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Abstract
Before the nervous system establishes its complex array of cell types and connections, multipotent cells are instructed to adopt a neural fate and an anterior-posterior pattern is established. In this report, we show that Smad10, a member of the Smad family of intracellular transducers of TGFbeta signaling, is required for formation of the nervous system. In addition, two types of molecules proposed as key to neural induction and patterning, bone morphogenetic protein (BMP) antagonists and fibroblast growth factor (FGF), require Smad10 for these activities. These data suggest that Smad10 may be a central mediator of the development of the frog nervous system.
Figure 1. Smad10 and Smad4 Are Distinct(A) Synthetic mRNA encoding Smad4, Smad10, Smad4/10/4, Smad10/4/10, Smad4-C, and Smad10-C (4 ng) was injected into one-cell embryos, and animal caps were analyzed for expression of molecular markers by RT-PCR. The lane marked Embryo contains total RNA from whole embryos; −RT is identical to Embryo except that reverse transcriptase (RT) was omitted; EF-1α, a ubiquitously expressed message, is a loading control (Krieg et al., 1989).(B) mRNA encoding GST-Hesr-1 (6 ng), myc-Smad10 (1 ng), and myc-Smad4 (1 ng) were injected into one-cell embryos and a GST pull-down assay was performed.(C) Animal caps expressing Hesr-1 (1 ng) were evaluated by RT-PCR.(D) Synthetic mRNA encoding DNS10 (4 ng) or Smad10 (S10, 4 ng) was injected into one-cell embryos alone or together and animal caps were analyzed by RT-PCR.
Figure 2. Smad10 Is Required for Formation of the Nervous System(A) Control MO (C MO, 5 ng/cell), Smad10 MO (S10 MO, 5 ng/cell), or DNS10 mRNA (4 ng/cell) were injected into the animal pole of both blastomeres of two-cell embryos. At stage 35, embryos were processed for histology and hematoxylin and eosin staining. NC, notochord; NT, neural tube; PE, pharyngeal endoderm. (B) Two-cell embryos were injected with 5 ng of morpholino or 4 ng of mRNA into the animal pole of each blastomere and then analyzed by whole-mount in situ hybridization for expression of the general neural marker NRP-1 (Knecht et al., 1995) and the mesodermal marker muscle actin (MA; Mohun et al., 1984).(C) Animal caps from embryos injected with control MO, Smad10 MO, and Smad10 MO with Smad10 mRNA (1 ng/cell, rescue) were analyzed by RT-PCR. U, uninjected; −, no RT. The phenotypes were highly penetrant (>80%) in multiple experiments (n > 5).
Figure 3. Smad10 Is Required for Expression of Anterior and Posterior Neural Markers and for Formation of Melanocytes(A) Embryos were injected and analyzed by in situ hybridization as in Figure 2B.(B) Albino embryos (albino mother, pigmented father) were examined for the appearance of melanocytes (MC; dark cells visible in the C MO embryo).(C) Embryos were injected and analyzed at early neurula by in situ hybridization as in Figure 2B, except that 12.5 ng of morpholino was injected per cell.(D) Animal caps were explanted at stage 8.5, prior to organizer signaling, and Sox-2 expression was analyzed by RT-PCR.(E) Two-cell embryos were injected with morpholinos and, at the 32-cell stage, β-galactosidase mRNA was injected into the B1 blastomere. At stage 27, the embryos were stained with X-gal (blue).
Figure 4. Smad10 Is Required for Formation of Organizer-Induced Tissues(A) Embryos were injected and analyzed by in situ hybridization as in Figure 2B. H, heart. (B) Two-cell embryos were injected with 5 ng morpholino into both animal poles and analyzed by RT-PCR at stage 33. WT-1 marks the kidney; NKX2.5 marks the heart. (C and D) Both animal poles of two-cell embryos were injected with morpholino and at stage 10.25 the embryos were processed for in situ hybridizations (C) or RT-PCR (D). GC, goosecoid; BU, brachyury. (E and F) Embryos were injected as in (C) and analyzed by in situ hybridization (E) with probes for XHex (stage 10.25) and endodermin (EDD, stage 27) or analyzed by RT-PCR (F) for the endodermal markers XHex (stage 10.25), Cerberus (cerb, stage 10.25), Xsox17 (both α and β isoforms, stage 10.25), and EDD (stage 27).
Figure 5. Smad10 Is Required for FGF-Mediated Neural Induction(A) One-cell embryos were uninjected or injected with Smad10 or control MO and then animal caps were treated with FGF protein at the gastrula stage. These caps, and uninjected/untreated caps (Control), were analyzed at stage 27 by whole-mount in situ hybridization.(B) β-gal-injected, β-gal-injected/FGF-treated (FGF + β-gal), and DNS10-injected/FGF-treated animal caps were analyzed by whole-mount in situ hybridization.(C) Animal caps treated as in (A) and (B) were analyzed by RT-PCR.(D) Uninjected, dominant-negative FGF receptor-injected (XFD, 1 ng), and DNS10-injected animal caps were cultured during the blastula stage in the absence or presence of FGF. These caps were then analyzed by RT-PCR at stage 11.
Figure 6. Smad10 Is Required for Noggin-Mediated Neural Induction(A) One-cell embryos were injected with noggin mRNA (Nog, 250 pg) alone, with Smad10 MO, or with control MO. Animal caps from these as well as uninjected embryos (control) were explanted, cultured, and analyzed by whole-mount in situ hybridization.(B) Animal caps treated identically to those described in (A) were processed for RT-PCR analysis.
