N-Cadherin Locks Left-Right Asymmetry by Ending the Leftward Movement of Hensen s Node Cells
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1 Developmental Cell, Volume 30 Supplemental Information N-Cadherin ocks eft-ight Asymmetry by Ending the eftward Movement of Hensen s Node Cells aquel V. Mendes, Gabriel G. Martins, Ana M. Cristovão, and eonor Saúde
2 Supplemental Figure S1 A Control MNCD2 AB B B C D E E F G 0h00m + 4h20m 0-4h20m 0h00m + 5h20m 0h-5h20m H I I J K M N 4h30m 7h00m 4h30m-7h00m 5h30m 8h20m 5h30m-8h20m Control MNCD2 AB Control MNCD2 AB O P U 5som V 5som shh shh Q W 3som X 3som fgf8 fgf8 cer1 cer1 S T Y 5som Z 5som snai1 snai1
3 Figure S1. oss of N-cadherin activity in the Hensen's node prolongs leftward cell movements and affects asymmetric gene expression in Hensen's node and PM. elated to Figure 2 and Figure 3. (A) A stage diagram showing photoconverted cells (red dots) on the right side of the node. Position of photoconverted cells at two time-points and their tracks in a control (B-C) and in a MNCD2 antibody-treated embryo (E-F). Overlay tracks of all cells analyzed from six controls (D) and five MNCD2 antibody-treated embryos (G) photoconverted at the beginning of stage and tracked until stage. (H) A stage diagram where photoconverted cells at stage (light-red dots) moved to the left and new cells were photoconverted (dark-red dots) on the right side of the node. Position of photoconverted cells at two timepoints and their tracks in a control (I-J) and in a MNCD2 antibody-treated embryo (-M). Overlay tracks of all cells analyzed from six controls (K) and five MNCD2 antibody-treated embryos (N) photoconverted at the beginning of stage and tracked until stage. In control embryos, 11 out of 20 labeled cells at stage crossed the midline and 0 out of 34 labeled cells at stage crossed the midline; in MNCD2 antibody-treated embryos, 7 out of 24 labeled cells at stage crossed the midline and 10 out of 16 labeled cells at stage crossed the midline. The expression of shh was asymmetric on the left side of the node in 93% (n=16) of controls (O) and in 100% (n=19) of MNCD2-treated embryos (P). The expression of fgf8 was asymmetric on the right side of the node in 80% (n=30) of controls (Q) and became bilaterally symmetric in 43% (n=37) of MNCD2-treated embryos (). In MNCD2-treated embryos the expression of
4 in the peri region was downregulated in 63% (n=30) of the embryos (T) in contrast to controls where asymmetric expression on the left peri region was found in 75% (n=12) (S). The expression of was found to be restricted to the left PM in 100% (n=5) of controls (U) and in 100% (n=5) in MNCD2-treated embryos (V). The expression of cer1 was asymmetric on the left side of the PM in 87% (n=15) of controls (W) and downregulated in 53% (n=15) of MNCD2-treated embryos (X). The expression of snai1 was asymmetric on the right side of the PM in 97% (n=31) of controls (Y) and became bilateral in 32% (n=38) of MNCD2-treated embryos (Z). Insets in all panels show a magnification of the node region. Black and white arrowheads indicate normal and abnormal expression, respectively., left;, right; AB, antibody.
5 Supplemental Figure S2 pcaggs-gfp pcaggs-n-cadyfp A B C D E F shh shh G H I J K fgf8 fgf8 M N O P Q S T 5som U V W 5som X cer1 cer1
6 Figure S2. N-cadherin overexpression on the right side of the node does not affect asymmetric gene expression. elated to Figure 3. Chicken embryos electroporated with pcaggs-gfp or pcaggs-n-cadyfp on the right side of the node at stage HH3 + and photographed at stage (A, G, M, S, D, J, P, V). The same embryos photographed at stage (B, H, N, E, K, Q) or at 5-somite stage (T, W) and in situ hybridized with shh (C, F), fgf8 (I, ) (O, ) and cer1 (U, X). Insets are magnifications of the node region. Arrowheads indicate normal gene expression. Black bracket, notochord width in a control embryo; red bracket, notochord width in a N-cadherin overexpressing embryo;, left;, right.
7 Supplemental Table S1 Table S1. Number of cells that crossed the midline at different stages of development in control vs Anti-N-cad treated embryos. elated to Figure 2. Beginning of cell tracks Nº of tracked cells Nº of cells that cross the midline during stages + Nº of cells that do NOT cross the midline # Control 27 16* Anti-N-cad 22 2* Anti-N-cad 13 n/a Control 28 n/a n/a Anti-N-cad 24 n/a n/a * The higher number of cells that cross the midline at stage in control embryos when compared to Anti-N-cad embryos is most probably due to a technical issue. In controls we managed to start the tracks at an earlier time point (spanning on average 3 hours before reaching stage + ), while in Anti-N-cad embryos we only managed to start the tracks at a later time point (spanning on average 2 hours before reaching stage + ). # The cells that did not cross the midline, either stayed on the right side of the node and were displaced anteriorly or died or divided or aggregated. n/a, not applicable.
8 Movie S1. Time-lapse movie of a chicken embryo electroporated on the right side with Kaede-NS photoconvertible fluorescent protein and treated with IgG2a control antibody. elated to Figure 2. Cells on the right side of the node were photoconverted from green to red at stage and followed for a period of 4 hours and 10 minutes after which some cells were found on the left side of the node. When the embryo reached stage, new cells on the right side of the node were photoconverted from green to red and followed over a period of 4 hours and 20 minutes, after which, no cells were found to cross to the left side of the node. Cell tracks of the photoconverted cells are shown in the end. The yellow dashed line in the first frame marks the position of the primitive pit and primitive groove, and the pink circle highlights the photoconverted region. Movie S2. Time-lapse movie of a chicken embryo electroporated on the right side with Kaede-NS photoconvertible fluorescent protein and treated with anti-n-cad antibody. elated to Figure 2. Cells on the right side of the node were photoconverted from green to red at stage and followed for a period of 2 hours after which some cells were found on the left side of the node. When the embryo reached stage, new cells on the right side of the node were photoconverted from green to red and followed over a period of 6 hours and 10 minutes, after which some cells were found to cross to the left side of the node. Cell tracks of photoconverted cells are shown in the end. The yellow dashed line in the first frame marks the position of the
9 primitive pit and primitive groove, and the pink circle highlights the photoconverted region. Movie S3. Time-lapse movie of a chicken embryo electroporated on the right side with full-length N-cadherinYFP. elated to Figure 2. The time-lapse starts at stage and finishes at stage, spanning a 7-hour period. Fluorescent cells overexpressing N-cadherin found on the right side of the node never crossed to the left side. Cell tracks of fluorescent cells are shown in the end. The yellow line in the first frame marks the position of the primitive pit and primitive groove. Movie S4. Time-lapse movie of a chicken embryo electroporated with Kaede-NS photo-convertible fluorescent protein on the right side and treated with PBS. elated to Figure S1 and Figure 2. Cells on the right side of the node were photo-converted from green to red at stage and followed for a period of 4 hours and 20 minutes after which some cells were found on the left side of the node. When the embryo reached stage, new cells on the right side of the node were photo-converted from green to red and followed over a period of 2 hours and 30 minutes, after which no cells were found to cross to the left side of the node. Cell tracks of the photoconverted cells at stage and later at stage are shown in the end. The yellow dashed line in the first frame marks the position of the primitive streak, and the pink circle highlights the region of photoconverted cells.
10 Movie S5. Time-lapse movie of a chicken embryo electroporated with Kaede-NS photo-convertible fluorescent protein on the right side and treated with the N-cadherin blocking MNCD2 antibody. elated to Figure S1 and Figure 2. Cells on the right side of the node were photo-converted from green to red at stage and followed for a period of 5 hours and 20 minutes after which some cells were found on the left side of the node. When the embryo reached stage, new cells on the right side of the node were photo-converted from green to red and followed over a period of 2 hours and 50 minutes, after which some cells were found to cross to the left side of the node. Cell tracks of the photoconverted cells at stage and later at stage are shown in the end. The yellow dashed line in the first frame marks the position of the primitive streak, and the pink circle highlights the region of photoconverted cells.
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