March 15, 2005;
To proliferate or to die: role of Id3 in cell cycle progression and survival of neural crest progenitors.
The neural crest
is a unique population of mitotically active, multipotent progenitors that arise at the vertebrate neural plate border
. Here, we show that the helix-loop-helix transcriptional regulator Id3
has a novel role in cell cycle progression and survival of neural crest
progenitors in Xenopus. Id3
is localized at the neural plate border
during gastrulation and neurulation, overlapping the domain of neural crest
induction. Morpholino oligonucleotide-mediated depletion of Id3
results in the absence of neural crest
precursors and a resultant loss of neural crest
derivatives. This appears to be mediated by cell cycle inhibition followed by cell death of the neural crest
progenitor pool, rather than a cell fate switch. Conversely, overexpression of Id3
increases cell proliferation and results in expansion of the neural crest
domain. Our data suggest that Id3
functions by a novel mechanism, independent of cell fate determination, to mediate the decision of neural crest
precursors to proliferate or die.
[+] show captions
Id3 is expressed in premigratory and migratory neural crest cells. (A) Id3 expression at the gastrula stage (stage 10.5). Id3 expression is seen in the ectoderm of the animal half of the embryo and starts to decrease in the region above the dorsal lip (arrow). (B-D) Dorsal views of embryos at stages 12.5-15 (anterior toward left). Id3 expression is progressively down-regulated through late-gastrula and early-neurula stages and maintained at the neural plate border. Arrows indicate blastopore (B), and neural fold (C,D). (D) The arrowhead indicates the hindbrain region. (E) Anterior lat eral view of Id3 expression at stage 15. Id3 is clearly expressed in the anterior neural plate (arrow), midbrain and hindbrain regions in two stripes, and at the neural plate border including the premigratory neural crest domain (arrowhead). (F) Double in situ hybridization with Id3 and FoxD3 RNA. The foxD3 expression (purple) overlaps with the Id3 expression (light blue) in the neural crest domain (arrowhead). The Id3-expressing region is slightly larger than the foxD3-expressing neural crest domain. (G) Dorsal view of Id3 expression at stage 21. Id3 is expressed in migrating cranial neural crest cells (arrow) and the trunk neural fold region (arrowhead). (H) Dorsal view of Id3 expression at stage 23. Id3 is expressed in the dorsal neural tube at cranial and trunk levels (arrowhead) as well as in the optic placode (arrow). (I) Section of an early-neurula-stage embryo showing Id3 expression in the superficial layer (arrow) at the border of neuronal and non-neuronal ectoderm. (J,K) Section of an embryo showing migrating neural crest cells (arrow) coming out of the cranial dorsal neural tube at stage 22. Id3 expression in the dorsal neural tube (J) is magnified in K. Id3 is also expressed in the optic placode (arrowhead). (NT) Neural tube. (L) Id3 expression in the trunk dorsal neural tube and neural crest cells (arrow) coming out of the dorsal neural tube as well as in the ectoderm above it. (NT) Neural tube; (n) notochord.
RT-PCR analysis in ectodermal explants of embryos injected with chordin and wnt8 RNA. Animal caps were dissected from the embryos injected with 100 pg of chordin (Chd) and/or 200 pg of wnt8 RNA, cultured until stage 18 and subjected to RT-PCR. Neural crest marker slug expression is induced only in the presence of both chordin and wnt8 as previously shown. Only Id3 is maintained under the neural crest-inducing conditions, while Id4 was maintained or up-regulated under neuralizing condition. EF1α expression is used as a loading control.
Id3 depletion leads to a loss of neural crest progenitors. (A) Morpholino antisense oligo sequence (Id3-MO) corresponding to a region 5′ upstream of the Id3 start codon (25 bp; -8 to -32) and its control oligo (control-MO) with seven mismatches (red letters). (B) Id3 protein synthesis was monitored in the absence of oligo (no MO) or in the presence of morpholino oligos (control, Id3 or Id2 oligo) in an in vitro translation system. Only Id3-MO specifically blocks Id3 protein translation. (C) Neurula-stage embryos injected with control-MO or Id3-MO. (Panel a) slug expression (arrow) is intact on the side injected with control-MO (C-MO, Red-Gal). (Panels b-d) Embryos injected with Id3-MO show the loss of early neural crest markers (arrows) on the injected side, which is marked by Red-Gal staining: slug (panel b), sox10 (panel c), and foxD3 (panel d). (D) slug expression is not affected by Id2 depletion (arrow). (E) The loss of neural crest progenitors by Id3 depletion (panel a) is rescued in the presence of chick Id3 (panels b,c).
Id3 depletion results in a loss of neural crest derivatives. (A) Dorsal (panel a) and ventral (panel b) views of an embryo injected with control-MO showing symmetrical head morphology. Dorsal (panel c) and ventral (panel d) views of an embryo injected with Id3-MO exhibiting structural loss on the injected side (arrow). The arrowhead indicates the midline. (B, panel a) Schematic diagram of cartilages in a stage 45 embryo. The different colors indicate the skeletal elements originate from different streams of migrating cranial neural crest. Meckel's (i), cerathoyal (ii), basihyal (iii), and branchial arch (iv) cartilages are shown here. (Panel b) Cartilage staining of a control embryo shows the symmetrical structures shown in the diagram. (Panels c,d) Whole or partial loss of cartilage on the Id3-MO-injected side. The dotted red line indicates the midline. (C) The dorsal fin of a control-MO-injected embryo is intact (panel a), but an Id3-MO-injected embryo exhibits a collapsed dorsal fin by depletion of the structure on the injected side (panel b). (D) Anterior and posterior patterning in Id3-MO-injected embryos is normal (panel b) compared with the control (panel a). (E) Stage 31 embryo injected with Id3-MO and subjected to in situ hybridization with a trp-2 probe. (Panels a,b) Eyes express trp-2. trp-2-positive pigment cells (purple) that are newly synthesized (dorsal neural tube; white arrow) or migrating superficially under the ectoderm (lateral; black arrowhead) are depleted or decreased on the Id3-MO-injected side (panels a,c), compared with the control side (panels b,d). (Panels a,c) Red dots (red arrow) are Red-Gal staining indicating Id3-MO-injected cells. (F) sox10 expression is lost in migrating neural crest cells destined to give rise to cranial ganglia (V, VII, IX, and X) on the Id3-MO-injected side that is marked by Red-Gal staining (panel a), compared with the control side (panel b).
Id3 is required for cell proliferation and survival of neural crest. (A) Neurula-stage embryos injected with morpholino oligos and subjected to double in situ hybridization with FoxD3 and Sox2 RNA probes. (Panel a) Dorsal view of a control embryo (anterior toward left) shows foxD3 expression (purple) intact on the side injected with control-MO (Red-Gal). (Panels b,c) Dorsal view of an embryo (anterior toward left) injected with Id3-MO and anterior view of another embryo injected with Id3-MO show that the neural crest marker foxD3 (purple, arrowhead) is lost on the injected side (Red-Gal), while the neural plate (border line, arrow) marked by sox2 expression (light blue) is not expanded (red dotted line) and does not replace the neural crest. The black dotted line indicates the midline. (B) Cell proliferation assay measured by immunostaining with antiphosphorylated histone H3 antibody in an embryo injected with Id3-MO. Dorsal view (panel a) and magnified anterior view (panel b) of an embryo show that mitotic cells (black dots at control side, arrowhead) are absent or decreased in the neural crest region injected with Id3-MO (arrow, Red-Gal). (C) Expression of cell cycle inhibitor in the embryos injected with Id3-MO. (Panel a) p27XicI (purple, arrow) is ectopically expressed in neural crest progenitors when Id3 is depleted (white arrow). (Panel b) Double in situ hybridization reveals that foxD3 expression (light blue, arrowhead) in neural crest is replaced by p27XicI expression (purple, arrow) at the side injected with Id3-MO (Red-Gal). (D) Differentiation markers in the embryos injected with Id3-MO. n-tubulin (panel a) and NeuroD (panel b) are not ectopically expressed in the neural crest at the side injected with Id3-MO at stage 13. (Panel b) Their expression is slightly increased in trigeminal ganglia at the side injected with Id3-MO (arrow) at stage 14. (E) TUNEL staining in the embryos injected with Id3-MO or control-MO. (Panels a,a′) Id3-MO-injected region (Red-Gal) of a stage 11 embryo is negative (arrow, no black dot) in TUNEL staining. (Panels b,b′) Dorsal view of a stage 14 embryo (anterior toward left) shows significantly increased cell death (black dots, arrow) on the neural crest region injected with Id3-MO (Red-Gal), compared with the uninjected side (arrowhead). (Panels c,c′) Anterior view of a stage 17 embryo also shows elevated cell death (black dots, arrow) on the neural crest region injected with Id3-MO (X-gal staining, turquoise color), compared with the uninjected side (arrowhead). (Panels d,d′) Anterior view of a stage 16 embryo injected with control-MO shows that cell death doesn't occur in the injected region (Red-Gal, arrow), compared with the uninjected side (arrowhead). The MO-injected region is magnified for each embryo in a′-d′. (F) The graph illustrating the results of TUNEL staining as percentages of TUNEL-negative (black bar) or TUNEL-positive (red bar) embryos out of the embryos injected with control-MO or Id3-MO.
Id3 overexpression expands the neural crest precursor domain. (A) Xenopus Id3 (panels a,b) and Id4 (panel c) ectopic expression (Red-Gal) expands the neural crest domain as shown by slug expression (purple). Chick Id2 (panel d), Id3 (panel e), and Id4 (panel f) also have similar activity. (B) The sections of cranial (panel a) and trunk (panel b) neural plates of the stage 14 embryos subjected to BrdU-labeling assay show that the number of proliferating cells (BrdU-labeled cells, brown) is increased upon overexpressing Id3 (X-Gal, turquoise color). (Arrow) Id3-injected side; (arrowhead) control side. (C) The number of pigment cells expressing trp-2 (purple) is increased at the side injected with Id3 RNA (panels a,c; Red-Gal), compared with the control side (panels b,d) at stage 33.