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Neurogenesis in Xenopus neural ectoderm involves multiple gene families, including basic helix-loop-helix transcription factors, which initiate and control primary neurogenesis. Equally important, though less well understood, are the downstream effectors of the activity of these transcription factors. We have investigated the role of a candidate downstream effector, Noelin-1, during Xenopus development. Noelin-1 is a secreted glycoprotein that likely forms large multiunit complexes. In avians, overexpression of Noelin-1 causes prolonged and excessive neural crest migration. Our studies in Xenopus reveal that this gene, while highly conserved in sequence, has a divergent function in primary neurogenesis. Xenopus Noelin-1 is expressed mainly by postmitotic neurogenic tissues in the developing central and peripheral nervous systems, first appearing after neural tube closure. Its expression is upregulated in ectopic locations upon overexpression of the neurogenic genes X-ngnr-1 and XNeuroD. Noelin-1 expression in animal caps induces expression of neural markers XBrn-3d and XNeuroD, and co-expression of secreted Noelin-1 with noggin amplifies noggin-induced expression of XBrn-3d and XNeuroD. Furthermore, in animal caps neuralized by expression of noggin, co-expression of Noelin-1 causes expression of neuronal differentiation markers several stages before neurogenesis normally occurs in this tissue. Finally, only secreted forms of the protein can activate sensory marker expression, while all forms of the protein can induce early neurogenesis. This suggests that the cellular localization of Noelin-1 may be important to its function. Thus, Noelin-1 represents a novel secreted factor involved in neurogenesis.
FIG. 3. Noelin-1 and -2 expression during development. Whole mount in situ hybridization with a probe to the Z exon reveals the distribution of Noelin-1 and Noelin-2 transcripts in the neural tube and cranial ganglia. All embryos are oriented with anterior to the right. A: Earliest expression is seen after neural tube closure in the spinal cord and trigeminal ganglia at stage 22. B: By stage 27 cells in the olfactory placodes and in the trigeminal (V) and geniculate (VII) ganglia are strongly reactive. Punctate staining is visible in the pineal gland and spinal cord. C: A stage 33 embryo shows increasing signal in the Vth and VIIth ganglia, olfactory pits, pineal gland and spinal cord. A population of cells in the retina is also positive for Noelin-1 and -2. D: By stage 35/36 the IXth and Xth ganglia are also positive for the transcripts. A small number of cells in the branchial arches express the Z exon (arrowhead). E-G: Dorsal views of embryos in A-C. E: Stage 22 embryo shows staining in the Vth ganglia and in the spinal cord. Transcripts are not detected in the brain at this stage. The black arrow marks the anterior extent of Noelin-1 and -2 signal in the neural tube. F: By stage 27, spinal cord staining extends into the brain and up to the level of the midbrain. The pineal gland and olfactory placodes are visible. G: At stage 33 the signal is increasingly intense in the cranial ganglia, spinal cord, and more rostrally into the forebrain. e, eye; hb, hindbrain; o, olfactory placode/pit; p, pineal gland; sc, spinal cord; V, trigeminal; VII, geniculate; IX, glossopharyngeal; X, vagal; asterisks mark the general position of otic vesicles. Brown color is due to melanocytes in the skin or retinal pigmented epithelium in the eye.
FIG. 4. Cranial expression of Noelin-1 and -2. 10mmsections through head regions of a stage 35 embryo similar to that in Fig. 3D, stained with a probe to the Z exon. A: A midbrain-region transverse section reveals Noelin-1 and -2-positive cells in the pineal gland and brain. Arrow points to the body of the gland, arrowhead denotes cells that are not positive for Noelin-1 or -2. B: A hindbrain section at the midotic vesicle level shows signal in the VIIIth ganglion (arrow) and faintly in the IXth ganglion (arrowhead) ventral to the otic vesicle. C: Parasagittal section through the eye, Vth and VIIth ganglia and the otic vesicle. The retinal ganglion cell layer also contains signal. D: A higher-power view of a transverse section through the eye reveals transcripts in the retinal ganglion cell layer and inner nuclear layer, but not in the pigmented epithelium, photoreceptor layer, ciliary marginal zone or lens. E: A hindbrain section showing signal in the IXth ganglion. e, eye; hb, hindbrain; inl, inner nuclear layer; le, lens; mb, midbrain; rgc, retinal ganglion cell layer; V, trigeminal; VII, facial nerve and geniculate; VIII, facial-acoustic; IX, glossopharyngeal; asterisks denote otic vesicle; brown pigment is seen in melanocytes and retinal pigmented epithelium.
FIG. 5. Caudal expression of Noelin-1 and and HNK-1 immunostaining. 10 mm sections through embryos stained with the Z exon. Arrowheads mark neuron populations in the spinal cord that express the gene. A: Stage 28 embryo with signal in Rohon-Beard cells and interneurons. B: The same section stained with HNK-1 shows that Noelin marks only a subset of the neurons present. Some of the HNK-1 signal is quenched by the in situ reaction product. C: Stage 28 embryo with signal in interneurons and motor neurons. D: HNK-1 staining of the same section as in C. E: Stage 33 spinal cord illustrates expanding domain of Noelin-1 and -2 expression. F: Stage 35 spinal cord shows neural crest cells migrating into the fin (arrow) expressing Noelin-1 and -2. In E and F, black line demarcates the ventral neural tube. Brown cells are pigmented neural crest cells (melanocytes). no, notochord.
FIG. 6. DiI labeling of premigratory medial neural crest. Noelin-1 and -2 are expressed in the cranial ganglia. Transverse section through stage 33 embryos with neural crest cells labeled by focal injections of DiI in the neural folds at stage 17. Whole mount in situ hybridization was performed against the Z exon. A: Schematic diagram of a stage 17 embryo illustrating relative locations of lateral neural crest (pink), placode domain (blue) and the medial neural crest which were labeled with DiI (red dots, arrow). B: Bright field view of a stage 33 embryo section at the caudal eye level, showing Noelin-1 and -2 expression in the Vth ganglion. The cells marked by the arrow are not Noelin-1 or -2-positive. C: Fluorescent view of section in B, arrow marks DiI positive neural crest cells. D: Bright field view of a hindbrain level section including the VIIth ganglia. Ganglion cells express Noelin-1 and -2 (arrowhead), migrating neural crest cells do not (arrow). E: Fluorescent image of section in (D) shows DiI-positive cells surrounding, but not mixed within the ganglion. Arrow marks a cell that is DiI positive and Noelin negative; arrowhead marks a cell that is Noelin-1 and -2 positive and DiI negative. F: Overlay of the fluorescent image shows nonoverlapping DiI and Noelin-1 and -2 cells. In both embryos (B-C and D-F), DiI positive neural crest cells surround the condensing ganglia but do not appear to mix with the placodal cells at this stage. Analysis at later stages was not possible due to dilution of the DiI. e, eye; hb, hindbrain; mb, midbrain; V, Vth ganglion (trigeminal); VII, VIIth ganglion, (facial nerve and geniculate).
FIG. 7. Neurogenin induces Noelin-1 and expression. Embryos injected with 100 pg X-ngnr-1 make ectopic neurons in the epidermis. Noelin-2 upregulation is represented by expression of the A exon and Noelin-1 is represented by expression of the B exon. These in situ results are identical to those performed for the Z exon, and results are identical for X-ngnr-1 and XNeuroD. Embryos shown are siblings from the same injection experiment, with anterior to the right. A: Noelin-2 is upregulated robustly in X-ngnr-1 injected embryos. An embryo probed for the A exon shows induced Noelin-2 expression in ectopic neurons (for example, see white arrow). Also note ectopic staining for the A exon in the head (arrowhead). B: Sibling injected embryo stained for the B exon. Ectopic neurons (arrow) express Noelin-1. C: Sibling embryo stained with N-tubulin shows massive ectopic induction of neurons in the epidermis (arrow). Note the extent of N-tubulin staining as compared to embryos in (A) and (B). Extra headtissue is positive for N-tubulin (arrowhead). D-G: Animal caps sectioned at 12 microns. Embryos were injected with X-ngnr-1 or b-galactosidase (control) at the 2-cell stage, caps were collected at stage 9 and cultured to stage 24. In situs were performed for N-tubulin or the separate Noelin exons, the Z pattern is representative of Noelin-1 and -2. D: Noelin-1 and -2 are upregulated weakly in animal caps as compared to the results in whole embryos (arrows). E: Animal caps stained with N-tubulin show robust activation of expression (arrows). F: Control animal cap stained with the Z exon shows no induction of Noelin expression in the absence of X-ngnr-1. G: Control animal cap stained with N-tubulin also fails to express this gene. e, shows region of eye, usually malformed or absent in injected embryos.
FIG. 8. Noelin-1 promotes neurogenesis in neuralized tissue. Animal caps were collected at stage 9 from embryos injected at the 2-cell stage with 100 pg noggin, 500 pg Noelin-1, or both. The explants were cultured to stage 27. Whole mount in situ hybridization was performed against N-tubulin. A: Animal caps injected with Noelin-1 alone do not express N-tubulin. B: Animal caps injected with noggin do not express N-tubulin. (Diffuse purple color is due to trapping of reaction components inside animal cap cavities, see black arrowhead.) C: Animal caps co-injected with noggin and Noelin-1 upregulate N-tubulin by stage 24 (caps shown in this figure were fixed at stage 27). At stage 24, a smaller proportion of the explants express N-tubulin (3/6, data not shown). In the representative experiment shown here, 14/17 explants express N-tubulin to varying degrees. In some cases, strongly positive N-tubulin regions were confined to small areas of the explant (arrowheads); the majority of explants with induced N-tubulin expression show positive cells scattered through regions of the
explants (arrows).
FIG. 9. Noelin-1 activates sensory and differentiation markers. RTPCR analysis shows Noelin-1 induces expression of several neural markers. 2-cell stage embryos were injected with 500 pg Noelin-1 constructs, 100 pg noggin, or both; control animal caps were injected with 600 pg green fluorescent protein mRNA. Animal caps were dissected at stage 9 and cultured to stage 24.A: Lane 1: whole embryo control at stage 24; lane 2: St 24 control injected animal caps; lane 3: noggin-injected animal caps; lane 4: noggin + Noelin-1 animal caps; lane 5: Noelin-1 injected animal caps. Animal caps injected with both noggin and Noelin-1 express SybII (SybII row, lane 4). HoxB9 is very slightly induced by Noelin-1 alone in this experiment. Krox-20 is induced by Noelin-1, but not by Noelin-11noggin. En-2 is not induced by any of the injections. Otx2 and NCAM are induced by explants injected with noggin (lanes 3 and 4), Noelin-1 alone slightly induces Otx2 (lane 5). EF1a is a loading control. Muscle actin (MA) is a control for mesoderm contamination of animal cap explants. B: Secretion or ER localization of Noelin-1 affects its inducing capabilities. Three different forms of Noelin-1 were expressed either alone or in conjunction with noggin. Noelin-1 is the endogenous form of the protein; Noelin-1-myc is a highly secreted form (see Fig. 2B); and Noelin-1-myc-KDEL is retained in the endoplasmic reticulum (ER, see Fig. 2D). Lane 1: st 24 whole embryo; lane 2: st 24 control injected animal caps; lane 3: noggin caps; lane 4: noggin + Noelin-1; lane 5: Noelin-1; lane 6: noggin + Noelin-1-myc; lane 7: Noelin-1-myc; lane 8: noggin + Noelin-1-myc-KDEL; lane 9: Noelin-1-myc-KDEL. SybII is upregulated in all samples with Noelin isoforms when explants are co-injected with noggin (lanes 4, 6, 8 of SybII row). Highly secreted form of Noelin-1 caused greatest SybII expression (lane 6). XBrn-3d is induced by noggin and by Noelin-1 (lanes 3 of XBrn-3d row). In conjuction with noggin, secreted forms of Noelin-1 cause up to 3.4-fold induction of this gene over noggin alone (lanes 4 and 6). XNeuroD expression is activated by noggin and by Noelin-1 co-expression (lanes 3, 4, 6 and 8 of XNeuroD row). Noelin-1-myc alone induces XNeuroD expression (lane 7). HoxB9, En-2 and NCAM are not induced by Noelin-1 (lanes 5, 7, 9 of appropriate rows). Otx2 is upregulated by Noelin-1 constructs by two-fold over control animal caps (lane 2 vs. lanes 5, 7, and 9); however noggin is several times stronger an activator of Otx2 than Noelin-1. Krox-20 expression is induced by all three constructs of Noelin-1 (lanes 5, 7, and 9 of Krox-20 row); however, addition of Noelin-1 is not sufficient to cause noggin-injected explants to express Krox-20. EF1a and MA are controls as described above.
