Tbx16 and Msgn1 are required to establish directional cell migration of zebrafish mesodermal progenitors

Abstract

The epithelial to mesenchymal transition (EMT) is an essential process that occurs repeatedly during embryogenesis whereby stably adherent cells convert to an actively migrating state. While much is known about the factors and events that initiate the EMT, the steps that cells undergo to become directionally migratory are far less well understood. Zebrafish embryos lacking the transcription factors Tbx16/Spadetail and Mesogenin1 (Msgn1) are a valuable system for investigating the EMT. Mesodermal cells in these embryos are unable to perform the EMT necessary to leave the most posterior end of the body (the tailbud) and join the pre-somitic mesoderm, a process that is conserved in all vertebrates. It has previously been very difficult to study this EMT in vertebrates because of the multiple cell types in the tailbud and the morphogenetic changes the whole embryo undergoes. Here, we describe a novel tissue explant system for imaging the mesodermal cell EMT in vivo that allows us to investigate the requirements for cells to acquire migratory properties during the EMT with high spatio-temporal resolution. This method revealed that, despite the inability of tbx16;msgn1-deficient cells to leave the tailbud, actin-based protrusions form surprisingly normally in these cells and they become highly motile. However, tbx16;msgn1-deficient cells have specific cell-autonomous defects in the persistence and anterior direction of migration because the lamellipodia they form are not productive in driving anteriorward migration. Additionally, we show that mesoderm morphogenesis and differentiation are separable and that there is a migratory cue that directs mesodermal cell migration that is independent of Tbx16 and Msgn1. This work defines changes that cells undergo as they complete the EMT and provides new insight into the mechanisms required in vivo for cells to become mesenchymal.

Keywords: EMT; Mesoderm; Morphogenesis; Tbx16; Zebrafish.

Figure 1

Tbx16 and Msgn1 act cell-autonomously in migration out of the tailbud. A. Diagram of transplant scheme. Labelled undifferentiated cells are removed from donor embryos and placed in the ventral margin (fated to become tail somites) of unlabeled gastrulating embryos. B–C,E–F. Fluorescently labelled donor cells (red) overlaid on bright field images of host embryos at 24 hours post fertilization. B. Wild-type donor cells in wild-type host. C. MO donor cells in wild-type host. E. Wild-type donor cells in MO host. F. MO donor cells in MO host. Brackets and arrow indicate locations of donor cells. D. Percentage of wild-type host embryos containing donor cells in somite, fin or epithelium, and undifferentiated groups. *: p<0.01 by χ² test. G. Percentage of MO host A-P body length containing donor cells. (Distance from posterior of embryo to anterior-most donor cell divided by total A-P body length.) *: p<0.01 by Anova. Bars show standard deviation. H. 24 hours post fertilization MO host with somite-like organization of wild-type donor cells. Left panel is a single frame bright field image; middle is a Z projection through 4 μm of fluorescent dextran-labeled donor cells; right is a Z projection through 4 μm of embryos stained with a muscle myosin antibody. Arrows show somite-like structures formed from donor cells. This is the same embryo as in Figure S1A. Scale bar is 50 μm.

Figure 2

A novel tailbud explant method allows for high spatio-temporal imaging of migrating cells in vivo. A. The posterior portion of an embryo in mid-somitogenesis is dissected away from the anterior tissue and yolk, mounted, and imaged. B. Time lapse image series of an explant from a wild-type host expressing a fluorescent membrane marker driven by the tbx16 promoter (white) with wild-type donor cells (red) taken at mid-somitogenesis. Anterior is to the left with the notochord running down the middle of the tissue. These images correspond to frames from Movie S1. Scale bar = 50 μm. C. Time lapse image series of two cells in an explant from a wild-type embryo mosaically expressing the fluorescent actin marker LifeAct driven by the tbx16 promoter. Scale bar = 10 μm.

Figure 3

Spt and Msgn have cell non-autonomous effects on speed. A. Charts show tracks of individual donor cells migrating over two hours. Each cell is in a different color. Anterior is to the left and each axis is 70 μm total. Vertical and horizontal scale bars in wt→wt plot are each 20 μm. Asterisk in MO→wt plot indicates a cell that moved significantly anteriorly and then reversed direction. B. Average speed of donor cells measured over two hours. C. Average net distance of donor cells from their starting points at the end of two hours. Bars in B, C show standard deviation. *: p<0.01 by pairwise Anovas. Data in B, C also displayed in box and whisker plots in Figure S3A, B.

Figure 4

Tbx16 and Msgn have cell autonomous effects on migratory persistence. A. The persistence of donor cell movement at two hours is equal to the ratio of the straight line distance between starting and ending points to the total distance travelled. Data is taken from the two-dimensional tracks in Figure 3A. Bars show standard deviation. Data analyzed by pairwise Anova and is also shown in box and whisker plot in Figure S3C. B. The percentage of time points that cells moved anteriorly, posteriorly, or did not move along the A-P axis. Data is taken from the one-dimensional A-P tracks in Figure S4. *: p<0.01 by χ² test.

Figure 5

tbx16;msgn1-deficient cells form protrusions fairly normally during axis elongation. A. Number of lamellipodia, filopodia, and blebs per cell measured in the MZ of embryos mosaically labelled with LifeAct and fixed at the 14 somite stage. Bars show standard deviation. *: p<0.01 by two-tailed heteroscedastic t-tests. B. Percent of protrusions in each body axis direction: lateral versus medial; ventral versus dorsal; and anterior versus posterior using the same labelling conditions as for A. Data were analyzed with z-tests.

Figure 6

tbx16;msgn1-deficient lamellipodia are less productive than wild-type lamellipodia. A. Diagram of how measurements of protrusion functionality were assessed. The direction of cell body movement was determined in the frame following the formation of each protrusion and the region of the cell (Front, Side, or Back) on which the protrusion formed relative to this direction was recorded. Then, at the last frame of a protrusion’s lifetime it was noted whether the protrusion was Unproductive (Retracted or Overtaken by the formation of another protrusion) or Productive (Overtaken by the cell body moving in that direction). Whereas the first measurement scores the initial trajectory of the cell, the later measurement reveals the action of the cell when the protrusion is no longer present. B. The percentage of protrusions formed in each direction, front, side, or back, relative to instantaneous cell body movement. *: p<0.01 by χ² test. C. The percentage of protrusions that were retracted, overtaken by another protrusion, or overtaken by the cell body. *: p<0.01 by χ² test. D. The percentage of unproductive and productive lamellipodia formed in the anterior and posterior directions. *: p<0.01 by z-test.

Figure 7

Model of mesodermal cell morphogenesis during gastrulation and somitogenesis. A. In gastrulating wild-type embryos, presumptive mesodermal cells transition through a highly blebbing intermediate state before becoming directionally migratory as they move from the epiblast (green) to the hypoblast (white). Yolk is gray. B. Later, during somitogenesis, neuromesodermal progenitors reside in a posterior pseudo-epithelium (PZ, blue) and transition to an anteriorward migratory state primarily driven by lamellipodia as they make the mesodermal fate choice and move into the MZ (orange). Pre-somitic mesoderm (PSM) is pink; notochord (N) is gray. C. During gastrulation, tbx16-deficient cells (which also don’t express Msgn1) begin to transition by becoming highly blebby but are never able to leave that state to become migratory. Colors are the same as in A. D. During somitogenesis, tbx16;msgn1-deficient cells leave the neuromesodermal progenitor epithelium and become highly motile, but never migrate anteriorly or leave the MZ despite the presence of directional cues. Colors are the same as in B. White is the region that lacks pre-somitic mesoderm. Red arrows and cell outlines denote aberrant behavior. A and C are lateral views; B and D are ventral views.