Table 1.
Acronyms Used. The list of vessel acronyms used in this paper.
Figure 1.
Example OPT view and slices from embryonic vascular mouse atlas data.
(A) An example OPT view from a Cy3-PECAM-1 immunostained 19 somite mouse embryo demonstrating the complexity of the vascular pattern at this stage of development. The coloured lines (green, blue, yellow) in this and subsequent subfigures indicate the slice taken from the 3D reconstruction. (B,C,D) Slices from the reconstructed 3D FDR-deconvolution OPT data set can be viewed along any dimension. B is sliced along the green line in A, C is sliced along the blue line in A, and D is sliced along the yellow line in B and C. (E) Magnified view of the boxed region in B, depicting the perineural vascular plexus (pink arrow). All scale bars represent 100 microns.
Figure 2.
Surface renderings of embryonic vascular structures.
(A) Reconstructed FDR-deconvolution OPT data of the 19 somite embryo is shown as a surface rendered object. (B) The surface rendered object can be zoomed in to any magnification, as in this magnified image of the vasculature in the mouse head. (C) The surface rendering can also be rotated so that it can be viewed from any angle. Viewing the rendering from the left side reveals structures in the heart (arrow) that are obscured by the tail in (A). (D) The 3D data can also be segmented as described in Materials and Methods. The DA, heart and ICAs are labelled yellow, the UV dark pink, and the unsegmented vasculature blue. (E) Segmentation of the data allows selective display of labelled structures. Exclusion of the unsegmented data provides better analysis of the ICAs and the pharyngeal arch arteries.
All scale bars represent 100 microns.
Figure 3.
Volume renderings of embryonic vascular structures.
(A) Reconstructed FDR-deconvolution OPT data can be visualized as a volume rendering. The vasculature of a 19 somite mouse embryo is visualized as a volume rendering using a hot metal colour map. (B) OPT data acquired for the mouse atlas include a co-registered 3D autofluorescence data set to visualize the vasculature in context of the rest of the embryo. The reconstruction from the OPT data of the autofluorescence channel of the 19 somite embryo can be visualized as a volume rendering using a blue colour map. (C) Since the data sets are co-registered, both volume renderings from (A) and (B) can be visualized in the same space. All scale bars represent 100 microns.
Figure 4.
Stages of vascular development from 5 to 20 somites.
(A) In the 5 somite embryo the vasculature, volume rendered with a hot metal colourmap, is confined mostly to a completed DA, a developing heart, the allantois, the extraembryonic circulation, and clusters of PECAM-1 expressing cells dispersed throughout the cephalic mesenchyme. The autofluorescence is volume rendered with a transparent red colour map for overall positioning. (B) The 8 somite embryo has a rudimentary vascular plexus permeating the cephalic mesenchyme, the UV is elongating and the heart has initiated looping. (C) Occipital intersomitic vessels have begun to develop in the 11 somite embryo. (D) After turning, the cervical intersomitic vessels emerge in the 14 somite embryo. (E) The intersomitic vessels have begun to branch and connect together in the 16 somite embryo. (F) The vasculature of the 19 somite mouse embryo is a complicated but stereotypic structure. All scale bars represent 100 microns.
Table 2.
Embryos Imaged.
Figure 5.
Development of the cephalic plexus between the 5 and 20 somite embryo.
(A) The vasculature in the 5 somite mouse embryo is a series of disconnected clusters of PECAM-1-expressing cells. The DA and the heart are surface rendered red, PECAM-1 expression throughout the cephalic mesenchyme is surface rendered orange, and the autofluorescence of the mouse embryo is volume rendered with a hot metal colourmap. (B) By 11 somites, the cells have aggregated into a rudimentary vascular plexus. Larger vessels such as the PHV (blue arrowhead), the PMA (yellow arrowhead) and the ICA (green arrowhead) are visible (see also Supplemental Video S1). The PHV at this stage is a single large vessel that runs in an anterior-posterior direction starting from the cephalic flexure down to the first intersegmental vessel. (C) The cephalic plexus has remodelled into a more stereotypic pattern by 15 somites. The cephalic veins are easily distinguishable (green bracket). (D) At 19 somites the cephalic plexus has become more refined into recognizable structures. The cephalic veins are still visible at this stage (green bracket). All scale bars represent 100 microns.
Figure 6.
Connections between the embryonic and extraembryonic circulation in the early mouse embryo.
(A) At 7 somites, the omphalomesenteric arteries and veins (green) are large structures. The UA (dark pink) connects to the DA and traverses the allantois, but is quite small. The DA and heart is surface rendered red, and the embryo autofluorescence volume rendered with a hot metal colourmap. The development of the UV is shown in Figure 6. (B) At 14 somites, the UA is accompanied by a series of smaller vessels connecting to nearby vasculature. At this point, it is still smaller than the OA and OV. After turning, the omphalomesenteric vessels branch off on one side of the embryo, and the umbilical vessels the other. (C) By 19 somites the UA has grown in size and is approximately equal in diameter to the OA. (D) By 26 somites, the OA is significantly smaller in diameter than the UA, suggesting that the balance of flow begins to favour the feto-maternal interface through the UA at some time between E9.0 and E9.5. All scale bars represent 250 microns.
Figure 7.
The development of the umbilical vein in the mouse embryo.
(A) Discrete clusters of PECAM-1 expressing cells (dark pink) were evident along the length of the body wall immediately next to the junction of the body wall to amnion. The DA and heart is surface rendered yellow, and the embryo autofluorescence volume rendered with a hot metal colourmap. (B) The cells aggregated in a primarily anterior-posterior fashion beginning at the SV. (C) By 11 somites, the UV was almost complete, and had begun to develop a plexus which extended dorsolaterally. At the end of turning, the UV was complete and joined to the extraembryonic components of the vessel. (D) By 15 somites the UV is the second largest vessel in the embryo trunk. All scale bars represent 100 microns.
Figure 8.
Development of the occipital intersomitic vessels.
(A) The various stages of occipital intersomitic vessel development can be visualized in the 8 somite embryo (inset). The vessels surrounding somites 1 through 5 are segmented as DA (red), intersomitic vessels (pink) and the early cardinal vein (blue). The vessels initiate bilaterally, and an AVS originating from the DA connects to the cardinal vein (yellow arrowheads). An ISV then develops connecting the distal tip of the ISA to the cardinal vein (blue arrowhead). The ISA soon regresses (white arrowhead) leaving the vertebral artery connected to the cardinal vein. Somite number 4 is labelled as S-4. (B) The transient AVS have regressed (yellow arrowheads) by 15 somites, leaving only the expected dorsal ISA (pink), connected by the VTA (green), which connects via ISVs (light blue) to the ACV or CCV (blue). Somites numbers 1 and 4 are labelled as S-1 and S-4. All scale bars represent 100 microns.
Figure 9.
Development of the cervical intersomitic vessels.
(A) The various stages of cervical intersomitic vessel development can be segmented as visualized as surface renderings in the 16 somite mouse embryo. The vessels along the right side of the embryo and surrounding somites 1 through 16 are labelled as: DA (red), ISA (pink), ISV (blue), VTA, DLAV and PNVP (green), ACV and CCV (cyan), UV (dark pink), UV plexus (purple), and PCV (blue). Somites 1, 5, 10 and 15 are numbered as S-1, S-5, S-10 and S-15. (B) Branches of PECAM-1 expression originating from the tips of the ISAs (yellow arrowheads) were observed to turn towards the location of the future PCV. (C) A second branch from the ISA was also observed to extend in a predominantly anterior direction (pink arrowheads) to connect up with other ISAs, eventually forming the DLAV. PECAM-1 expression along the location of the expected PCV was observed to lag development of the ISAs and is discontinuous (yellow arrowheads). (D) The PNVP develops through remodelling of the VTA and DLAV. Branches initiate medially from the DLAV (pink arrowhead), begin to remodel into simple mesh (blue arrowhead), and eventually remodel into a fine structured capillary plexus surrounding the neural tube. Note at this stage that the first ISA has regressed. Scale bars represent 100 microns.