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First published online January 23, 2009
doi: 10.1242/10.1242/dev.026211

Development 136, 677-688 (2009)
Published by The Company of Biologists 2009
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Actomyosin stiffens the vertebrate embryo during crucial stages of elongation and neural tube closure

Jian Zhou, Hye Young Kim and Lance A. Davidson*

Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15260, USA.


Figure 1
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  		Fig. 1. Dorsal isolates increase in stiffness with stage. (A) Schematic of microsurgery involved in making the dorsal isolate. The dorsal isolate contains neural ectoderm (ne), notochord (no), paraxial somitic mesoderm (so) and endoderm (en). Removal of the two-cell layered neural ectoderm reveals a distinct boundary between the notochord and paraxial mesoderm. (B) A microsurgically isolated dorsal isolate (DI) contains an identical configuration of tissues to dorsal tissues within a whole embryo (WE), shown with rhodamine-dextran (red) and fibrillar fibronectin matrix (green). Scale is the same for both panels. (C) The nanoNewton Force Measurement Device (nNFMD) measures resistive forces generated by the explant in response to compression by a computer controlled force-calibrated optical fiber. (D) Stiffness (defined at the Young's modulus after 180 seconds of unconstrained compression) is measured from a stress-relaxation protocol wherein the explant is compressed, a trace of the resistive force is measured, and a stiffness (Pa) is calculated. (E) Dorsal isolates were cut from a single clutch of embryos and their stiffness measured. Samples younger than stage 13 show little change in stiffness. However, stiffness increases sharply once the dorsal axis begins rapid elongation. The number of explants in each set is indicated in parentheses below the graph. Stars above pair-wise sets of explants indicate significant (one asterisk; P<0.05) and highly significant (two asterisks; P<0.01) differences.

 

Figure 2
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  		Fig. 2. Mechanics of the notochord.(A) Schematic of microsurgical construction of two-plus, zero-notochord and (A')two-notochord explants lacking medial fragments of paraxial notochord. (B) Frames and explant lengths over time from a single representative time-lapse (n=4) demonstrate that two-plus and zero-notochord explants elongate the same as dorsal isolate explants. (C) Maximum projection of confocal sections of fibronectin fibrils illustrates the transverse architecture of control explants, two-plus notochord (asterisk, medial fragment of paraxial mesoderm), zero-notochord and (C') two-notochord explants (lacking medial fragments of paraxial mesoderm. Scale bar is the same for all panels in C and C'. (D) Comparison of stiffness between explants bearing two-plus notochord and zero-notochord. (D') Comparison of the stiffness of two-notochord explant (lacking medial paraxial fragment) compared with zero-notochord explant. (E) LiCl strategy for generating supersized notochords in dorsal tissues as shown (F) in DAI 7 to DAI 8 embryos. (G) Chordin RNA in situ showing increased notochord within dorsal isolates made from DAI 7 to 8 embryos. (H) Transverse confocal section of a rhodamine dextran-labeled (red) and fibrillar fibronectin labeled (green) dorsalized embryo reveals enlarged notochord (no) when compared with Fig. 1B. (I) Comparison of stiffness of dorsal isolates with LiCl enlarged notochord to dorsal isolates made from untreated cohort reveal no significant difference. **P<0.05.

 

Figure 3
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  		Fig. 3. Mechanics of paraxial-medial and paraxial-lateral tissues.(A) Schematic diagram of explants carrying complete sets of paraxial tissues (lateral-medial-medial-lateral or LMML) and those carrying only paraxial lateral tissues (lateral-lateral or LL) of the dorsal axis. (B) Fibronectin fibril localization in transverse sections of LMML and LL explants shows the location of paraxial mesoderm outlined by fibronectin. (C) A view of XmyoD expression through the endodermal face of explants reveals a distinct boundary between paraxial-medial (XmyoD expressing) and paraxial-lateral (non-expressing) tissues of control dorsal isolates and LMML explants. XmyoD is absent from most of the length of LL explants that can occasionally express a small `wedge' of XmyoD in their most posterior third (arrowhead). All explants are positioned so that anterior is at the top of the panel. (D) Frames and explant lengths over time from a single representative time-lapse (n=6) demonstrate that LMML and LL explants elongate to the same degree as control explants. (E) The stiffness of LL and MM isolates show that MM explants are significantly more stiff than LL explants. *P<0.05; **P<0.01.

 

Figure 4
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  		Fig. 4. Mechanics of endodermal and neural tissues. (A) Schematic showing removal of either neural layers or endoderm layers and histology of transverse sections collected from the resulting rhodamine dextran-labeled explants. (B) The stiffness of explants without the neural plate is compared with the stiffness of control dorsal isolates. We carried out two additional trials after a highly significant difference was found in one of the first three sets of explants. The last trial found a significant but small increase in explants lacking a neural plate. (C) The stiffness of explants without endoderm is compared with the stiffness of control dorsal isolates. In all cases, there were significant reductions in the stiffness of explants bearing endoderm. *P<0.05; **P<0.01.

 

Figure 5
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  		Fig. 5. Role of ECM in tissue stiffness. (A) Representative transverse maximum projections of confocal sections show fibronectin (FN), laminin (LAM), and fibrillin (FIB) are all present within the dorsal tissues by stage 16. FN surrounds both axial (n, notochord) and paraxial mesoderm (s, prospective somitic mesoderm). LAM and FIB surround the axial mesoderm but do not assemble more laterally (asterisk). (B) The stiffness of explants injected with fibronectin morpholinos (FNMO) does not differ from uninjected explants at either stage 13 or at stage 16. Seven to nine explants from each of three separate clutches of embryos were tested. (C) Representative maximum projections of FNMO-injected dorsal isolates exhibit severe reduction in fibronectin fibrils and also show defects in assembly of fibrillin and laminin compared with control isolates sectioned with identical confocal settings. (C') Intensity profiles collected mediolaterally across the midline (indicated by transparent line across the midline in xz-projections shown in C indicate as expected that FN assembly is severely reduced but FIB and LAM also exhibit significant reductions.

 

Figure 6
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  		Fig. 6. The role of actomyosin contractility in tissue stiffness.(A) A transverse confocal section of F-actin labeled with bodipy-FL phalloidin shows that latrunculin B (latB) significantly reduces the amount of F-actin in the whole embryo. (B) Treatment of explants for 20 minutes with latB reduced tissue stiffness by up to 70% in a dose-dependent manner. The entire tissue dissociates at higher concentrations with treatments lasting more than 60 minutes. (C) Incubation of explants for 40 to 60 minutes in Y27632 reduced tissue stiffness up to 50% in a dose-dependent manner. (D) Combined treatment of explants cultured 40 to 60 minutes in Y27632 and 20 minutes in latB showed that explants cultured in Y27632 dropped in stiffness to the level of explants incubated in latB. Dose dependence of latB and Y27632 was carried out with embryos from three different clutches. Results from three clutches were scaled to the control stiffness and combined in the single chart shown. Whisker box bounds in B and C indicate 1st quartile, median and 3rd quartile. Error bars indicate standard deviations. 1Control dorsal isolates treated with DMSO carrier were pooled in B and C. **P<0.05.

 

Figure 7
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  		Fig. 7. Differences in F-actin and pMLC density may underlie spatial differences in tissue stiffness. (A,B) Maximum projection images from representative embryos (projected from 20 sections at 0.5 µm intervals), show F-actin (green; bodipy-FL phallacidin) and fibronectin localization (red; mAb 4H2) across the dorsal region of embryos at stage 13 (A) and stage 16 (B). Neural, endoderm, pre-somitic mesoderm and notochord regions (labeled boxes) are shown at higher resolution in separate panels below. Cellular architecture at stage 16 within the pre-somitic mesoderm differs considerably from stage 13 with the formation of cell `buttresses' (double-headed arrows) and a pre-myocoel (arrowheads) in the stage 16 embryo. The endoderm in both stages have considerably less F-actin than adjacent tissues. Fibronectin staining was carried out to allow identification of overall tissue morphology. A and B are shown at the same scale. (C) Maximum projection images (from 10 sections at 0.5 µm intervals) show pMLC localizes to the interfaces between mesoderm, endoderm and neural ectoderm, as well as the interface between notochord and prospective somitic mesoderm in both stage 13 and stage 16 embryos. pMLC is strongly localized to the pre-myocoel interface between the dorsal and ventral leaflets of the somitic mesoderm and lateral plate (arrowheads) at stage 13 and 16. Samples in A-C were each from the same clutch of embryos fixed and processed in the same vial to allow direct comparison of either F-actin or pMLC levels. Non-specific background staining is shown in embryos processed without addition of primary antibody.

 

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© The Company of Biologists Ltd 2009