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Delta/Notch signaling promotes formation of zebrafish neural crest by repressing Neurogenin 1 function

Robert A. Cornell*,{dagger} and Judith S. Eisen

Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403, USA
* Present address: Department of Anatomy and Cell Biology, 1-530 Bowen Science Building, University of Iowa Medical School, Iowa City, IA 52242, USA



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  		Fig. 1. ngn1 is expressed in RB neurons and in dorsal root and cranial ganglia. (A) Dorsal view of 6-somite stage embryo, processed to reveal ngn1 RNA expression (cytoplasm, dark blue) and anti-Islet immunoreactivity (nuclear, brown). Cells expressing ngn1 but not Islet have a clear nucleus (asterisk, A'). In all figures, embryos are shown with anterior towards the left, except in transverse sections. In caudal neural plate (inset A'), RBs (white arrowheads) and a few cells adjacent to RBs (asterisk) express high levels of ngn1. By contrast, more rostrally, in developmentally older neural plate (inset A''), cells surrounding RBs do not express high levels of ngn1. (B) Lateral view of somites 5-12 of an embryo fixed at 34 hpf and processed to reveal ngn1 expression in DRGs (arrowheads). Expression is also visible in neural tube (NT). (C) Transverse section of the embryo in A showing ngn1 expression in DRGs (arrowheads) and neural tube (NT). (D) Lateral head view of 34 hpf embryo showing ngn1 expression in nascent cranial ganglia (arrowheads). Scale bars: 50 µm.

 


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  		Fig. 2. RBs but not PMNs are eliminated by ngn1 morpholinos. (A-D) Lateral views of 18 hpf wild-type embryos at somites 15-20, processed to reveal isl2 RNA expression. In this and subsequent figures, the portrayed phenotype was observed in at least 80% of injected embryos. (A) Uninjected embryo. Expression of isl2 can be seen in RBs in dorsal neural tube and in a subset of primary motoneurons (PMN) in ventral neural tube. (B) Embryo injected at the two-cell stage with approximately 1.5 ng of ngn1 MO that straddles the start AUG of the ngn1 transcript. As revealed by isl2 expression, RBs were absent or highly reduced, whereas PMNs were normal (40 embryos scored). Note: in embryos injected with 7.5 ng of ngn1 MO, PMNs in the tail were reduced in some embryos; however, it is unclear whether this phenotype resulted from targeting of the ngn1 transcript, because target specificity may not be maintained at this concentration. (C) Embryo injected at two-cell stage with approximately 3.0 ng of ngn1mismatch MO, which is identical to ngn1 MO except at four base positions (28 embryos scored). RBs and PMNs are normal, indicating that sequence complementarity is essential for protein knockdown at this morpholino concentration. At 6.0 ng of injected ngn1mismatch MO, embryos resembled ngn1 MO-injected embryos (20 embryos scored), suggesting that at this high dose, the ngn1 mismatch MO targets the ngn1 transcript, despite the lack of complementarity at four out of 25 positions. We therefore ignored effects of morpholinos at this high dose, because of the uncertainty of the transcript being targeted. (D) Embryo injected at two-cell stage with approximately 1.2 ng of ngn15'UTR MO, that complements an upstream region of the 5'UTR. The phenotype appeared identical to that in ngn1 MO-injected embryos at this dose (25 embryos scored). At 3.6 ng and higher, embryos showed patchy necrosis in the CNS. This phenotype was presumed to result from toxicity of the morpholino at this dose, as opposed to further reduction of Ngn1 function. Scale bar: 50 µm.

 


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  		Fig. 3. Early markers of RBs are eliminated by ngn1 MO. (A,C,E,G,I) Uninjected embryos. (B,D,F,H,J) Embryos injected at the one- to two-cell stage with 1.5 ng of ngn1 MO. (A,B) Embryos fixed at the five-somite stage (about 12 hpf), processed to reveal isl1 RNA expression. RBs (arrowheads, RB) and trigeminal ganglion precursors (tri) were highly reduced, whereas PMNs and hatching gland (HG) were still present in injected embryos (25 embryos scored). (C,D) Embryos fixed at the five-somite stage, processed to reveal neurod (nrd) RNA expression. RBs (arrowheads) and trigeminal ganglion precursors (tri) were highly reduced in injected embryos (24 embryos scored). (E,F) Embryos fixed at the three-somite stage (about 11 hpf), processed to reveal dla expression (left half of neural plate is shown). High level expression, identifying RB precursors (arrowheads) and trigeminal ganglia (tri) was eliminated, whereas low level expression in lateral neural plate and trigeminal ganglia was still present in injected embryos. Medial neural plate expression at high and low levels was present in injected embryos (43 embryos scored). (G,H) Embryos fixed at 24 hpf and processed to reveal zn12 antibody immunoreactivity in RBs. RBs were highly reduced or completely absent from injected embryos, although residual labeling was still visible in unidentified cell bodies and axon tracts within the intermediate spinal cord and hindbrain (28 embryos scored). (I,J) Embryos fixed at 24 hpf and processed to reveal zn1 and znp1 antibody immunoreactivity. PMN axons are labeled and appear normal in number in injected embryos (26 embryos scored). Scale bars: 100 µm in A-F; 50 µm in G-J.

 


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  		Fig. 4. Injection of ngn1 MO rescues trunk neural crest in embryos with reduced Delta/Notch signaling. (A,D,G,J,M) Wild-type embryos; (B,E,H,K,N) mib mutants; (C,F,I,L,O) mib mutants injected with 1.5 ng ngn1 MO. (A-C) Lateral views of 20 hpf embryos processed to reveal isl2 expression. (A) In wild types, isl2 expression is visible in RBs and a subset of PMNs. (B) In mib mutants, there are supernumerary RBs and PMNs. (C) In ngn1 MO-injected mib mutants, RBs are highly reduced but supernumerary PMN are still present (nine mutant embryos). (D-F) Dorsal views of flat-mounted, 11 hpf embryos processed to reveal fkd6 expression (black) and distribution of epitope-tagged ngn1 MO (red). (D) In wild types, fkd6 expression is visible in premigratory neural crest in head and trunk. (E) In mib mutants, fkd6 expression is highly reduced in the trunk domain. (F) In ngn1 MO-injected mib mutants, fkd6 expression is restored on the injected side (an epitope-tagged morpholino was used in this experiment, see Materials and Methods) (five mib mutants with morpholino in lateral neural plate scored). (G-I) Lateral trunk views of 20 hpf embryos processed to reveal crestin expression. (G) In wild types, crestin expression is visible in premigratory and migratory neural crest. (H) In mib mutants, crestin expression is all but absent from the trunk. (I) In ngn1 MO-injected mib mutants, crestin expression is restored, although in an abnormal pattern. (J-L) Lateral trunk views of live 4 dpf embryos. (J) In wild types, neural-crest-derived black melanophores are visible in dorsal, intermediate and ventral stripes. (K) In mib mutants, melanophores are virtually absent from the trunk and tail. (L) In ngn1 MO-injected mib mutants, melanophores are restored in the trunk and tail. Melanophores are abnormally distributed, however, often present between the spinal cord and somites (12 mib mutants scored). (M-O) Higher magnification lateral views of the embryos shown in J-L, respectively. Neural crest-derived yellow xanthophores are present in wild-type embryos (M), are highly reduced in mib mutants (N) and are restored in ngn1 MO-injected mib mutants (O) (12 mib mutant embryos scored). (P) Dorsal view of a flat-mounted, 11 hpf mib mutant embryo processed to reveal fkd6 expression (black) and distribution of epitope-tagged ngn1 MO (red). ngn1 MO was injected into one cell at the 32-cell stage. A few cells in trunk lateral neural plate express fkd6 (box and arrowhead), and these all contain the ngn1 MO, suggesting a cell autonomous of conversion of RBs to neural crest by the morpholino (four mib mutants with dispersed clones of morpholino-containing cells scored). (P',P'') Higher-magnification view of boxed area in P in bright field (P') to show fkd6 expression, and under fluorescent optics (P'') to show distribution of the morpholino. (Q,R) Dorsal view of flat mounted, 11 hpf embryos processed to reveal fkd6 expression (blue) and ß-gal activity (turquoise). (Q) Embryos co-injected with RNAs encoding dominant negative X. laevis Supressor of Hairless [dnSu(H)] and ß-gal have reduced fkd6 expression in the trunk neural plate (10/85 injected embryos had X-gal stain in the trunk neural plate, all of these had highly reduced fkd6 expression). (R) Embryos injected with these RNAs in addition to 1.5 ng ngn1 MO have restored fkd6 expression in this domain (arrowheads) (six out of six embryos with co-localization of ß-gal activity and ngn1 MO in lateral neural plate). Scale bars: in A, 100 µm in A-C; in D, 200 µm in D-F; in G, 100 µm in G-I; in J, 100 µm in J-L; in M, 50 µm in M-O; in P, 200 µm; in Q, 200 µm in Q,R.

 


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  		Fig. 5. Sensory neurons, but not autonomic neurons, are eliminated by ngn1 MO. (A,C,E,G,I) Uninjected, wild-type embryos; (B,D,F,H,J) embryos injected with 1.5 ng of ngn1 MO. (A,B) Lateral trunk views of 4 dpf embryos, processed to reveal anti-Hu immunoreactivity. Embryos have been deliberately under-developed to avoid strong staining in spinal cord underlying DRG neurons. Hu-labeled DRG neurons are absent from injected embryos, although spinal cord stain is beginning to become visible. (C,D) Lateral view just posterior to eye of 28 hpf embryos processed to reveal zn12 and anti-acetylated tubulin immunoreactivity. Trigeminal (tri), anterior lateral line (all), acoustic (ac) and posterior lateral line (pll) cranial ganglia are all highly reduced in injected embryos (28 embryos scored). (E,F) Lateral views of gut lumen of 4 dpf embryos processed to reveal anti-Hu immunoreactivity. Enteric neurons are present in both untreated and injected embryos. (G,H) Transverse sections, near axial level of pectoral fins, of 5 dpf embryos processed to reveal anti-tyrosine hydroxylase immunoreactivity. Label ventral to notochord (n) is in sympathetic neurons (arrowheads), which are present in both untreated and injected embryos. Asterisk indicates autofluorescence in a blood cell. (I,J) Lateral head view of live embryos at 5 dpf. Asterisk indicates permanently open mouth of injected embryos. Scale bars: in B, 50 µm in A,B; in D, 50 µm in C,D; in F, 50 µm in E,F; in H, 50 µm in G,H; in J, 100 µm in I,J.

 


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  		Fig. 6. RBs and DRG neurons are not necessarily derived from the same neural plate precursor cell. Embryos were injected with rhodamine-dextran into one cell at the 32-cell stage. Of 60 successfully labeled embryos, 10 had labeled RBs at 24 hpf, a frequency that is consistent with earlier experiments (Kimmel and Warga, 1987Go). We fixed all embryos at 48 hpf and processed them to reveal Hu-immunoreactivity. (A-F) Transverse sections near somite 10 of 2 dpf embryos. (A) Bright field image of an embryo with a labeled RB. The neural tube has been outlined to make it more obvious. (B) Lineage-tracer distribution in the section shown in A. Asterisk indicates a labeled RB, recognizable by its large cell body and dorsal position. (C) Anti-Hu immunoreactivity in the section shown in A. Although a DRG neuron (arrowhead) is present in this section, it does not contain lineage tracer, nor did any other DRG neurons in this embryo (not shown). (D) Bright field image of a different embryo that had a labeled RB at 24 hpf. This section was torn in processing. The neural tube has been outlined to make it more prominent. (E) Lineage-tracer distribution in the section shown in D. Arrowhead indicates a lineage-labeled DRG. (F) Anti-Hu immunoreactivity in lineage-labeled cell indicates that it is a neuron (arrowhead). Scale bar: 25 µm.

 

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© The Company of Biologists Ltd 2002