in the organizer region and transiently in axial mesoderm. Injection of zebrafish chordin mRNA to the ventral side of Xenopus embryos induced secondary axes. Ectopic overexpression in zebrafish resulted in an expansion of paraxial mesoderm and neurectoderm at the expense of more lateral and ventral derivatives, producing a range of defects similar to those of dorsalized zebrafish mutants (Mullins et al., 1996). In accordance with the proposed function of chordin in D-V patterning, dorsalized zebrafish mutants showed expanded domains of chordin expression by midgastrulation, while some ventralized mutants had reduced expression; however, in all mutants examined, early organizer expression was unaltered. In contrast to Xenopus, zebrafish chordin is also expressed in paraxial mesoderm and ectoderm and in localized regions of the developing brain, suggesting that there are additional roles for chordin in zebrafish embryonic development. Surprisingly, paraxial mesodermal expression of chordin appeared unaltered in spadetail mutants that later lack trunk muscle (Kimmel et al., 1989), while axial mesodermal expression was affected. This finding reveals an unexpected function for spadetail in midline mesoderm and in differential regulation of chordin expression during gastrulation.
FIG. 1. Sequence conservation and expression of zebrafish chordin. (A) The alignment compares the amino acid sequence of zebrafish
Chordin with Xenopus Chordin and Drosophila Short gastrulation. Stars and dots indicate identical amino acids and conservative amino acid
substitutions, respectively. Cysteine-rich repeats are outlined in boxes and the region corresponding to the CR1 PCR amplification product is
shown in gray. (B) The numbers indicate the percentages of amino acid identity within and outside the CR repeats (shaded) in the three proteins
(proteins are not drawn to scale). (C) Northern blot analysis demonstrates that a single chordin transcript is present at low levels in the late
blastula, increases during gastrulation, and persists through early somitogenesis. RNA was collected fromembryos prior to midblastula transition
(mat) and at sphere stage (sph), 30%epiboly (30%), 50% epiboly (shld), 70%epiboly (70%), 6 somites (6s), 20 somites (20s), and 24 h postfertilization
(24h). For this blot, the probe was a HinfII restriction fragment (601 bp) that included CR3 and CR4.
FIG. 2. Dynamic expression of chordin in the developing zebrafish. (A, B) Zebrafish chordin was detected at the future dorsal side of
the late blastula (sphere stage: A, animal pole view; B, dorsal view). (C, D) By early gastrulation, expression was confined to deep cells in
the embryonic shield (arrowhead). D is a sagittal section through the shield at 50% epiboly. (E–H) As gastrulation proceeded, expression
was found transiently in the axis (arrowhead) and new bilateral domains of expression appeared (open arrowhead) in all superficial
(ectoderm) and deep (mesoderm) cell layers, except the layer closest to the yolk (open arrow, H). E is shield stage at 50% epiboly, F is 30
min after E, and G and H are at 70% epiboly. H is a transverse section through the axis and flanking regions. (I–K) In late gastrulae,
chordin was expressed in cells adjacent to the notochord and downregulated in the axis (open arrowheads). Bilateral expression domains
persisted in the most posterior region only. I is at 90% epiboly and J and K are at 100% epiboly. (L, M) At late gastrulation, chordin was
also expressed in discrete domains in the forebrain, midbrain, and hindbrain (in blue). Double labeling with a probe for krox-20, which is
expressed in rhombomeres 3 and 5 (in pink), indicated that chordin expression in the hindbrain is very dynamic, present in different
rhombomeres over time. Scale bars 150 mm (A–C, E–G, and I–M), 25 mm (D), and 50 mm (H).
FIG. 3. Dorsalizing activity of zebrafish Chordin in Xenopus and zebrafish embryos. (A) Uninjected Xenopus embryo (stage 37/38); (B)
Xenopus embryo coinjected with 10 nl of zebrafish chordin and GFP RNA (20 ng/ml) on the ventral side at the two-cell stage; (C) Xenopus
embryo coinjected ventrally at the one-cell stage with 5 nl of zebrafish chordin and GFP RNA (20 ng/ml). (D–I) In situ hybridization using
pax2 and myoD probes (D–F) or pax2 and ntl probes (G–I) of uninjected zebrafish embryos (D and G) or embryos injected with chordin
RNA (E, F, H, and I) at the one- to four-cell stage during segmentation. All injected embryos shown correspond to class d of Table 2. (E,
F) Severely dorsalized embryos were characterized by their elongated shape, laterally expanded domain of pax2 expression at the midbrain–
hindbrain junction (arrow), and ventrally extending somites (open arrows). (H) In some injected embryos, the ntl expression domain
(arrowheads) in the notochord was widened relative to controls (G). D–H are dorsal views. (I) A ventral view of the same embryo as H
shows the expansion of pax2 expression in the brain around the entire circumference of the embryo (arrow). Pronephric duct expression
of pax2 (arrow, G) was often missing in injected embryos.
