Sherwood V , Manbodh R , Sheppard C , Chalmers AD .

Medical Research Council , MRC_G120/844 Medical Research Council , G120/844 Medical Research Council

	Figure 1. RASSF7 belongs to a novel family of proteins that co-localize with Ras genes in the human genome. (A) Schematic representations of the classical and N-terminal (NT) RASSF families: Classical human family RASSF1-6 and classical Drosophila RASSF (dmRASSF), NT family RASSF7–10 and Drosophila RASSF7/8-like (dmRASSF7/8). See materials and methods for accession numbers. C1, protein kinase C conserved region 1 (grey); SARAH, Sav/RASSF/Hippo (spotted); zinc finger (checked); coiled-coil (black); and RA, Ras-association domain (striped). The NT RASSF represent a distinct family of proteins from the classical RASSFs. (B) Graphical representation of human chromosomes 11 and 12 (modified from Ensembl), showing that the NT RASSF genes map near various Ras isoforms. The exception is P-CIP1/RASSF9, where we can not find a Ras gene nearby.
	Figure 2. RASSF7 expression in developing Xenopus embryos. (A) RT-PCR analysis of early Xenopus development for RASSF7 and as a control, the ornithine decarboxylase (ODC) gene. Thirty cycles were used in the PCR. RASSF7 exhibits both maternal and zygotic expression. (B) Tissue-specific expression patterns of RASSF7 analyzed by in situ hybridization at stages 16, 20, and 30. A wholemount and transverse section is shown for each stage. ep, epidermis; np, neural plate; ey, eye; br, brain; nt, neural tube; pn, pronepheros; lm, lateral plate mesoderm; ba, branchial arches; ov, otic vesicle; and tg, trigeminal ganglion. There is prominent expression of RASSF7 in many embryonic tissues.
	Figure 3. Knockdown of RASSF7 expression causes developmental defects. (A) Immunoblot of HA-tagged RASSF7 (56 kDa) from uninjected and MO injected embryos at stage 16. Injected embryos were injected with 20 ng of MO and 2 ng of HA-RASSF7 RNA into both cells at the two-cell stage. α-tubulin is shown as a loading control (50 kDa). MO1 is able to eliminate tagged RASSF7 in the embryos. (B) Lateral and dorsal views of Xenopus embryos injected with morpholinos (MOs), two RASSF7 MOs (MO1 and MO2), and a control MO (Con MO). RASSF7 knockdown embryos display a short and bent body axis, with reduced eye pigmentation and developmental delay. (C) Percentages of affected embryos for each MO at stage 39, calculated from four independent experiments (Con MO, n = 375; MO1, n = 359; MO2, n = 345). (D) Phenotypic rescue was achieved for MO2 by injection of RASSF7 RNA. (E) Quantification of the rescue experiment (percentages calculated from three independent experiments; RNA, n = 157; MO2, n = 171; RNA plus MO2, n = 166).
	Figure 4. RASSF7 knockdown causes pronounced neural tube defects. (A) Schematic representation of a stage 39 Xenopus tadpole indicating the anterior/posterior level that sections were taken. Diagram adapted from a Xenbase image, based on previous work (Nieuwkoop and Faber, 1967). The sections cut through the otic vesicle (OV). (B) Stage 39 embryos injected with either Con MO or MO1 were sectioned and stained with hematoxylin and eosin. The ventricular cavity (▴) is reduced in MO1 injected embryos compared with control sections and is termed “moderately affected” or is completely lost and is therefore termed “severely affected.” In MO1-injected embryos, cells spread from the pial regions of the brain into areas normally occupied by connective tissue (arrows). (C) Developing brain tissue was stained with polarity markers: anti-laminin (green), phalloidin (red), and counterstained with DAPI (blue). Images were from an AP position similar to that in A and focus on the neural tube, just above the notochord (NC). Tissue polarity is disrupted in both the moderately and severely affected tissues. All bars, 10 μm.
	Figure 5. RASSF7 knockdown results in nuclear fragmentation and cell death in the developing neural tube. (A) Sytox Green staining of neural cells in MO injected embryos. Nuclear fragments of varying sizes (arrows) are observed in RASSF7 knockdown tissues, which are not present in the control tissues. (B) Nuclear fragments are not observed in the notochord, somites, and developing gut (endoderm) of RASSF7 knockdown embryos, as indicated by sytox green staining. These are tissues where RASSF7 expression was not observed. Affected neural tissue spreading toward the notochord is highlighted by an arrow in the MO1 notochord image. (C) Sections of neural tissue were TUNEL stained (red) or anti-active caspase 3 (green), and DAPI counterstained (blue). Arrows indicate fragmented nuclei that are not apoptosis-positive. (D) Graphs indicate percentage of cell death. *** p< 0.001, n = 9 from three experiments for both MOs (TUNEL); and n = 3 from three independent experiments for both MOs (active caspase 3). More than 7000 cells were counted for each individual specimen. Error bars, SD. All bars, 10 μm.
	Figure 6. RASSF7 is required for spindle formation and completion of mitosis in the neural tube. (A) Centrosome number is not affected by RASSF7 knockdown. γ-tubulin staining (red) was used to examine the centrosomes (highlighted by arrows) of the neural tube of Con MO– and MO1-injected embryos and were counterstained with DAPI (blue). VC, ventricular cavity. (B) Centrosome number in the neural tube is unaffected by RASSF7 knockdown. No significant difference was found between Con MO and MO1; n = 6 specimens for each MO from three independent experiments. (C) Cellular proliferation was examined in the neural tube of MO-injected embryos by staining with anti-phospho-S10 histone H3 (green) and DAPI (blue). (D) The number of phospho-histone H3 proliferating cells as a percentage of the total number of cells counted (DAPI stain). Proliferation is similar between the two MO tissues and is not significantly different. n = 9 from three injection experiments for both MOs, >7000 cells were counted for each individual specimen. (E) Counts of dividing cells in the following phases: prophase (also includes prometaphase), * p< 0.05; metaphase, no significant difference; and anaphase (also includes telophase), ** p< 0.01. Mitotic phases were distinguished by DAPI staining using the following criteria: prophase/prometaphase, condensed DNA; metaphase, genetic material aligned on the metaphase plate; and anaphase/telophase, chromosome separation. Counts were from three experiments (Con MO, n = 90; MO1, n = 90). All error bars, SD. Knockdown cells arrest in early mitosis. (F) The total number of spindles counted as a percentage of the total number of dividing cells. * p< 0.05, n = 6 specimens for each MO, from three experiments. The number of mitotic spindles was greatly reduced in the dividing RASSF7 knockdown cells. (G) α-tubulin staining (green) was used to visualize spindles in mitotic cells of the neural tube, whereas γ-tubulin staining was used to mark the centrosomes (red). Sections were counterstained with DAPI (blue). Mitotic spindles were missing, or occasionally abnormal, in the dividing RASSF7 knockdown cells. All bars, 10 μm. RASSF7 knockdown cells fail to progress through mitosis because of a deficiency in spindle formation.
	Figure 7. RASSF7 localizes to the centrosomes. (A) Embryos were injected with GFP-RASSF7 at the two-cell stage, cultured until stage 10, fixed, sectioned, and stained as described in materials and methods. GFP-RASSF7 (green) colocalized with γ-tubulin (red) during interphase and prophase (arrow head and arrow, respectively) and during metaphase and anaphase (arrows). Sections were counterstained with DAPI (blue). The localization has been repeated in more than three independent experiments. (B) RASSF7-HA (red) and GFP-RASSF7 (green) exhibit the same localization in stage 10 embryos. Interphase cells shown. (C) Later stage embryos (stage 20) also have centrosome localized GFP-RASSF7 (green), as indicated by γ-tubulin (red). Image shows the neural tube of a stage 20 embryo. VC, ventricular cavity. All bars, 10 μm. RASSF7 colocalizes with γ-tubulin throughout the cell cycle.
	Figure 8. The centrosome localization of RASSF7 requires microtubules. (A) Microtubules are lost in cells treated with 20 μg/ml of nocodazole for 2 h. Microtubules of the animal cap from stage 10 embryos were stained using α-tubulin (red), and the cell nuclei were visualized using DAPI (blue). (B) Nocodazole treatment resulted in a loss of GFP-RASSF7 (green) localization at the centrosome. γ-tubulin was used to stain the centrosomes (red) and nuclei counterstained with DAPI (blue). All bars, 10 μm.

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