Knapp D et al. (2006), Neurotrophin receptor homolog (NRH1) proteins r...

XB-ART-34735

Dev Biol 2006 Dec 15;3002:554-69. doi: 10.1016/j.ydbio.2006.09.038.

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Neurotrophin receptor homolog (NRH1) proteins regulate mesoderm formation and apoptosis during early Xenopus development.

Knapp D , Messenger N , Ahmed Rana A , Smith JC .

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Recent experiments suggest that Xenopus Neurotrophin Receptor Homolog 1 (NRH1) proteins act through the planar cell polarity pathway to regulate convergent extension movements during gastrulation and neurulation. We show in this paper that NRH1 proteins are also required for the proper expression of mesodermally expressed genes such as Xbra and Chordin, and to a lesser extent, of Xwnt11. Loss of NRH1 function is followed, during gastrula and neurula stages, by a dramatic increase in apoptosis. Apoptosis is delayed by injection of Xbra RNA, suggesting that cell death is a consequence, at least in part, of the down-regulation of this gene, and it is also delayed by expression of activated forms of Rho, Rac and Cdc42. These small GTPases have previously been implicated in the planar cell polarity pathway in Xenopus and, in other systems, in the regulation of apoptosis. We conclude that the effects of NRH1 proteins include the regulation of mesodermal gene expression and that the disruption of gastrulation that is caused by their loss of function is a consequence of the down-regulation of Xbra and other genes, in addition to direct interference with the planar cell polarity pathway. The apoptosis observed in embryos lacking NRH1 function is not an indirect consequence of the disruption of gastrulation, and indeed it may contribute to the observed morphological defects.

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Species referenced: Xenopus laevis
Genes referenced: akt1 cdc42 chrd dvl2 fn1 gsc itgam mob1a nradd rac1 rho tbx2 tbxt wnt11b
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	Fig. 1. Expression pattern of NRH1a and the design of NRH1 antisense morpholino oligonucleotides (MOs). (A) Expression pattern of NRH1a. RNA was extracted at the indicated stages from animal pole (AP), marginal zone (MZ) and vegetal pole (VP) regions, and the expression of NRH1a and ornithine decarboxylase (ODC) was analysed by real-time RT-PCR and normalised to levels of total RNA, with maximum observed expression defined as 100. Note significant levels of NRH1a expression in animal and vegetal pole regions. The inset shows expression of NRH1a at early gastrula stage 10 analysed by in situ hybridisation. There are high levels of NRH1a expression in the marginal zone, as previously reported (Bromley et al., 2004). (B) Expression pattern of Xbra, analysed and normalised as described for (A). Note that expression of this gene in animal pole and vegetal pole regions is lower than that of NRH1a. (C) The design of antisense morpholino oligonucleotides directed against NRH1 gene products in X. laevis and X. tropicalis. The initiating ATGs of X. laevis NRH1a and NRH1b and of X. tropicalis NRH1 are shown in red, preceded by their 5′ untranslated sequences. The sequences targeted by the indicated antisense morpholino oligonucleotides are coloured blue or green. A control MO differs by four bases (starred) from Xl MOb1. (D) Efficacies of the antisense morpholino oligonucleotides. Embryos were injected with 40 ng of the indicated MOs at one-cell stage, and 500 pg RNA encoding HA-tagged versions of NRH1a or NRH1b was injected at the two-cell stage. Embryos were cultured until mid blastula stage 8, and extracts were subjected to western blotting using an anti-HA antibody. All four X. laevis MOs block the translation of their cognate RNA while the control MO has little or no effect.
	Fig. 6. Gene expression in embryos lacking NRH1 function. (A–E) Xenopus embryos were injected at the one-cell stage with 80 ng of a control MO or of Xl MOa1 or Xl MOb1. They were then assayed by real-time RT-PCR at stages 10, 11, 13 or 16 for expression of Xbra (A), Chordin (B), Xwnt11 (C), Goosecoid (D) or NRH1a (E). All values were normalised to those of ornithine decarboxylase and are presented as arbitrary units. Note that levels of Xbra, Chordin and NRH1b are substantially reduced by inhibition of NRH1 activity. (F) Scatter graphs summarising the results of four independent experiments investigating gene expression levels in embryos in which NRH1 activity is inhibited. The results show the fold reduction in gene expression caused by NRH1 MOs (such that a value of 1 would indicate no difference between control embryos and embryos injected with Xl MO1a or Xl MOb1). The ratios were calculated at the stage at which each gene was expressed at its maximum level (for example, at stage 11 for Xbra and stage 13 for Chordin). (G) Xenopus embryos were injected at the two-cell stage with 40 ng control MO into one blastomere and 40 ng Xl MOb1 into the other. The control and experimental sides of the embryos were distinguished by co-injection of 4 ng Lissamine-labelled control MO or fluorescein-labelled control MO. Embryos were allowed to develop to early gastrula stage 10 when they were analysed by in situ hybridisation using a probe specific for Xbra. Note that levels of Xbra are lower in the descendants of the blastomere injected with Xl MOb1 (right). Nine experiments of this kind have been carried out, together with five where one side of the embryo was injected with Xl MOb1 and the other left uninjected and six where one side of the embryo was injected with control MO and the other left uninjected. Only in the latter group were levels of Xbra similar in the two halves of the embryo. (H) Western blot of extracts from embryos injected at the cell stage with 80 ng of a control MO or Xl MOb1, probed with an antibody specific for Xbra (upper panel) or GAPDH (lower panel). Note that levels of Xbra are greatly reduced by inhibition of NRH1 protein function.
	Fig. 2. Down-regulation of NRH1 proteins causes defects in gastrulation. (A) X. laevis embryos were injected at the one-cell stage with 80 ng of a control MO, or Xl MOa1 or Xl MOb1. Embryos shown are at the equivalent of neurula stage 16. Note that embryos injected with control MO develop normally, while those injected with Xl MOa1 (B) or Xl MOb1 (C) do not complete gastrulation normally. (D) Embryos injected with 40 or 80 ng Xl MOb1 as indicated, shown at the equivalent of stage 28. Embryos injected with Xl MOb1 tend to disaggregate and arrest development so as to resemble those at gastrula or neurula stages (E, F). (G) X. tropicalis embryos injected with Xt MO show a similar phenotype to that observed in X. laevis embryos. (G, H) Embryos injected with 30 ng of a control antisense morpholino oligonucleotide develop normally, while those injected with 30 ng Xt MO do not complete gastrulation normally (I) and go on to exhibit anterior and posterior truncations (J).
	Fig. 3. Antisense morpholino oligonucleotide Xl MOa1 causes defects in nuclear division. (A, B) One cell of an X. laevis embryo at the two-cell stage was injected with 40 ng control MO together with fluorescein-dextran (green), and the other cell was injected with Xl MOa1 together with rhodamine-dextran (red). Note that Xl MOa1 causes retardation of cell division. (C, D) By neurula stage 14, embryos injected with Xl MOa1 have many fewer cells than control embryos and larger nuclei. DNA is stained with SYTOX green. (C) Embryo previously injected at the one-cell stage with 80 ng control MO. (D) Embryo injected with 80 ng Xl MOa1; note the smaller number of much larger nuclei. (E, F) Higher magnification of cells in the animal pole regions of such embryos at mid gastrula stage 11. DNA is stained by SYTOX green and tubulin by means of an antibody against β-tubulin (red). (E) Embryo injected with 80 ng control MO. Note normal metaphase figures (arrowheads). (F) Embryo injected with Xl MOa1. Note mitoses with multiple spindles (arrowheads). (G, H) In a separate experiment, embryos injected with 80 ng Xl MOa1 also show abnormal mitosis (G), but cells in an embryo injected with the same amount of Xl MOb1 appear normal.
	Fig. 4. Effect of loss of NRH1 function on cell spreading and convergent extension. (A, B) Cell spreading on fibronectin is not prevented by Xl MOb1, but cells detach prematurely at neurula stage 15. Embryos of X. laevis were injected at the one-cell stage with 80 ng control MO or Xl MOb1 together with fluorescein-dextran (green) or rhodamine-dextran (red), respectively. Animal caps derived from the two type of embryo were pooled, and cells were dissociated, treated with activin and seeded onto fibronectin-coated dishes. (A) Stage 13; both control cells (green) and cells lacking NRH1 activity (red) adhere to fibronectin. (B) Stage 15; control cells (green) remain adherent, but cells lacking NRH1 activity (red) detach from the substrate. (C) Loss of NRH1 function interferes with cellell adhesion. Cells derived from the animal pole regions of embryos injected at the one-cell stage with the indicated MOs were treated with activin, seeded onto a fibronectin-coated substrate and cultured to the equivalent of late neurula stage 17. Cells derived from control embryos detach from the substrate and form clumps, but those derived from embryos in which NRH1 activity is inhibited fail to aggregate in a dose-dependent manner. (G, H) Dorsal marginal zone regions (eller sandwiches derived from embryos injected with Xl MOb1 tend to shed cells and elongate only poorly. Embryos were injected at the one-cell stage with 80 ng control MO or Xl MOb1. Dorsal marginal zone regions were dissected from such embryos at early gastrula stage 10, assembled into Keller sandwiches and cultured to the equivalent of late neurula stage 18. Note the dramatic elongation of control sandwiches (G) compared with those in which NRH1 activity is inhibited (H). Note also that cement glands form in control dorsal marginal zone regions (arrowheads in G) but not those in which NRH1 function is inhibited (H). The insets in (G) and (H) show Keller sandwiches before the removal of detached cells. Note that dorsal marginal zone regions derived from embryos injected with Xl MOb1 shed many cells.
	Fig. 5. Apoptosis in embryos injected with NRH1 MOs. (A) Whole-mount TUNEL assays of X. laevis embryos at stage 14 reveal low levels of apoptosis in embryos injected with 80 ng of a control MO and high levels in embryos injected with 80 ng Xl MOa1 (B) or Xl MOb1 (C). (D) An embryo injected with 80 ng Xl MOb1 and bisected at stage 14. Anterior is to the right. Note high levels of apoptosis in dorsal and ventral mesoderm and in ectoderm. (E) Apoptosis in Xenopus embryos analysed by a tPARP cleavage assay. Arrows indicate the major tPARP cleavage product, which indicates the presence of active caspases. Note that apoptosis is detectable in embryos injected with a control MO at stage 20, but occurs earlier in embryos injected with NRH1 MOs and, as a positive control, in embryos treated with cycloheximide (CHI). (F) The premature onset of apoptosis in embryos injected with Xl MOb1 is delayed by subsequent injection of 500 pg RNA encoding NRH1b. (G) Inhibition of NRH1 function in X. tropicalis also causes apoptosis. Embryos were injected with our control MO (G), with Xt MO (H) or with MOs designed to prevent correct splicing of NRH1 mRNA in X. tropicalis (I, J; see Fig. 7) and were analysed by TUNEL staining at stage 13. Note increased levels of apoptosis in panels H, I and J. This experiment has been carried out three times using Xt MO and once using +intron 1 MO and δexon 2 MO, with the same result obtained each time.
	Fig. 7. Antisense morpholino oligonucleotides designed to interfere with the correct splicing of X. tropicalis NRH1 RNA cause down-regulation of Xbra and Chordin and morphological defects that resemble those caused by MOs designed to inhibit NRH1 translation. (A) The first three exons (of a total of six or seven) of X. tropicalis NRH1. The figure shows the position of the MO designed to interfere with NRH1 translation (ATG MO) and the positions of the forward (F1) and reverse (R1, R2) PCR primers used to assess the efficacy of the MOs designed to interfere with splicing. The figure is not drawn to scale. Exon 1 is 326 base pairs (bp), intron 1 is 9013 bp, exon 2 is 134 bp, intron 2 is 740 bp, and exon 3 is 363 bp. If the mRNA is correctly spliced, the combination of F1 and R1 should give no PCR product and F1 and R2 should yield a product of 490 bp. (B) The + intron 1 MO is designed to cause intron 1 to be retained, so that the first 15 amino acids are followed by 67 missense amino acids before a stop codon is reached. If intron 1 is retained in this way, the combination of F1 and R1 should give a PCR product of 280 bp and F1 and R2 should yield a product in excess of 9.0 kb. (C) The δexon 2 MO is designed to exclude exon 2 from the mature RNA. In this case, exon 3 would no longer be in frame with exon 1, and a truncated protein consisting of 15 correct amino acids followed by 25 missense residues would be formed before a stop codon is encountered. If exon 2 is deleted in this way, the combination of F1 and R1 should yield no PCR product and F1 and R2 should yield a product of 360 bp. (D) Verification of the efficacy of MOs + intron 1 and δexon 2. Comparisons of lanes 2 and 3, and 6 and 7, indicate that both MOs reduce levels of correctly spliced transcript by ≥ 50%. Note that the predicted 9.5 kb band in lane 4 is absent, probably because our PCR conditions do not efficiently amplify products of this size. (E) Embryos were injected with 30 ng of the indicated MOs and cultured to stage 11.5 before being assayed for expression of Xbra and Chordin. Note that all three MOs cause down-regulation of both genes. Analysis of Xbra expression revealed that similar results were obtained at stages 10.5 and 12.5, and the same down-regulation was observed when the experiment was repeated. (F) MOs + intron 1 and δexon 2 yield similar phenotypes in whole embryos (compare with Fig. 2). The phenotype observed with MO + intron 1 is more severe than that observed with δexon 2 (indeed, such embryos do not survive to stage 32); the reason for this is unclear.
	Fig. 8. Apoptosis in embryos injected with NRH1 antisense morpholino oligonucleotides is delayed or prevented by injection of RNA encoding Xbra. (A, B) Inhibition of Xbra function by injection of a specific MO causes limited apoptosis. (A) TUNEL staining of embryos injected at the one-cell stage with 80 ng control MO and cultured to stage 14. Note the absence of apoptosis. (B) TUNEL staining of embryos injected with 80 ng Xbra MO. Note apoptotic cells. (C) Injection of RNA encoding Xbra delays or rescues apoptosis induced by NRH1 MOs. Embryos were injected at the one-cell stage with 80 ng of a control MO or with Xl MOb1 or Xl MOa1 as indicated. They then received, at the 2- cell stage, a total of 50 pg Xbra RNA injected at four positions in the equatorial region. Embryos were cultured for equivalent times to the indicated stages, and extracts were assayed for the ability to cleave tPARP. Note that the onset of caspase activity is delayed or prevented by Xbra. This experiment has been carried out twice, with the same result both times.
	Fig. 9. Apoptosis in embryos injected with NRH1 antisense morpholino oligonucleotides is delayed or prevented by injection of RNA encoding active forms of the small GTPases Rho, Rac or Cdc42. (A) Embryos were injected at the one-cell stage with 80 ng of a control MO or of Xl MOb1 as indicated, and at the 2- to 4-cell stage, they were injected at four equatorial positions with RNA encoding constitutively active forms of Rho, Rac or Cdc42 (5, 50 and 30 pg per embryo, respectively). They were cultured to the indicated stages, and extracts were assayed for the ability to cleave tPARP. Note that the onset of caspase activity is delayed by active forms of all three small GTPases. With the exception of the experiment involving Rac, these experiments have been carried out twice, with the same results both times. (D). Inhibition of the planar cell polarity pathway, and the resulting disruption of gastrulation, does not cause dramatic apoptosis. Embryos were injected at the one-cell stage with 80 ng of a control MO (D), or with 80 ng of Xl MOb1 (E). In panel F, 3.6 ng RNA encoding Dsh-DEP+, a dose that is sufficient severely to disrupt gastrulation, was delivered by four equatorial injections made at the 2- to 4-cell stage. They were cultured to stage 14 and subjected to whole-mount TUNEL staining. Note that, in contrast to embryos injected with Xl MOb1 (E), little apoptosis occurs in embryos injected with RNA encoding Dsh-DEP+ (F). Note that the experiments in panels D were carried out at the same time as those illustrated in Figs. 8A, B, and the control embryos in Fig. 8A and panel D are identical.