Altered Noggin Expression in Neighboring Blastomeres Changes the Neural Fate of C-Tier Blastomeres
Transcripts for several signaling proteins and receptors are found in Xenopus cleavage stage embryos, indicating that they may influence cell fate decisions prior to interactions occurring during gastrulation (reviewed in Sullivan et al., 1999). Previous studies reported that noggin transcripts are present in animal blastomeres (Sive 1993; Pandur et al., 2002), suggesting Noggin as a candidate for the previously described animal-to-vegetal signal that promotes a primary neuronal fate in C-tier lineages (Bauer et al., 1996). To characterize this further, we dissected 32-cell embryos into A, B, C, and D-tiers (Fig. 1), isolated mRNA and semi-quantified by PCR the relative levels of maternal noggin and bmp4 mRNAs in each tier. Because cleavage furrows vary between embryos and our samples contained material pooled from about 25 embryos, we verified the accuracy of our dissections by showing that a vegetally localized mRNA (Vg1; Weeks and Melton, 1987) was primarily detected in the vegetal (C-tier and D-tier) samples and an animal-enriched mRNA (Wnt8b; Cui et al., 1995) was excluded from the vegetal-most (D-tier) samples (Fig. 2A). On average, A-tier and B-tier blastomeres contained significantly more noggin mRNA than C-tier or D-tier blastomeres (Fig. 2B) and all tiers contain about the same amounts of bmp4 mRNA (Fig. 2B). These results are consistent with previous in situ hybridization studies performed at blastula and early gastrula stages that show that noggin mRNA is concentrated in the B-tier descendants, with some detected in C- and D-tier descendants (Vodicka and Gerhart, 1995; Kuroda et al., 2004). Together, these data suggest that C-tier blastomeres and their progeny may be subject to Noggin signals from their animal blastomere neighbors, which could modulate their contributions to neural progeny.
Figure 2. Maternal noggin mRNA is differentially distributed across the 32-cell blastomere tiers. A: A representative PCR experiment demonstrating that mRNA for noggin is abundant in the A-tier and B-tier and present at a low concentration in the C-tier and D-tier. bmp4 mRNA is uniformly distributed (see B). To confirm that tiers were dissected correctly, samples were also processed for detection of an animal-enriched (Wnt8b) and a vegetal-enriched (Vg1) mRNA. B: The density of the bands was quantified and normalized to H4 expression. The results from 4 separate experiments were averaged and expressed relative to whole embryo (WE) expression, which was set at 100%. -, minus reverse transcription. Bars indicate SEM.
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If animal-tier Noggin promotes C-tier primary spinal neuron fate, then increased levels of Noggin in animal blastomeres should increase the numbers of primary spinal neurons produced in C-tier lineages, whereas decreased levels should reduce those numbers. To test this, we focused on the two C-tier blastomeres (C2, C3; Fig. 1) that produce the largest numbers of primary spinal neurons (Moody, 1989). First, noggin transcripts were injected into the neighboring B-tier blastomeres (B2, B3; Fig. 1), and the numbers of PMN and RBN produced by their C-tier neighbors were counted. PMN numbers were significantly increased in both the C2 and C3 lineages (Fig. 3A). There were small increases in the number of RBN produced in both lineages that did not reach a significant level (Fig. 3A). To antagonize a potential animal-tier-derived Noggin signal, expression of BMP4, which binds Noggin at high affinity (Zimmerman et al., 1996), was increased by mRNA injection in neighboring blastomeres. When this was done in B-tier neighbors, which are major progenitors of the neural plate (Moody, 1987), neural plate formation (83%, n = 48, sox2 or n-tubulin expression; Fig. 4F) and midline mesoderm (41%, n = 27, chd expression) were so significantly repressed that the resulting patterning defects made it difficult to analyze differentiated primary neurons. Therefore, we injected bmp4 mRNA into a non-neural region that neighbors the C-tier cells, i.e., the D-tier blastomeres (D2, D3; Fig. 1) so that the protein could diffuse into the C-tier environment with minimal direct effects on neural plate formation. In these cases, PMN and RBN numbers were significantly reduced in both C-tier lineages (Fig. 3B). Together, these experiments indicate that the local levels of extracellular Noggin in the equatorial region regulate the numbers of primary spinal neurons produced by the C-tier lineages.
Figure 3. The numbers of primary neurons produced by C-tier blastomeres are affected by altering levels of BMP signaling in neighboring blastomeres. A: Overexpression of Noggin in B-tier neighbors by mRNA injection significantly increases the numbers of PMN produced by C-tier lineages, but does not significantly alter RBN numbers. B: Overexpression of BMP by mRNA expression in D-tier neighbors significantly reduces the numbers of both PMN and RBN produced by C-tier lineages. Asterisks indicate significant difference from control numbers at the P < 0.05 level.
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Figure 4. Altering Noggin levels in single B-tier or D-tier blastomeres alters the size of the neural plate progenitor pool, indicated by sox2 expression (A–F), and the numbers of nascent primary neurons, indicated by n-tubulin expression (G–T). A: A βgal mRNA-injected (B-tier) embryo demonstrating that the width of the neural plate (purple) on the injected side (white line) is indistinguishable from the width of the neural plate on the uninjected control side (black line). B: A noggin mRNA-injected (B-tier) embryo has an expanded neural plate on the injected side (white line). C: A bmp4 mRNA-injected (D-tier) embryo has a reduced neural plate on the injected side (white line). D: Injection of the control MO (B-tier) on one side does not alter the width of the neural plate (white line). E: Injection of Noggin MO (B-tier) reduces the width of the neural plate (white line) on the side of injection. F: The entire neural plate is grossly reduced when bmp4 mRNA is injected into a B-tier blastomere. G: Control embryo demonstrating the three stripes of nascent primary neurons on each side of the neural plate. Asterisk indicates side injected with control (β-gal) mRNA. H: A noggin mRNA-injected (B-tier) embryo has expanded numbers of cells in the PMN (large arrow) and RBN (small arrow) stripes. I: D-tier expression of BMP4 represses the size of the RBN (small arrow) and interneuron (large arrow) progenitor stripes on the injected side. The PMN stripe is not visible in this neural groove stage embryo. J: Lineage labeling of an A-tier blastomere (red cells) after D-tier bmp4 mRNA injection demonstrates that many A-tier cells continue to express n-tubulin (arrows). J': βgal mRNA-injected A-tier control. In contrast, many fewer B-tier-derived cells (arrows in K) and no C-tier-derived cells (red in L) express n-tubulin following bmp4 mRNA injection of the D-tier neighbor. K': Many βGal-labeled cells (between arrows) in B-tier control; L': Several n-tubulin expressing C-tier cells (arrows) in βgal mRNA-injected control. M: Injection of a control MO on one side (asterisk) does not alter the three primary neuron progenitor stripes in the neural plate. N: Injection of Noggin MO into a dorsal B-tier blastomere reduces the PMN stripe (large arrow) on the side of the injection. O: Injection of Noggin MO into a ventral B-tier blastomere reduces the RBN stripe (small arrow) on the side of the injection. P,Q: Lineage labeling shows that after nMO injection of a B-tier blastomere, many B-tier-derived cells (between arrows) continue to express n-tubulin (P); in contrast, very few C-tier-derived cells (arrows) express n-tubulin after nMO injection of the B-tier neighbor (Q). R–T: Lineage labeling shows that after nMO injection of a C-tier blastomere, many A-tier-derived cells (R, between arrows) and B-tier-derived cells (S, between arrows) continue to express n-tubulin; in contrast, very few C-tier-derived cells (arrows) express n-tubulin (T). All examples are dorsal views. A–F are stage 14/15 (neural plate); G,H, J–N, P–T are stage 16/17 (neural fold); I, O are stage 19/20 (neural groove/tube).
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To determine whether the changes in primary spinal neuron numbers occurred early in the neural plate when the primary neuron progenitors are first established (Lamborghini, 1980; Jacobson and Moody, 1984; Chitnis, 1999), the extent of the neural plate domain was measured by sox2 expression. In control βgal mRNA-injected embryos, the size of the injected side of the neural plate was not significantly different from the uninjected side (Fig. 4A; mean difference between sides = 5.67% ± 1.3; n = 32; P > 0.05). Increased expression of Noggin in B-tier blastomeres caused the neural plate to expand on the injected side (Fig. 4B; 72.7% of embryos); the width of the neural plate on the Noggin-injected side was significantly larger than that on the uninjected side (mean increase = 24.46% ± 4.24, n = 22, P < 0.001). To lower Noggin signaling in the C-tier environment without directly inhibiting neural plate progenitors, bmp4 mRNA was injected in D-tier blastomeres. In these embryos, the neural plate was significantly smaller on the injected side (Fig. 4C; mean decrease = 25.6% ± 3.1, n = 31, P < 0.001). Thus, modulating the levels of Noggin signaling alters the numbers of cells in the neural plate that ultimately give rise to the primary spinal neurons.
To determine whether the numbers of nascent PMN and RBN were consequently altered, the expression of a neural-specific β-tubulin gene that is expressed by primary neurons near the time of their terminal mitosis (Fig. 4G; Richter et al., 1988; Good et al., 1989; Moody et al., 1996) was monitored after the same manipulations. In those embryos in which Noggin expression was increased in B-tier blastomeres, the n-tubulin expression domain was expanded (Fig. 4H; 72.6%, n = 106), whereas in those embryos in which bmp4 mRNA was injected in D-tier blastomeres, the n-tubulin expression domain was reduced (Fig. 4I; 74.1%, n = 104). Single blastomeres were labeled with lineage tracer (βGal) to determine whether these reductions were confined to the descendants of the C-tier. In A-tier labeled embryos (n = 29), numerous βGal-labeled cells continued to express n-tubulin (Fig. 4J), whereas fewer cells derived from B-tier (n = 23; Fig. 4K) and no cells derived from C-tier (n = 27; Fig. 4L) expressed n-tubulin. Thus, the effects of increased vegetal BMP4 are not confined to the C-tier-derived cells, but are most effective in cells that are in closest proximity to the source of the signal. Together, these experiments demonstrate that altering Noggin/BMP4 levels in the vicinity of C-tier blastomeres affects the production of primary spinal neurons at the earliest stages that they can be distinguished.
To determine whether the promotion of primary spinal neurons derived from the C-tier lineages is specific to Noggin originating from animal tiers, the level of Noggin protein was reduced in B-tier cells in the intact embryo by injection of antisense noggin morpholino oligonucleotides (nMO). No changes were observed in the size of the neural plate (sox2 expression) after unilateral injection of a control morpholino (cMO; Fig. 4D). However, injection of nMO in the B2 or B3 lineage caused a significant reduction (mean decrease = 14.0% ± 1.35, n = 32, P < 0.001) on the injected side (Fig. 4E). Likewise, although there were no changes after injection of the cMO (Fig. 4M), the majority of B2-injected embryos showed a reduction in the expression of n-tubulin in the PMN stripe (74.4%, n = 90; Fig. 4N), and the majority of B3-injected embryos showed a reduction in staining intensity in the RBN stripe (76.8%, n = 69; Fig. 4O) after nMO injection. This differential effect on PMN and RBN is due to the differential population of ventral (B2, C2) versus dorsal (B3, C3) regions of the neural tube (Moody, 1989; Fig. 3). To determine whether these decreases are due to repression of primary neural fate in the B-tier lineage (autonomous effect) or in the C-tier lineage (through signaling), nMO was injected into a B-tier cell and βgal mRNA injected into either the same B-tier cell or its C-tier neighbor. In all B-tier labeled embryos (n = 33), about half of the descendants still expressed n-tubulin (Fig. 4P), whereas in 87.5% (n = 40) of C-tier labeled embryos, nearly no descendants were n-tubulin positive (Fig. 4Q). Thus, the repression of primary neurons resulted from small reductions in the nMO-injected B-tier lineage and large reductions in the neighboring C-tier lineage. Although the cell counts of C-tier lineages (Fig. 3) demonstrate that B-tier Noggin levels affect the numbers of primary spinal neurons produced, C-tier blastomeres and their progeny also contain noggin mRNA at cleavage, blastula, and gastrula stages (Fig. 2; Vodicka and Gerhart, 1995; Kuroda et al., 2004), and thus may promote neural fates in their own and neighboring lineages. To test this, we injected nMO in the C-tier lineage, which resulted in a reduced n-tubulin domain in 84.7% of embryos (n = 92). Lineage labeling demonstrated that in all A-tier labeled (n = 21) and B-tier labeled (n = 24) embryos, large numbers of βGal-labeled cells continued to express n-tubulin (Fig. 4R, S), whereas in 72.5% of C-tier labeled embryos (n = 40) very few βGal-labeled cells expressed n-tubulin (Fig. 4T). These data demonstrate that reducing Noggin in C-tier blastomeres primarily affects only the C-tier lineage.
The Neural Fate-Promoting Signal Occurs in Blastomere Explants
We tested the neural-promoting signaling of B-tier blastomeres in explants that were dissected at cleavage stages and cultured in simple medium without growth factors to determine if the putative interaction occurs in the absence of gastrulation movements and their associated signaling interactions. Explants were assayed for elongation, a morphological indicator of dorsal axial tissue differentiation (Wilson and Keller, 1991), and zygotic markers that are expressed during gastrulation and/or neurulation: Xbra, a pan-mesodermal gene; chd, a dorsal mesoderm gene; sox2, a neural plate gene; and n-tubulin, a primary neuron gene. C-tier explants cultured alone (C-tier; Fig. 1) elongated slightly (Fig. 6A) in 45.7% of cases (n = 127). Nearly all expressed abundant Xbra and about half expressed abundant chd (Fig. 6A; Table 1). sox2 and n-tubulin were expressed at lower levels and frequency (Fig. 6A; Table 1). To test whether any of these genes were expressed as a result of endogenous noggin mRNA (Fig. 2), 8-cell precursors of the C-tier cells were injected with nMO to reduce the translation of noggin transcripts, and C-tier explants were made 1 hr later at the 32–64 cell stage. The frequency and intensity of Xbra and chd expression were not altered, but sox2 expression intensity was reduced and n-tubulin expression was extinguished (Fig. 6B; Table 1). These data indicate that by the time the explants were removed from the cleavage embryo, C-tier cells are autonomously capable of expressing a mesodermal fate, with some bias to express a neural (sox2) program independent of further signaling, in accord with some earlier reports (Cardellini 1988; Gallagher et al., 1991; Pandur et al., 2002). However, primary neuron development (n-tubulin) requires further exposure to Noggin.
Figure 6. Blastomere explant expression of mesodermal (Xbra, chd) and neural (sox2, n-tubulin) genes. A: C-tier explants express abundant levels of Xbra and chd, albeit the latter at a lower frequency (see Table 1 for numbers and statistical comparisons). About half of the explants express low levels of sox2 (*) and less than half express n-tubulin (arrows). B: When C-tier blastomeres were injected with nMO, sox2 expression (*) is reduced in intensity and n-tubulin expression is extinguished. C: When C-tier blastomeres were co-cultured with B-tier blastomeres, the explants elongate more elaborately than C-tier (A) or B-tier (D) explants. Although mesoderm gene expression is not altered, the frequency and intensity of neural gene expression increases. D: B-tier explants express mesodermal genes at low levels and in fewer explants (*). sox2 expression (*) is less broad and n-tubulin expression (arrow) is less frequent. E,F: Injection of βgal mRNA into a B-tier cell demonstrates that both sox2-expressing (E) and n-tubulin-expressing (F) cells are derived in part from the B-tier lineage. G,H: Injection of βgal mRNA into a C-tier cell demonstrates that both sox2-expressing (G) and n-tubulin-expressing (H) cells are derived in part from the C-tier lineage. I: When C-tier blastomeres are co-cultured with D-tier blastomeres, elongation is reduced, but the expression of mesodermal genes is unaffected. However, sox2 expression is confined to small domains (arrows) and n-tubulin expression is greatly reduced (arrow).
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Table 1. Neighboring Blastomeres and Noggin Levels Affect Explant Expression of Neural Genesa
| ||Xbra % (n)||chordin % (n)||sox2 % (n)||n-tubulin % (n)|
|C-tier alone||91.9 (37)||58.1 (43)||52.0 (50)||38.1 (21)|
|C-tier-nMO||93.3 (30)||57.7 (45)||43.5 (92)||0 (26)**|
|C + B-tiers||100 (35)||50.0 (48)||84.8 (59)**||75.0 (56)**|
|B-tier alone||10.0 (30)||30.9 (42)||38.8 (67)||9.3 (43)|
|C + D-tiers||98.0 (50)||54.8 (42)||46.3 (54)||13.3 (45)**|
|C-tier + Noggin||ND||64.1 (39)||68.3 (41)*||50.0 (30)*|
|C + B-tiers + BMP||ND||37.8 (37)*||22.0 (50)**||19.4 (36)**|
|C + B-nMO||96.4 (28)||45.2 (62)*||26.1 (46)**||23.9 (67)**|
|C + B-nMO + Noggin||ND||64.2 (19)**||55.2 (29)**||59.3 (27)**|
|C + B-cMO||ND||62.2 (37)||65.9 (41)||77.3 (44)|
|C-nMO − B||100 (33)||67.9 (28)*,†||44.1 (34)**,***||34.4 (32)**,***|
When C-tier blastomeres were cultured in continued association with their B-tier neighbors (C+B-tier; Fig. 1), neural fate was strongly promoted. These explants elongated at a significantly higher frequency (80.2%, n = 106; P < 0.005) and to a greater morphological complexity compared to C-tier explants (Fig. 6C). Xbra and chd expression was comparable in frequency and intensity to C-tier explants, whereas sox2 and n-tubulin were expressed at significantly higher frequencies and levels compared to either C-tier alone or B-tier alone explants (compare Fig. 6C to A,D; Table 1). Because Xbra and chd expression was similar in C+B-tier and C-tier explants, the increase in neural gene expression in C+B-tier explants probably is not due to increased mesoderm formation, consistent with the report that BCNE-derived neural induction is independent of mesoderm signaling (Kuroda et al., 2004). Analysis of B-tier explants cultured alone (B-tier; Fig. 1) demonstrated that their endogenous ability to express mesodermal or neural genes also does not account for the increase in C+B-tier explants; all four markers were expressed at significantly lower frequencies than in C+B-tier explants (Fig. 6D: P < 0.001, Table 1). However, when either B-tier (Fig. 6E,F) or C-tier (Fig. 6G,H) blastomeres were marked with a lineage tracer prior to co-culture, both sox2 (92.5%, n = 80) and n-tubulin (100%, n = 38) expressing cells were derived from both blastomeres. Thus, it is possible that the enhanced neural gene expression derives from mutual blastomere interactions.
Since neighboring blastomeres on the animal pole side promote C-tier neural fate, we asked whether neighboring blastomeres on the vegetal pole side repress it, as suggested by experiments in which BMP4 expression was increased in D-tier cells (Fig. 3B) and studies showing that vegetal pole blastomeres contain maternal molecules that repress a neural fate (Fig. 2; Kessler, 1999; Moore and Moody, 1999). Consistent with this proposal, explants made from C+D tier blastomeres (Fig. 1) elongated less frequently than C-tier controls (37.8%; n = 98) and the extent of elongation was less extensive than in C+B-tier explants (Fig. 6I). Although the frequency of sox2 expression was comparable to that of C-tier explants (Table 1), it was significantly reduced in 2 out of 3 independent assays (8 and 36%), and levels of expression were clearly reduced (compare Fig. 6I to 6A). n-tubulin expression was significantly reduced in frequency and intensity (Fig. 6I; Table 1). Since Xbra and chd expression was comparable to that in C-tier explants (Fig. 6I; Table 1), the effects on neural gene expression are likely independent of mesoderm formation. These data demonstrate that association with D-tier blastomeres represses the neural, including primary spinal neuronal, fate of C-tier blastomeres.
To determine whether these effects are due to Noggin expressed prior to gastrulation, blastomeres were cultured under conditions that either increased or decreased Noggin during cleavage/morula stages. First, C-tier explants were cultured in Noggin-containing medium for 2 hr, washed and then returned to factor-free medium shortly before MBT. These explants expressed sox2 and n-tubulin at significantly higher frequencies and greater staining intensities compared to C-tier explants cultured alone, whereas chd expression did not change (Fig. 7A; Table 1). To test whether the putative blastomere signal could be Noggin, C+B-tier explants were cultured in the presence of BMP4 protein for 2 hr, washed and then returned to factor-free medium shortly before MBT. All three marker genes were significantly repressed in frequency in these explants (Table 1), and the expression levels of the neural genes were markedly reduced (Fig. 7B). Blockade of endogenous Noggin was additionally achieved by injecting the 8-cell precursors of either the B-tier or C-tier with nMO to reduce the translation of noggin transcripts. Injection of a control MO (C+B-cMO) had no effect on any of the monitored genes (Table 1). In those explants in which the nMO was injected into B-tier precursors (C+B-nMO), the frequency of chd expression was reduced (Table 1), but not the intensity of the staining (Fig. 7C), and Xbra expression was unaltered (Table 1). In contrast, both the frequency and intensity of sox2 and n-tubulin expression were significantly reduced (Fig. 7C; Table 1). Lineage labeling showed that in 82.4% of sox2-positive explants (n = 34) and 63.0% of n-tubulin-positive explants (n = 46), the neural cells were derived from both B-tier and C-tiers; in the remaining explants, the cells were derived from the B-tier alone. Adding Noggin to the culture medium of C+B-nMO-injected explants for 2 hr prior to MBT reversed these reductions (Fig. 7D; Table 1). As shown in whole embryos (Fig. 4T), Noggin expression in the C-tier blastomeres also is required for the expression of the neural genes. In C+B-tier explants in which the C-tier was injected with nMO (C-nMO+B), neither mesoderm gene was altered, but both neural markers were reduced in frequency and intensity (Fig. 7E; Table 1). Lineage labeling showed that those cells expressing either Xbra or chd were derived from both blastomeres in every explant; in only 40% of sox2-positive explants (n = 15) and 34.4% of n-tubulin-positive explants (n = 32) were neural cells derived from the C-tier. In fact, comparison of the expression of chd, sox2, and n-tubulin to the C+B-nMO explants demonstrated a significantly higher frequency of expression (Table 1), suggesting that C-tier nMO injection had mostly an autonomous effect. Thus, in C+B-tier explants, blocking endogenous noggin translation in the B-tier lineage affects both B- and C-tier derived neural progeny whereas blocking endogenous noggin translation in the C-tier lineage predominantly affects C-tier derived neural progeny, consistent with the whole embryo data (Fig. 4).
Figure 7. The expression of a dorsal mesoderm gene (chd) and neural genes (sox2, n-tubulin) are altered by manipulating the levels of BMP signaling in explant cultures. A: Adding Noggin to C-tier cultures for 2 hr prior to MBT significantly increases elongation and intensifies the expression of sox2 (*) and n-tubulin (arrows) (compare Fig. 6A); chd expression is not significantly altered in frequency or intensity. B: Adding BMP4 protein to C+B-tier co-cultures for 2 hr prior to MBT significantly reduces the expression of all three genes (*).C: Reducing Noggin translation by nMO injection in B-tier blastomeres does not alter chd expression in C+B-tier explants, but reduces both sox2 (*) and n-tubulin (arrows) expression. D: The effect of the B-tier nMO injection is rescued by culturing the C+B-tier explants in exogenous Noggin for 2 hr prior to MBT. chd expression is not altered, but sox2 (*) and n-tubulin (arrows) expression is more intense and detected in more explants. E: Reducing Noggin translation by nMO injection in C-tier blastomeres does not alter chd expression in C+B-tier explants, but reduces both sox2 (*) and n-tubulin (arrows) expression.
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