QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00446

This Article Summary Full Text Full Text (PDF) Movie Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cayouette, M. Articles by Raff, M. Search for Related Content PubMed PubMed Citation Articles by Cayouette, M. Articles by Raff, M.

The orientation of cell division influences cell-fate choice in the developing mammalian retina

Michel Cayouette^*, and Martin Raff

MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, London WC1E 6BT, UK
^* Present address: Stanford University School of Medicine, Fairchild Building, 299 Campus Drive, Stanford, CA 94305-5125, USA

View larger version (54K): [in a new window]   Fig. 1. Experimental setup for time-lapse video recording. (A) Retinal explants from newborn rats were cultured on organotypic filters, which allow the culture medium to diffuse through the explant by capillary action. The edge of the explant was folded over, which allowed RNECs infected with a GFP-encoding retrovirus to be observed along their apico-basal axis. Areas of the folded edge are shown after (B) 3 hours and (C) 6 days. Note that all mitotic cells are at the apical surface of the neuroepithelium in B and that two, distinct cell layers have developed in C, just as observed in vivo. However, the retinal ganglion cells die in the explant because they lack neurotrophic support from their normal target cells. Scale bar: 20 µm.

View larger version (78K): [in a new window]   Fig. 7. The effect of Numb overexpression on clonal development in newborn rat retinal explants. (A) Immunostaining of Numb in the newborn rat retina using polyclonal antibodies (Upstate Biotech). Punctate Numb staining is concentrated at the apical side of both interphase and mitotic RNECs (arrow in inset). (B) Control retinal section, stained as in A but without the primary anti-Numb antibodies. (C) The retroviral vector encoding Numb and alkaline phosphatase. The full-length open-reading frame for Numb was cloned in front of an internal ribosome entry site (IRES), which is upstream of the coding sequence for placental alkaline phosphatase (PLAP). The bicistronic mRNA encoding Numb and PLAP is transcribed from the Xenopus EF1 promoter. (D-G) Examples of clones in frozen sections of a newborn retinal explant infected with a control retrovirus encoding alkaline phosphatase (PLAP) alone and cultured for 10 days. Four clones containing a photoreceptor (D), an amacrine cell (E), a bipolar cell (F) and a Müller cell (G) are shown. (H) Composite of a z-series of confocal images showing Numb immunostaining in a frozen section of a newborn retinal explant infected with the Numb-expressing retrovirus. There is strong Numb staining throughout the two interphase RNECs shown. This contrasts with the apical concentration of endogenous Numb in nontransfected RNECs, shown in A. (I,J) Wholemounts of newborn retinal explants 10 days after infection with either the control retrovirus (I) or the Numb-expressing retrovirus (J). Note that the control explant contains a larger diversity of cell morphologies. Scale bars: 20 µm in A,B; 10 µm in D-G; 5 µm in H; 100 µm in I,J.

View larger version (32K): [in a new window]   Fig. 2. GFP+ RNECs undergoing interkinetic nuclear migration. (A) A single GFP+ RNEC is seen at the edge of the folded explant, 24 hours after infection with a GFP-encoding retrovirus. (B) Snapshots from a time-lapse video recording of a GFP+ RNEC undergoing interkinetic nuclear migration along its apico-basal axis. The cell divided horizontally at the apical surface of the neuroepithelium at 16.5 hours. Arrowheads indicate the plane of the cell division, perpendicular to the orientation of the mitotic spindle. Scale bars: 20 µm in A; 40 µm in B.

View larger version (33K): [in a new window]   Fig. 3. Horizontal and vertical divisions of GFP+ RNECs. (A) Snapshots from a time-lapse video recording showing two cells in the same field, one of which divided horizontally (cell 1) and one of which divided vertically (cell 2). The dashed line indicates the apical surface of the neuroepithelium and arrowheads the plane of cell division (which is perpendicular to the orientation of the mitotic spindle). See also Movie 1 at http://dev.biologists.org/supplemental/. Scale bar: 40µm. (B) Higher magnification view of cell 1 dividing horizontally in time frame 12h08. (C) Higher magnification view of cell 2 dividing vertically in time frame 13h18. (D) Quantitative analysis of the distribution of mitotic spindle orientations relative to the plane of the neuroepithelium in live retinal explants, as determined by time-lapse video microscopy of GFP+ RNECs.

View larger version (61K): [in a new window]   Fig. 4. Horizontal divisions producing two daughter cells that acquire a similar morphology and cell body size. (A-E) Five examples of daughter cells produced by horizontal divisions of newborn RNECs, seen 60-90 hours after the division. Note the similarity in size and morphology of the two daughter cells in each case. In the example shown in E, it was possible to fix the explant after the recording, stain the nuclei with propidium iodide and find the two GFP+ daughter cells again in a confocal microscope. Both daughter cells have a similar body size and morphology and are located in the photoreceptor layer. (F) A two-photoreceptor clone in a frozen section of a newborn retinal explant, 10 days after infection with a retrovirus encoding GFP. (G) Quantification of cell body size of GFP-infected photoreceptor cells (PR) and interneuron layer cells (INL), measured in cryosections of retinal explants 10 days after infection with a GFP-encoding retrovirus. Results are mean±s.d. of 35 PR and 28 INL cells. (H) Cell-body sizes of pairs of daughter cells produced by horizontal divisions of GFP-infected RNECs, followed by video recording. The daughters of all 12 horizontal divisions followed are shown. Note that in 11 out of 12 cases the daughter cells are similar in size to photoreceptors. Scale bars: 20 µm in A-E; 10 µm in F.

View larger version (50K): [in a new window]   Fig. 5. Vertical divisions producing two daughter cells that acquire different morphologies. (A-E) Five examples of daughter cells produced by vertical divisions in newborn RNECs, seen 70-96 hours after the division. Note that the two daughters differ in size and morphology. In the example shown in E, it was possible to fix and stain the explant and find the two GFP+ daughter cells in a confocal microscope as described for Fig. 4E. The larger daughter cell (arrow) is located in a cell layer that contains big nuclei (presumably the interneuron layer), whereas the smaller daughter cell (arrowhead) is located in a cell layer that contains small nuclei (presumably the photoreceptor layer). (F) A z-series confocal projection of a clone containing a Müller cell (arrow) and a photoreceptor (arrowhead) in a frozen section of a newborn retinal explant, observed 10 days after infection with a retrovirus encoding GFP. The section was imaged in a confocal microscope and a stacked z-series is shown. (G) Quantification of cell body size of GFP-infected photoreceptors and interneuron layer cells as measured in cryosections of retinal explants. This is the same graph as shown in Fig. 4G. (H) Cell-body sizes of pairs of daughter cells produced by vertical divisions of GFP-infected RNECs followed by video recording. The daughters of all 11 vertical divisions that were followed are shown. Note that in 9 out of 11 cases the daughter cells are different in size. The larger daughter is much larger than a photoreceptor cell and is similar in size to an interneuron layer cell. The smaller daughter is usually similar in size to a photoreceptor cell. Scale bars: 10 µm in A-D; 2 µm in E,F.

View larger version (87K): [in a new window]   Fig. 6. Maintenance of a basal process during RNEC division. (A-D) Snapshots of a time-lapse video recording showing a GFP+ RNEC that maintained its basal process (arrows) during division. This cell divided with a horizontal spindle. The cleavage plane is indicated with arrowheads in D. The basal process seems to be asymmetrically inherited by the daughter cell on the left. (E-G) Confocal micrographs of a different GFP+ RNEC in metaphase, labeled with propidium iodide (PI). The PI label is shown in E, GFP in F and a merged image in G. The arrow in F points to the metaphase plate and the arrowhead in G points to the basal process.

View larger version (13K): [in a new window]   Fig. 8. Quantitative clonal analysis of Numb-infected and control retinal explants. Newborn explants were infected and analyzed as in Fig. 7. A total of 1891 control clones and 1704 Numb clones in nine different retinal explants were analyzed. The results are shown as mean±s.d. (A) Clone size. (B) Proportion of photoreceptor-only clones. (C) Proportion of photoreceptors in the total population of infected cells analyzed. (D) Proportion of clones containing at least one neuron, one Müller cell or more than one cell type. (E) Proportion of interneurons or Müller cells in the total population of infected cells analyzed. (F) Proportion of two-cell clones containing either two photoreceptors (PR) or a photoreceptor and another cell type. In (B-F), all of the differences between the control and Numb clones are significantly different when analyzed by a non-parametric statistical test (P<0.01).