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Dev Dyn
2006 Dec 01;23512:3268-79. doi: 10.1002/dvdy.20979.
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Cell behaviors associated with somite segmentation and rotation in Xenopus laevis.
Afonin B
,
Ho M
,
Gustin JK
,
Meloty-Kapella C
,
Domingo CR
.
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During vertebrate development the formation of somites is a critical step, as these structures will give rise to the vertebrae, muscle, and dermis. In Xenopus laevis, somitogenesis consists of the partitioning of the presomitic mesoderm into somites, which undergo a 90-degree rotation to become aligned parallel to the notochord. Using a membrane-targeted green fluorescent protein to visualize cell outlines, we examined the individual cell shape changes occurring during somitogenesis. We show that this process is the result of specific, coordinated cell behaviors beginning with the mediolateral elongation of cells in the anteriorpresomitic mesoderm and then the subsequent bending of these elongated cells to become oriented parallel with the notochord. By labeling a clonal population of paraxial mesoderm cells, we show that cells bend around their dorsoventral axis. Moreover, this cell bending correlates with an increase in the number of filopodial protrusions, which appear to be posteriorly directed toward the newly formed segmental boundary. By examining the formation of somites at various positions along the anteroposterior axis, we show that the general sequence of cell behaviors is the same; however, somite rotation in anteriorsomites is slower than in posteriorsomites. Lastly, this coordinated set of cell behaviors occurs in a dorsal-to-ventral progression within each somite such that cells in the dorsal aspect of the somite become aligned along the anteroposterior axis before cells in other regions of the same somite. Together, our data further define how these cell behaviors are temporally and spatially coordinated during somite segmentation and rotation.
Fig. 1. Cell shapes associated with somite segmentation and rotation.A: A schematic of the paraxial mesoderm indicating the nomenclaturefor prospective somites in the presomitic mesoderm (PSM) and recentlyformed somites. B: A dorsal scan of a fixed and cleared stage 24 embryoinjected with mRNA encoding GAP43-GFP captured on a confocal mi-croscope. C: A lateral scan of another embryo at the same stage. Imageswere captured with the embryo’s anterior end oriented toward the top ofthe image and dorsal end toward the right of the image. D: A graphrepresenting the length/width ratio of cells located in S-I (n ⫽ 40 cells)and S-III (n ⫽ 40 cells) regions of the PSM. GFP, green fluorescentprotein.
Fig. 2. Cells maintain an elongated cell shape during rotation. A: Anillustration of the transplant procedure used to obtain the images in B, C,and D. Host embryos received transplantations of rhodamine- and flu-orescein dextran-labeled cells and were allowed to develop to stage 28and then were fixed and cleared for image analysis. B: The resultingembryo exhibited a mixture of red and green fluorescent cells scatteredthroughout the paraxial mesoderm. The z-stack collected from thisimage was rendered into a three-dimensional (3-D) image using theAmira software package. C: A lateral view of the 3-D image reveals thatcells in the dorsal portion of S0 complete rotation before cells positionedin other regions of the same somite. D: A ventral view of the same imageshows that cells maintain an elongated shape as they bend toward theanterior and posterior directions during rotation.
Fig. 3. Somite rotation consists of the move-ments of cells around the dorsoventral axis. A:Lateral view of a fixed and cleared stage 28 embryo that was labeled with green fluorescent protein (GFP) in the B1 blastomere at the 32-cell stage. The subset of labeled cells is positioned in the central region of the presomitic mesoderm (PSM) and in mature somites. A: The same image as shown above, but pseudo-colored to highlight the relative position of cells located within the central region of the paraxial mesoderm along the length of the anteroposterior axis. B: Lateral view of a fixed and clearedstage 24, GFP-labeled embryo, which receiveda transplant of Mini-Ruby (molecular probes)-labeled cells, seen in red. Bⴕ: The same image,inverted, where the Mini-Ruby cells have beenindividually pseudocolored. A clonal populationof cells remains restricted to the dorsal or ven-tral portions of the PSM (S-I blue, S-II, yellow)and in more advanced somites (S0 to SIV). Forall panels, anterior is to the left and dorsal is tothe top.
Fig. 4. Somite rotation is associated with anincrease in filopodial protrusions. A: Dorsalview of a stage 28 embryo in which the B1blastomere at the 32-cell stage was injectedwith mRNA encoding GAP43-GFP. The embryowas fixed, cleared, and mounted such that an-terior is to the top of the image. B: Close-up ofS0 (white box) showing the formation of filopo-dial protrusions that are posteriorly directed.Cells in the presomitic mesoderm (PSM) makebroad cell contacts with the adjacent notochord(red star). C: A graph representing the percent-age of cells with protrusions seen in the myo-tome (SI; n ⫽ 150 cells), rotating somites (S0;n ⫽ 159 cells), and PSM (S-I; n ⫽ 39 cells). GFP,green fluorescent protein.
Fig. 5. Temporal differences associated withthe formation of anterior and posterior somites.A,B: Dorsal (A) and lateral (B) views of a fixedand cleared five-somite embryo (stage 19)show that somite rotation is relatively slow incomparison to segmentation (arrow) and that itis not complete until the somite matures to S-3.C,D: An older fixed and cleared embryo with 22somites (stage 28) scanned from both the dor-sal (C) and lateral (D) perspectives shows thatsomite rotation is coincident with segmenta-tion.
Fig. 6. Temporal differences in the formation of anterior and posterior somites. A: Graph showingthe total number of somites formed over time. An R2value of 0.9911 indicates a strong linearpattern of somite formation, and suggests that somites are added at regular time intervals(approximately 50 min) regardless of their position along the anteroposterior axis. B: Graphshowing the average number of somites in rotation at given time intervals over a 950-min timeperiod.
Fig. 7. Somite formation occurs in a dorsal-to-ventral progression.A,B: Comparison of cell shapes between a dorsal view at the level of theneural tube (A) and a ventral view at the level of the notochord (B) in thesame fixed, cleared, and green fluorescent protein (GFP) -labeled five-somite embryo (stage 19). C,D: In an older fixed, cleared, and GFP-labeled embryo (stage 27), during the formation of the 19th somite,dorsal (C) and ventral (D) differences in cell morphology become moreapparent. E: An illustration of cell angle measurements as cells in S0rotate 90 degrees to form myotome fibers. The angle of rotation wasmeasured for cells in S0 relative to the starting position of cells in thepresomitic mesoderm (PSM; S-I). F: A graph showing the angle ofrotation for individual cells within S0 at three different axial regions of thedeveloping paraxial mesoderm: (1) anterior, consisting of embryos with4 to 6 somites; (2) middle, consisting of embryos with 9 to 12 somites;and (3) posterior, consisting of embryos with 18 to 20 somites.
Fig. 8. Proposed four-step model for cell shape changes occurringduring somite morphogenesis. A1: Cells elongate mediolaterally in theanterior presomitic mesoderm (PSM; S-I). A2: The number of posteriorlydirected filopodial protrusions increases. A3: Elongated cells bendaround the dorsoventral axis such that the anterior cell moves to thelateral edge of the somite (shown in brown) and a more posterior cellmoves to the medial region of the somite (shown in gray). A4: Cellsachieve a parallel alignment along the anteroposterior axis. B: Theoverall pattern of somite morphogenesis occurs in a dorsal-to-ventralgradient. Colored cells indicate that movement occurs around the dor-soventral axis during somite segmentation and rotation. C: During theformation of posterior somites, rotation and formation (S0) occur simul-taneously, such that cells positioned in the dorsal (red) and middle(yellow) regions of the somite have completed rotation. Thus by the timethe somite matures to SI, almost all of the cells have completed rotation.
