XB-ART-4318Dev Biol 2003 Dec 15;2642:339-51. doi: 10.1016/j.ydbio.2003.08.017.
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Cloning and characterization of Xenopus Id4 reveals differing roles for Id genes.
We have identified Xenopus Id4, a member of the Id (inhibitor of differentiation/DNA binding) class of helix-loop-helix proteins. Id factors dimerize with general bHLH factors, preventing their interaction with tissue-specific bHLH factors, to inhibit premature differentiation. The presence of several Id proteins could reflect simple redundancy in function, or more interestingly, might suggest different activities for these proteins. During embryonic development, Xenopus Id4 is expressed in a number of neural tissues, including Rohon-Beard neurons, olfactory placode, eye primordia, and the trigeminal ganglia. It is also expressed in other organs, such as the pronephros and liver primordium. As embryogenesis progresses, it is expressed in the migrating melanocytes and lateral line structures. We compare the expression of Id4 mRNA with that of Id2 and Id3 and find that the Id genes are expressed in complementary patterns during neurogenesis, myogenesis, kidney development, in the tailbud, and in the migrating neural crest. To examine the regulation of Id gene expression during Xenopus neural development, we show that expression of Id3 and Id4 can be induced by overexpression of BMP4 in the whole embryo and in ectodermal explants. Expression of Id2, Id3, and Id4 in these explants is unaffected by the expression of FGF-8 or a dominant-negative Ras (N17ras), suggesting that Id genes are not regulated by the FGF signaling pathway in naive ectoderm. We also show that Notch signaling can activate Id2 and Id3 expression in the whole embryo. In contrast, Id4 expression in the Rohon-Beard cells is inhibited by activated Notch and increased by a dominant-negative Delta. This may reflect an increase in Rohon-Beard cells in response to inhibition of Notch signaling rather than transcriptional regulation of Id4. Finally, to compare the activities of Id2, Id3, and Id4, we use animal cap explants and in vivo overexpression to show that Id proteins can differentially inhibit the activities of neurogenin and neuroD, both neurogenic bHLH molecules and MyoD, a myogenic bHLH protein. Id4 is able to inhibit the activity all these bHLH molecules, Id2 inhibits MyoD and neuroD, while Id3 blocks only neuroD activity in our assays.
PubMed ID: 14651922
Article link: Dev Biol
Species referenced: Xenopus
Genes referenced: actc1 actl6a bmp4 id2 id3 id4 myod1 neurod1 neurog1 nog notch1
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|Fig. 2. mRNA expression of X. laevis Id2 (A, B, C) and Id3 (D, E, F). In all cases, anterior is to the left. (A, B, D, E) are dorsal views, (C and F) are lateral views. (A) Stage 15 embryo stained with Id2. Bilateral stripes at midline marked by black asterisk. Expression in the lateral plate mesoderm (red arrowheads) extends ventrally away from the viewer. (B) Stripe in the rhombencephalon (blue arrowhead) and population of cells that could be migrating crest (blue arrow). Staining in the lateral plate mesoderm has coalesced (red arrowheads). (C) Id2 is expressed in the eyes, branchial arches, brain structures, kidneys (red arrowhead), segmented myotome (m), and anterior blood islands (b). (D) Expression of Id3 at stage 11 is throughout the ectoderm but not in the blastopore (at right). (E) Stage 18 embryo stained with Id3. Expression is strong in the anterior neural plate, extending posteriorly to a stripe in the hindbrain (open red arrowhead) and a smaller stripe posterior (closed red arrowhead). Expression extends posteriorly throughout the neural plate. (F) Id3 is expressed in the most dorsal and ventral portions of the myotome (m).|
|Fig. 3. Id4 mRNA expression pattern in the early embryo. (A, C, E, G) Dorso-anterior views at stage 13, 15, 17, and 19. (B) Lateral view stage 13. (D, F) Dorsal view stage 15 and 17. (H) Lateral view stage 19. (I, K) Lateral and dorsal views, stage 24. (J, L) Lateral view of head staining, stages 32 and 34. (M) Lateral view stage 37. Embryos show staining in the anterior region of the embryo corresponding to the cement gland (yellow arrowhead). By stage 19, expression is also obvious bilaterally in the olfactory placode (black arrow), eye primordia (blue arrowhead), and in the trigeminal ganglia (red arrow). Dorsal and lateral views show expression in the Rohon–Beard neurons (black arrowhead). In later stages, Id4 is expressed in the liver (marked with a yellow L), branchial arches (black b), kidney (red arrowhead marks tubules in J, L, and M, red arrow marks duct in M), and in migrating melanophores (small red arrowheads in M).|
|Fig. 4. mRNA expression of X. laevis Id2 (A–D), Id3 (E–H), and Id4 (I–L). Yellow lines mark plane of section. (A, E, I) Both Id2 and Id3 are expressed in the nasal pit (blue arrowhead) and in the otic vesicle (left of asterisk). Migrating cells (small red arrowheads) mark ventrally migrating muscle cells in (A) (Id2), the leading edge of migrating muscle cells in (E) (Id3), and migrating melanophores in (I) (Id4). (D, H, L) Id2 is expressed in both the dorsal and ventral portions of the myotome, in a region corresponding to the nuclei of the muscle cells (D–M). Id3 is expressed in the most dorsal and ventral portions of the myotome (H–M). Id4 is expressed weakly in a similar pattern to Id2. Both Id2 and Id3 are expressed in punctate cells in the fin mesenchyme. Id4 is not expressed in the fin mesenchyme. (The chordoneural hinge is marked with a red arrowhead, the inner fin with an open yellow arrowhead and the outer fin with a closed yellow arrowhead.) Id4 is also expressed in the lateral line (io: infraorbital, a: aortic) marked in (I).|
|Fig. 5. The effect of BMP signaling on Id gene expression. (A) RT-PCR analysis on stage 20 ectodermal explants. E, whole embryo control; −RT, minus reverse transcriptase control; C, uninjected explant; Noggin doses: 37.5 pg, 25 pg, 12.5 pg; BMP4 doses: 125 pg, 250 pg, 374 pg. Injection of Noggin mRNA blocks expression of Id2 and Id4. Sox 3 expression is induced, indicating the presence of neural tissue, while epidermal keratin (EpiK) is also lost. BMP-4 injection does not increase the amount of Id2, Id3, or Id4 RNA in explants. (B–D) Lateral views of stage 15 embryos, anterior to left. (B, C) In situ hybrization for Id3. (D, E) In situ hybrization for Id4. (B) and (D) are uninjected controls. Overexpression of BMP-4 in the lateral ectoderm induces ectopic bottle cells (red arrowheads) which appears at the source of ectopic BMP (lineage tracing data not shown). (D) Ectopic Id4 RNA is present at a border of cells expressing BMP (open red arrowhead), rather than in the cells with the highest amount of injected BMP mRNA. (E) Id3 is expressed throughout the region surrounding the ectopic bottle cells but also stops abruptly, creating a sharp border.|
|Fig. 6. Id genes are differentially regulated by Notch signaling. (A–B) Id3 is induced in embryos injected with 500 pg of NotchICD (NICD). (C–F) ld4 expression is perturbed in embryos injected with NICD and enhanced in embryos injected with 1 ng of Deltastu, a truncated Delta which blocks Notch signaling. All views are dorsal with anterior to the left, except (F), which is a lateral view. mRNAs were injected in one cell at the two-cell stage. Injected side is marked with yellow arrowhead.|
|Fig. 7. Distinct activities of ld proteins. (A–H) Lateral views of stage 28 tadpoles, anterior to left. Embryos have been stained for expression of cardiac actin, which is expressed in the heart and somites. (A–D) Injection of 1 ng each of Id2, Id3, and Id4 has no effect on the expression of cardiac actin, suggesting that the mRNAs used in our study are unable to disrupt endogenous myogenic bHLH complexes. (E) Injection of 500 pg of MyoD induces ectopic expression of cardiac actin. (F, H, J, lanes 8 and 10) Id2 and Id4 can block the activities of ectopic MyoD, while Id3 does not do so (G and J, lane 9). Ectopic cardiac actin is marked with red arrowheads. (J) RT-PCR analysis of ectodermal explants injected with Id mRNAs with and without MyoD. mRNA doses are as above. (K) Id4 blocks expression of neural β-tubulin induced by neurogenin (lanes 7–10) and neuroD (lanes 11–14). Neurogenin and neuroD (100 pg each) were injected. Then 1 ng of each Id gene was injected. E, whole embryo control; RT, no reverse transcriptase control; C, uninjected ectoderm control.|
|id4 (inhibitor of DNA binding 4, dominant negative helix-loop-helix protein ) gene expression in Xenopus laevis embryos, NF stage 24, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.|
|id4 (inhibitor of DNA binding 4, dominant negative helix-loop-helix protein ) gene expression in Xenopus laevis embryo, NF stage 37, assayed by in situ hybridization, lateral view, anterior left, dorsal up.|
|id4 (inhibitor of DNA binding 4, dominant negative helix-loop-helix protein) gene expression in Xenopus laevis embryos, NF stage 19, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.|
|id2 (inhibitor of DNA binding 2) gene expression in Xenopus laevis embryos, NF stage 15-33, as assayed by in situ hybridization. Upper panels: NF stage 13 ( left) and NF stage 20 (right), dorsal view, anterior left; lower panel, NF stage 30, lateral view, anterior left, dorsal up.|
|id3(inhibitor of DNA binding 3, dominant negative helix-loop-helix protein ) gene expression in Xenopus laevis embryos, NF stage 11-37, as assayed by in situ hybridization. Upper panels, dorsal view, anterior left. Lower panel:, lateral view, anterior left, dorsal up.|