Journal: Development (Cambridge, England)

Article Title: Not just inductive: a crucial mechanical role for the endoderm during heart tube assembly

doi: 10.1242/dev.073486

Figure Lengend Snippet: Geometry for computational model. (A) Bright-field and OCT images of HH stage 8+ embryo. OCT sections were taken through medial (green), mediolateral (orange) and lateral (purple) locations around the AIP. On each section, endoderm (blue) and cardiogenic mesoderm (red) were resolved by visual inspection. Arrows indicate orientation of each OCT section within the embryo. Scale bars: 300 μm (black); 100 μm (white). (B) OCT sections shown in A arrayed in 3D space. We consider a 2D slice through the tissue. Note that the thickness of the mesoderm (red) is greater than that of the adjacent endoderm (blue). (C) 2D projection of this slice overlaid with a schematic of HH stage 8+ embryo. (D) For our model geometry, we consider an idealized 2D representation of the tissue, and both tissue layers are modeled as concentric circular rings of pseudoelastic material. We assume bilateral symmetry relative to the embryonic midline, and the model geometry includes only the yellow boxed region in C. A polar coordinate system (r, θ) has its origin at the center of the rings. See text for further details.

Article Snippet: Computational model Model geometry To help interpret our tissue cutting experiments, we constructed a nonlinear 2D finite element model of the endoderm and mesoderm around the AIP using COMSOL Multiphysics (Version 3.5, COMSOL AB, Providence, RI, USA).

Techniques:

Journal: Development (Cambridge, England)

Article Title: Not just inductive: a crucial mechanical role for the endoderm during heart tube assembly

doi: 10.1242/dev.073486

Figure Lengend Snippet: Tracking motion of endoderm and cardiogenic mesoderm around the AIP during heart tube assembly. (A) Schematic of representative HH stage 7+ embryo shown in B. Overlapping mesodermal (red arrowhead) and endodermal (blue arrowhead) fluorescent labels were injected in the lateral region of the AIP, and a single fluorescent label was placed in the endoderm at the medial point of the AIP (blue arrowhead). Embryos were cultured ex ovo and labels were tracked in time as the heart tube formed. The distance of both lateral labels from the midline (dM and dE for the mesoderm and endoderm, respectively) was measured at each time point. The length L between the two endodermal labels, and the separation distance dS between the (initially) adjacent labels in the endoderm and mesoderm were also measured. (B-D) Representative embryo after 0, 3 and 9 hours of incubation ex ovo. Red and blue tracks (and arrowheads) represent mesodermal and endodermal trajectories (and labels), respectively. Scale bar: 200 μm. (E) Distance of lateral labels from the midline (mesoderm, red line; endoderm, blue line), and separation distance between the labels (dashed black line) plotted as functions of time (n=5). (F) Endodermal stretch ratio around the AIP as a function of time (n=5). The distance between endodermal labels at 0 hour (L0) is used as the reference length. Error bars indicate s.d. During heart tube formation, the endoderm and cardiogenic mesoderm move together towards the midline, as the endoderm shortens around the AIP.

Article Snippet: Computational model Model geometry To help interpret our tissue cutting experiments, we constructed a nonlinear 2D finite element model of the endoderm and mesoderm around the AIP using COMSOL Multiphysics (Version 3.5, COMSOL AB, Providence, RI, USA).

Techniques: Injection, Cell Culture, Incubation

Journal: Development (Cambridge, England)

Article Title: Not just inductive: a crucial mechanical role for the endoderm during heart tube assembly

doi: 10.1242/dev.073486

Figure Lengend Snippet: Myosin-II-dependent contraction drives endodermal shortening around AIP. (A,B) Fluorescent labels were injected into the endoderm at medial and lateral locations around the AIP (blue arrowheads); these labels were separated by a distance L. Representative embryo cultured in 100 μM blebbistatin after 0 (A) and 3 (B) hours of incubation. After 3 hours of incubation, blebbistatin was washed out and culture was resumed. (C,D) Same embryo after 6 and 10 hours of total incubation. In this embryo, mesodermal cells adjacent to the lateral endoderm were also incidentally labeled. After wash-out, the lateral label separated into distinct mesodermal (red arrowhead) and endodermal (blue arrowhead) portions. (E) Endodermal stretch ratio around the AIP as a function of time for both blebbistatin-treated (solid line, n=4) and normal (dashed line, n=5) embryos. The distance between endodermal labels at 0 hour (L0) is used as the reference length. Error bars indicate s.d. The dashed line is identical to that shown in Fig. 2F. These results suggest that cytoskeletal contraction drives endodermal shortening around the AIP. Scale bar: 200 μm.

Article Snippet: Computational model Model geometry To help interpret our tissue cutting experiments, we constructed a nonlinear 2D finite element model of the endoderm and mesoderm around the AIP using COMSOL Multiphysics (Version 3.5, COMSOL AB, Providence, RI, USA).

Techniques: Injection, Cell Culture, Incubation, Labeling

Journal: Development (Cambridge, England)

Article Title: Not just inductive: a crucial mechanical role for the endoderm during heart tube assembly

doi: 10.1242/dev.073486

Figure Lengend Snippet: Computational model indicates endoderm as primary contractile tissue layer. (A,A′) Deformed shape of the AIP before (A) and after (A′) cutting (same images as in Fig. 6A,B). (B-D) When contraction is specified in the endoderm only (B) and an incision is simulated at the midline (C), the cut opens as observed experimentally (D). The model AIP curls posteriorly and qualitatively matches the deformed contour of the AIP in our cutting experiments (compare white contour inside dashed red box in A with geometry in D. (E) When the endoderm contracts, the (convected) circumferential Cauchy stresses are compressive in the mesoderm and tensile in the endoderm (computed along blue line in C. (F-H) When contraction is simulated in the mesoderm only (F), the model cut (G) fails to open up (H), and the shape of the AIP is not curled posteriorly as it is in experiments. (I) In this case, the endoderm is in compression and the mesoderm is in tension (computed along red line in G). Agreement between the experiments and model in D, but not in H, indicates that the endoderm (not the mesoderm) is the primary contractile tissue layer. r is the normalized radial distance across the rings, where 0 represents the inner curvature.

Article Snippet: Computational model Model geometry To help interpret our tissue cutting experiments, we constructed a nonlinear 2D finite element model of the endoderm and mesoderm around the AIP using COMSOL Multiphysics (Version 3.5, COMSOL AB, Providence, RI, USA).

Techniques:

Journal: Development (Cambridge, England)

Article Title: Not just inductive: a crucial mechanical role for the endoderm during heart tube assembly

doi: 10.1242/dev.073486

Figure Lengend Snippet: Endoderm actively contracts to pull the cardiogenic mesoderm towards the midline. (A) Schematic of HH stage 7 embryo. The cardiogenic mesoderm (red) is organized as a pair of bilateral epithelia that are separated on either side of the embryonic midline and remain in close contact with the underlying endoderm (blue). (B) Schematic of HH stage 8+ embryo. As the AIP descends, the cardiogenic mesoderm moves towards the midline and fuses to begin forming the early heart tube. Dashed black lines represent the neural tube. (C) Investigators have suggested that the endoderm serves primarily as an inductive substrate for the actively crawling mesoderm (red arrows). (D) Our results suggest that the endoderm also has a distinct mechanical role in early cardiogenesis; it actively contracts (blue arrows) to pull the cardiogenic mesoderm towards the midline. Although relative motion (red arrows) occurs between the endoderm and mesoderm during this process (probably owing to collective migration), this motion is much less than the convection caused by contraction.

Article Snippet: Computational model Model geometry To help interpret our tissue cutting experiments, we constructed a nonlinear 2D finite element model of the endoderm and mesoderm around the AIP using COMSOL Multiphysics (Version 3.5, COMSOL AB, Providence, RI, USA).

Techniques: Migration, Convection