Developmental Cell Supplemental Information Mitotic Membrane Turnover Coordinates Differential Induction of the Heart Progenitor Lineage Christina D. Cota and Brad Davidson
Supplemental Figures S1-S7 S1. FGF signaling is necessary for unequal cell division but is not required for polarized FGFR trafficking. Related to Figure 1. (A) Diagram of Fox>RFP induction assay as described in Materials & Methods. (B-C) Representative micrographs showing heart progenitor induction (FoxF>RFP, arrowheads) in control (B) and Mesp>FGFR:Venus transgenic embryos (C). Arrows indicate un-induced cardiac founder lineage cells (Mesp>GFP). Targeted expression of FGFR>Venus had no discernable impact on induction (5 trials, n>19/trial, P=0.805 (n.s.). (D-F) Representative micrographs of staged Mesp>FGFRDN:Venus founder cells, stained as indicated. Dashed lines in lateral sections (DE) indicate founder cell membranes (red) or epidermal border (white, Epi). Membrane associated FGFR:Venus foci (arrows) and intracellular vesicles (arrowheads) in mitotic founder cells. Solid lines in lateral projection (F) indicate dorsally (orange) and ventrally (white) positioned founder cell daughters. (G) Box-and-whisker plot of dorsal/ventral volume ratios derived from founder cell daughters (2 trials, n>12, GFP Control mean = 2.35±0.295 s.e.m, FGFRDN:Venus mean = 1.47±0.359 s.e.m, *P=0.00008). HP= heart progenitor, ATM= anterior tail muscle cell. Error bars represent the s.e.m. Scale bars are indicated in µm. Embryos are oriented anterior to the left.
S2. Selective rescue of heart progenitor induction is mediated by Intβ2 distal NPxF motif. Related to Figure 3. (A-F ) Representative micrographs showing single channel and merged images representative of heart progenitor induction (FoxF>RFP, arrowheads) in embryos co-transfected as indicated. Arrows indicate un-induced cardiac founder lineage cells (Mesp>GFP). Scale bars are indicated in µm. Embryos are oriented anterior to the left.
S3. Adhesion-dependent retention of Caveolin:GFP and Caveolin-dependent retention of FGFR:Venus in mitotic founder cells. Related to Figure 4. (A-D ) Representative micrographs showing heart progenitor induction (FoxF>RFP, arrowheads) in embryos cotransfected as indicated. Arrows indicate un-induced cardiac founder lineage cells (Mesp>GFP). (E-G) Lateral sections and graph (C-G) depicting comparative analysis of Caveolin:GFP enrichment along founder cell membranes in control (LacZ, E) and RapS17N (F) transgenic founder cells. Ventral/Dorsal (V/D) Ratio for cell cortex is shown. (VInt/DInt=2.62±0.10 for Caveolin:GFP) For calculation of Ventral/Dorsal (V/D) Ratios see Supplemental Materials & Methods. Error bars represent the s.e.m in (G). *P=0.040 for LacZ versus RapS17N. Significance was determined using two-tailed unpaired t-test. (H-K ) Lateral sections of FGFR:Venus and Caveolin:RFP distribution in pre-mitotic (H, J ) and mitotic (I, K ) founder cells transfected with either Caveolin:RFP or CaveolinP189L:RFP as indicated to the left. FGFR:Venus and Caveolin:RFP foci along the epidermal boundary indicated by arrows in H-J. Dashed lines (red) outline transgenic founder cells. D=dorsal, V=ventral, Epi=ventral epidermis (dashed line, white). Scale bars are indicated in µm. Embryos are oriented anterior to the left.
S4. Caveolin is required for heart progenitor induction. Related to Figure 5. (A-B ) Lateral sections and corresponding diagrams of FGFR:Venus distribution in pre-mitotic transgenic founder cells co-transfected as indicated. Dashed lines (red) outline founder cell membranes. Epi=ventral epidermis (dashed line, white). (C-F' ) Single channel and merged images showing representative heart progenitor induction phenotypes (FoxF>RFP, arrowheads) in embryos co-transfected as indicated. Arrows indicate un-induced cardiac founder lineage cells (Mesp>GFP). Scale bars are indicated in µm. Embryos are oriented anterior to the left.
S5. Caveolin-mediated rescue of heart progenitor induction in RapS17N transgenic embryos. Related to Figure 6. (A-C ) Single channel and merged images showing representative heart progenitor induction phenotypes (FoxF>RFP, arrowheads) in embryos co-transfected as indicated. Arrows indicate un-induced cardiac founder lineage cells (Mesp>GFP). Scale bars are indicated in µm. Embryos are oriented anterior to the left.
S6. Proposed model for anchorage dependent asymmetric cell fate determination. Related to Figure 7. (A) Diagrams illustrating how anchored mitosis may bias the turnover and redistribution of adherent membrane compartments (in red) to promote asymmetric fate specification. (A ) Prior to cytokinesis, a decreased rate of endocytic recycling drives a reduction in cell surface area and volume associated with mitotic rounding (Boucrot and Kirchhausen, 2007). (A ) In cells that remain anchored during rounding, persistent adhesion may maintain premitotic recycling rates or suppress internalization leading to asymmetric enrichment of adherent membrane compartments. (A ) Upon entry into cytokinesis, internalized vesicles enriched with Caveolin and associated proteins are re-distributed (Boucrot et al., 2011). Recycling pathways re-distribute Integrin receptors to the cytokinetic ring (Pellinen et al., 2008) establishing a central domain of adherent membrane where Caveolin may also be retained (Boucrot and Kirchhausen, 2007). (A ) Persistent, asymmetric adhesion during anchored division may bias the re-distribution of Caveolin and associated integral membrane proteins, including receptors.
Supplemental Tables S1-S2 Table S1. Caveolin CRISPR primers Related to Figure 5. Name grna(20-mer) a PAM CavA.39gRNA ACAACACGAAGCTGCAACCC CGG CavA.39gRNAmm1 ACAACcCGAAGCTGCAACCC CGG CavA.39gRNAmm2 ACAACACGAAGCTGCcACCC CGG CavA.219gRNA GGACAATGTGGAACTTGATT CGG CavA.219gRNAmm1 GGACAcTGTGGAACTTGATT CGG CavA.219gRNAmm2 GGACAATGTGGcACTTGATT CGG a For inverse PCR cloning: forward primers: 5 -(grna)tttaagagctatgctggaaacagcatagc-3 ; Reverse primer: 5 - TGAGACCCTCTCTCGGTCTCTATCTATACCA-3 Table S2. Primers Related to Figures 3 & 5. Name Primer sequence (5-3 ) Intβ1 Y694F forward TCTACCTTTAAAAATCCAATGTTTTCTGGTGGGAAAACTGCTGGA Intβ1 Y694F reverse TCCAGCAGTTTTCCCACCAGAAAACATTGGATTTTTAAAGGTAG A Intβ2 F801Y forward ACGAGGTTCGAAAACCCAACCTACCACGGAACGTAGACATCACG C Intβ2 F801Y reverse GCGTGATGTCTACGTTCCGTGGTAGGTTGGGTTTTCGAACCTCGT Caveolin(CavA) forward ACTGCGGCCGCAATGGCAGAAAACGGAGAA Caveolin(CavA) reverse ACTGGATCCTGCGCGTCTCTCCTCAGTGACA Caveolin P189L forward CATTTGGTGGATCATGCTATGTATTAGATGCCTCG Caveolin P189L reverse CGAGGCATCTAATACATAGCATGATCCACCAAATG Caveolin exon1 forward TGGCAGAAAACGGAGAACAG Caveolin exon1 reverse GGCATGATCCACCAAATGC
Supplemental Methods Equatorial cell volume measurements. For measurements of equatorial cell volume, confocal stacks of postmitotic cardiac founder lineage cells were analyzed using ImageJ. Equatorial cell volumes were approximated as rectangular cell volumes. Consistent positioning for cell measurements was achieved by identifying the section within a stack where the nuclear diameter was the largest. To avoid concerns about changes in division orientation, equatorial cell volume ratios were calculated as the ratio of the larger two cardiac founder lineage cells in a cluster versus the smaller two cells. FGFR:Venus measurements. For measurements of ventral/dorsal staining ratios, 1uM confocal stacks were analyzed using ImageJ. Only ventral views were analyzed. For each founder cell analyzed a lateral optical section was obtained. Consistent positioning for measurements was defined by finding the center of the DRAQ5-stained chromatin with the founder cell being analyzed. For exterior cell volume measurements, a ROI was set at the perimeter of each founder cell. Interior cell volume measurements were determined by setting a second ROI within the exterior cell ROI. ROIs were positioned such that subtraction of measurements obtained for the interior ROI from the exterior ROI represents values for a 2.5µm region of the founder cell cortex. Ventral and dorsal regions for each founder cell were set in relation to the axis of cell division (red rectangle; Fig. 1C -C ). Generation and use of the ROI and background subtraction tools achieved consistent outlining and detection of GFP-stained regions. The corrected total fluorescence (CTCF) was calculated as integrated density (area of selected cell mean fluorescence of background reading) and determined for the dorsal and ventral, interior and exterior founder cell volumes. To approximate the plasma membrane-associated fluorescence from these measurements for both the dorsal and ventral regions of each founder cell; plasma membrane-associated fluorescence was defined by exclusion such that for each dorsal and ventral region, the interior CTCF was subtracted from the exterior CTCF. From these values ratios were calculated comparing the ventral plasma membrane-associated fluorescence to the dorsal plasma membrane-associated fluorescence (V/D for cell cortex) or comparing the ventral intracellular fluorescence to the dorsal intracellular fluorescence (V Int /D Int for cell cortex). For measurements of the percent of FGFR-associated membrane (Fig. 2H) a lateral optical section was obtained each founder cell analyzed. In each section the circumference of the cell as well as the distance along that circumference associated with FGFR staining was measured. Results are expressed as a percentage equal to the distance along that circumference associated with FGFR staining/ the circumference of the cell. We also attempted to directly examine FGF receptor distribution using an Ciona FGFR antibody. FGFR mabs were prepared via custom antibody services (Abmart). Specificity was determined by western blot analysis of protein lysates prepared from Stage 14 C.intestinalis embryos. Antibodies obtained from immunization with either NGSKYDPVSK or PLLNPQPDAN epitopes detected a single band consistent with Ci-FGFR (predicted molecular weight: 76kD). Specificity was confirmed by colocalization of anti- FGFR signal and Venus fluorescence in Mesp>FGFR:Venus embryos. However, our FGFR antibody was not sufficiently sensitive to monitor in vivo localization (data not shown).
Statistical analysis. In all graphs, error bars represent standard error of mean (SEM). Significance was determined using Student s t-tests with two-tailed distributions in Microsoft Excel. Data expressed as a percentage or ratio was transformed prior to statistical analysis.
Supplemental References Boucrot, E., and Kirchhausen, T. (2007). Endosomal recycling controls plasma membrane area during mitosis. Proceedings of the National Academy of Sciences of the United States of America 104, 7939 7944. Boucrot, E., Howes, M.T., Kirchhausen, T., and Parton, R.G. (2011). Redistribution of caveolae during mitosis. Journal of Cell Science 124, 1965 1972. Pellinen, T., Tuomi, S., Arjonen, A., Wolf, M., Edgren, H., Meyer, H., Grosse, R., Kitzing, T., Rantala, J.K., and Kallioniemi, O. (2008). Integrin Trafficking Regulated by Rab21 Is Necessary for Cytokinesis. Developmental Cell 15, 371 385.