Table 1.
Limits of region A and B in early primitive streak embryos.
Figure 1.
Ingression and early migration of cells from region A and B.
(A) Bright field and fluorescent image of an early primitive streak stage embryo (HH3+, PS4) labelled with DiI (red) and DiO (green) along the anterior-middle primitive streak. (B) Close up image of fluorescently labelled cells within region A and B. (B–F) Time-lapse microscopy of the embryo in (A) revealed early movement patterns of labelled cells in region A and region B (n = 7). Panels represent still images obtained from time-lapse microscopy at the time points indicated in hours (h). (C–F) Cells from region A and B were found to migrate away from the primitive streak bilaterally in an anterior-lateral direction. Arrows in (E–F) indicate movement trajectories of cells from region A (red arrow) and region B (green arrow). Scale bars (A) and (F) = 100 µm, (B–E) = 50 µm.
Figure 2.
Location of fluorescent descendants of cells from region A and B at stage HH8.
(A–B) Bright field and fluorescent images of labelled embryo at stage HH8 and HH8+ showing the location of fluorescently labelled cells. Descendants from region A are labelled with DiI (red) and descendants from region B are labelled with DiO (green). White arrow heads in (A) mark where the intestinal portals are situated. At HH8 cells from region A were located in the intermediate mesoderm and head mesenchyme, whilst cells from region B were located in the intestinal portals and in the heart forming region. (Bi–Biv) Sections of embryo in (B) demonstrated that cells from region A (red) were located in the cranial paraxial mesoderm (CPM) and cells from region B (green) were located in the splanchnic mesoderm (SpM). (Bi–Biii) Colour channels of sections have been separated into DiI/red on the left hand side panels and DiO/green on the right hand side panels for clearer observation of cell location. (C) Labelled cells from region B were also observed in the dorsomedial region of the splanchnic mesoderm, marked with an oval (n = 10). (D) DiI labelled descendants of cells from a region of the primitive streak just caudal to the limits of region B were located in the extraembryonic mesoderm (red cells), (n = 21). Scale bars = 50 µm.
Figure 3.
Descendants of cells from region B are located within the splanchnic mesoderm and express Isl1.
(A) Bright field and fluorescent image showing the location of fluorescently labelled descendants of cells from region B at stage HH8 (red cells). (Ai–Aii) Sections through embryo in (A) were stained for Isl1 protein expression (green). DAPI signal (blue) has been superimposed to provide a clearer picture of the morphology of the tissue. White arrows in (Ai–Aii) point to the splanchnic mesoderm where fluorescent cells (DiI intercalated within the cell membrane) that express Isl1 are located. Neural tube (nt), (n = 11). Scale bar = 50 µm.
Figure 4.
Descendants of cells from region A are located within the CPM at stage HH9-10.
(A) Bright field and fluorescent image showing the location of fluorescently labelled descendants of cells from region A at stage HH9-10 (red cells). (Ai) Section through embryo in (A) showing location of labelled cells and Isl1 protein expression (green). DAPI signal (blue) has been superimposed to provide a clearer picture of the morphology of the tissue. White arrows in (Ai) point to the CPM where fluorescent cells are located. (B) Bright field and fluorescent image of embryo at stage HH9-10 showing location of CAG-GFP labelled cells (green cells) which were grafted within region A of the primitive streak at stage HH3 though to HH3+. (Bi–iv) Section of grafted embryo (B). (Biii–iv) Sections stained for Isl1 protein expression (red). Arrows indicate the location of CAG-GFP cells in the CPM. At stage HH9-10 CAG-GFP labelled cells grafted to region A were located within the CPM (n = 5). Splanchnic mesoderm (SpM), neural tube (nt), heart (ht). Scale bars (Ai), and (Biii–iv) = 50 µm, (Bi–ii) = 100 µm.
Figure 5.
Descendants of cells from region B are located within the splanchnic mesoderm and heart.
(A–Bi) Sections from labelled stage HH11-12 embryos. (A–B) Sections at the level of the caudal pharynx showed that fluorescent descendants of cells from region B (red cells) were located in the splanchnic mesoderm and within the heart (ht). (B–Bi) Isl1 protein expression is indicated (green). Images were taken on a confocal microscope, (C) DAPI signal (blue) has been superimposed to provide a clearer picture of the morphology of the tissue. (Bi) Higher magnification of the boxed region in (B). Labelled cells located in the splanchnic mesoderm with DiI intercalated within the cell membrane express Isl1. Neural tube (nt), (n = 20). Scale bars (A) = 50 µm, (B) = 60 µm, (Bi) = 13 µm.
Figure 6.
CAG-GFP cells grafted within region B are located in the splanchnic mesoderm and heart.
(A–B) Bright field and fluorescent images of embryos at stage HH6 (A) and HH8- (B) showing location of CAG-GFP labelled cells (green cells) which were grafted within region B of the primitive streak at stage HH3 though to HH3+. White arrow heads in (B) mark the location of the intestinal portals. (C) Section of grafted embryo stained for Isl1 protein expression (red). (D) DAPI signal (blue) has been superimposed to provide a clearer picture of the morphology of the tissue. At stage HH11-12 CAG-GFP labelled cells grafted to region B were located within the pharyngeal endoderm (cells marked with white asterix), splanchnic mesoderm and the heart (ht). Images were taken on confocal microscope. Neural tube (nt), (n = 19). Scale bar = 60 µm.
Figure 7.
Region B is the ingression site of CPCs located within the splanchnic mesoderm and heart.
(A–Bi) Sections of labelled embryos at stage HH11-12 stained for Isl1 protein expression (green). (A) Fluorescent descendants of cells (red cells) labelled in rostral subdivisions within region B (Br) are located within the splanchnic mesoderm (indicated with white arrow heads) and the heart (ht). (B) Fluorescent descendants of cells (red cells) labelled in caudal subdivisions within region B (Bc) are located within the splanchnic mesoderm (indicated with white arrow heads) and the heart (ht). (Ai, Bi) DAPI signal (blue) has been superimposed to provide a clearer picture of the morphology of the tissue. Images taken on confocal microscope, neural tube (nt) (n = 31). Scale bars = 60 µm.
Figure 8.
Fate mapping of fluorescent descendants of cells from region A and B to stage HH15.
(A) Bright field image of an embryo grown in MC culture. (B) Black and white fluorescent image showing the location of fluorescently labelled descendants of cells from region A. (Bi–Bii) Sections of (B) demonstrate that cells from region A are located in the first branchial arch (ba1), (n = 5). (C) Black and white fluorescent image showing the location of fluorescently labelled descendants of cells from region B (Ci–Cii) Sections of (C) show that cells from region B are locate in the splanchnic mesoderm and the outflow tract (oft), (n = 6). (Cii) Isl1 protein expression is shown in green and (Ciii) DAPI signal (blue) has been superimposed to provide a clearer picture of the morphology of the tissue. (C–Ciii) Images were taken on confocal microscope. Neural tube (nt), heart (ht), otic vesicle (oc), pharynx (ph).Scale bars (Bi–Bii, Ci) = 50 µm, (Cii) = 60 µm.
Figure 9.
Model illustrating the fate of cells derived from regions A and B.
The location of regions A (red) and B (green) within the anterior primitive streak of HH3 and HH3+embryos and their migration patterns are indicated by red and green arrows. At HH8 ‘A’ derived cells are located in the CPM and ‘B’ derived cells are found in splanchnic mesoderm. This is schematically shown in a section. By HH11-12 region ‘B’ descendants have segregated into Isl1 positive cells, which are found in the pharyngeal endoderm and splanchnic mesoderm, and an Isl1 negative lineage found in the linear heart tube. Region ‘A’ descendants continue to be found in CPM (not indicated). By HH15 the region ‘B’ derived cells are found in the outflow tract, heart and splanchnic mesoderm, whilst region ‘A’ descendants are seen in the cranial paraxial mesoderm, pharyngeal arches and outflow tract.