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Dev Biol
2003 May 15;2572:329-42. doi: 10.1016/s0012-1606(03)00109-x.
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The function of Xenopus germ cell nuclear factor (xGCNF) in morphogenetic movements during neurulation.
Barreto G
,
Reintsch W
,
Kaufmann C
,
Dreyer C
.
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The germ cell nuclear factor (GCNF, NR6A1) is a nuclear orphan receptor first described in the mouse testis and subsequently identified as an essential transcription factor in vertebrate embryogenesis. Here, we analyze the phenotype of Xenopus embryos after depletion of embryonic GCNF (xEmGCNF) protein using a specific morpholino antisense oligonucleotide. Morphological defects after xEmGCNF knockdown became obvious from neurulation onward. Among the abnormalities observed, defective formation of the neural tube and a short and curved main body axis were the most remarkable traits. Histological analysis, lineage tracing of injected blastomeres, and Keller sandwich explants revealed that xEmGCNF function is required for different patterns of cell intercalation during neurulation and consequently for the sequence of morphogenetic movements leading to formation of the neural tube. Further characterization of the phenotype at the molecular level showed an abnormal distribution of the extracellular matrix protein fibronectin and a reduction in the expression level of the integrin subunits alpha5 and alpha6, the limiting components of the laminin and fibronectin receptors, respectively. We propose integrin-mediated cell-matrix interaction as a process that requires xEmGCNF function and provides, in concert with cadherins-mediated cell-cell interactions, a molecular basis for morphogenetic cell movements during neurulation.
Fig. 1. The Morpholino antisense oligonucleotide specifically inhibits the translation of the xEmGCNF mRNA. Embryos were injected at the two-cell stage with 42 ng of a standard morpholino oligonucleotide as control (STA: lane 2) or a xEmGCNF-specific antisense Morpholino oligonucleotide (MOR: lanes 3 and 4) into each blastomere. Additionally, 100 pg per blastomere of a mRNA encoding an oocyte-specific GCNF isoform were coinjected (xOoGCNF; lane 4). Protein extracts of injected embryos at stage 17 were analyzed by Western blotting, using an antiserum against xEmGCNF [DEF] (David et al., 1998). The positions of exogenous xOoGCNF (arrowhead) and endogenous xEmGCNF (arrow) are indicated at the left. The lowest band detected by the antiserum is nonrelated to GCNF and can be used as loading control. Note that, in the presence of the MOR, the level of xEmGCNF protein decreased, whereas the xOoGCNF protein was still translated.
Fig. 2. Phenotypic effects of xEmGCNF loss-of-function. Embryos were injected at the two-cell stage into each blastomere with 42 ng of either the standard morpholino oligonucleotide as control (STA; A) or the xEmGCNF-specific antisense Morpholino oligonucleotide (MOR; D). Injected embryos were fixed at stage 18 (A, D), stage 28 (B, E), and stage 40 (C, F). Loss- of-function of xEmGCNF resulted in curved and reduced main body axis at tailbud (E) and tadpole stages (F), failure of the neural tube to close (arrow in E), atypical cloacae formation (arrowhead in E), and defective pigmentation of the eyes (arrows in F).
Fig. 3. Sequence of events giving rise to the neural tube was impaired after xEmGCNF knockdown. Shown are dorsal parts of 5-μm transverse sections from the midbody of STA- injected (A, C, E, G) and MOR-injected (B, D, F, H) embryos at neurula stages 16 (A), 18 (C), and 20 (E). The neural folds (black arrows) and medial neural groove (black arrowhead) were properly formed in STA-injected (A), but not in MOR-injected embryos (B). After neural fold fusion, a slight groove remained in the ectoderm (white arrows), and the lumen of the neural tube (white arrowheads) started to reopen in control (C), but not in experimental embryos (D). Note the irregularly shaped neural tube after xEmGCNF knockdown at stage 20 (F). In (G) and (H), embryos were subjected to whole-mount in situ hybridization with a nerve- specific tubulin (NST) probe before sectioning. The NST-expressing cells remained laterally spread at a greater distance from the dorsal midline after MOR injection (H). no, notochord; nt, neural tube; so, somitgenic mesoderm. Bar, 200 μm.
Fig. 4. Convergence and extension by cell intercalation during neurulation were affected after xEmGCNF knockdown. Embryos were coinjected at the eight-cell stage into each animal dorsal blastomere with 250 pg of β-gal mRNA and either 21 ng of the STA (A, C) or 21 ng of the MOR (B, D). Injected embryos were fixed at stage 20 and stained for β-gal activity as lineage tracer (A). (A) and (B) show dorsal aspects of stage 20 embryos. Anterior is at the top. (C) and (D) show details of (A) and (B), respectively. The widespread distribution of the β-gal staining in the MOR-injected embryos demonstrates the defective medial migration of the neural plate cells during neurulation. (E) and (F) show Keller sandwich explants at stage 25 equivalent. For these explants, the embryos were injected at two-cell stage into both blastomeres with STA (E) or MOR (F). Knockdown of xEmGCNF affected radial cell intercalation, thus reducing the elongation of mesoderm and neuroectoderm.
Fig. 5. Depletion of xEmGCNF perturbs fibronectin deposition in ECM. Embryos were injected with 21 ng of the STA (A, D, G) or 21 ng of the MOR (B, C, E, F, H, I) at eight-cell stage into either both animal dorsal or both animal ventral blastomeres as indicated in the top panels. At stage 18, embryos were fixed and transverse frozen sections were stained with anti-fibronectin antibodies (Winklbauer, 1998). (A) Overviews of transverse sections through the middle of the embryo. Details show the dorsal parts (D) or ventral regions (G) at higher magnification. Note depletion of fibronectin matrix between notochord (no) and archenteron roof in a dorsally injected embryo (B, E), and the thicker epidermis (ep) in a ventrally injected embryo (C, I). Somites (so), neural tube (nt). Bar, 200 μm.
Fig. 6. Expression of integrins α5 and α6 is reduced after depletion of xEmGCNF. (A) Schematic representation of the experimental system. Embryos were injected at the two-cell stage into each blastomere. Animal caps were explanted at stage 8.5 and cultivated until stage 17 equivalent. Total RNA was extracted and semiquantitative RT-PCR was performed. (B) Knockdown of xEmGCNF reduces expression level of integrins α5 and α6. Injection of noggin mRNA directed the ectoderm explants to an anterior neural fate (nogg, lanes 2). Further posterior specification of the neuroectoderm was achieved by an enhanced RA signaling (RA, lanes 3). In addition, embryos were coinjected with 42 ng of the STA (lanes 1) or 42 ng of the MOR (lanes 4). Expression levels of integrin subunits α6, β1, α5, α5 truncated (α5INT tr), and α3 were monitored by RT-PCR using gene-specific primers. Positive control, ODC +RT; negative control, ODC −RT; whole embryo, WE. (C) Depleted expression of integrins α5 and α6 after MOR injection is rescued by ectopic expression of xOoGCNF. Explants of anterior (lane 1) or posterior neuroectodermal explants (lanes 2) in the absence (lanes 1 and 2) or in the presence of MOR (lanes 3 and 4) and after injection of xOoGCNF mRNA (lane 4) were prepared and analyzed by RT-PCR as previously described. Note that products representing integrins α5 and α6 were reduced by GCNF knockdown (lane 3) and reconstituted by addition of xOoGCNF (lane 4).
