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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00669

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Spatial and temporal patterns of ERK signaling during mouse embryogenesis

Laura Beth Corson¹, Yojiro Yamanaka¹, Ka-Man Venus Lai^1,2,* and Janet Rossant^1,2,

¹ Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
² Department of Molecular and Medical Genetics, University of Toronto, ON, Canada

View larger version (58K): [in a new window]   Fig. 1. Spatial and temporal patterns of phosphorylated ERK in postimplantation mouse embryos. (A-J) Embryos from indicated stages (5.0-10.5 dpc) were fixed and stained for dp-ERK immediately upon dissection to preserve endogeneous domains of signaling. (K,L) Embryos (9 dpc) were cultured in the absence (K) or presence (L) of 50 µM U0126 (MEK inhibitor) for 45 minutes prior to dp-ERK labeling to confirm staining was specific for the phosphorylated form of ERK. (A-F) Monochrome epifluorescence images of Cy3-dpERK labeled embryos. (G-L) DAB-HRP-dp-ERK stained embryos. Sustained ERK activation (color-coded with red lettering) is observed in the ectoplacental cone (epc), extra-embryonic ectoderm (exe), branchial arches (ba), frontonasal processes (fnp), tailbud (tb), limb buds (lb), forebrain (fb), midbrain-hindbrain boundary (mhb), foregut (fg) and liver primordia (l). Brief ERK activation (pink lettering) is seen in the distal tip of the epiblast (dt), allantoic bud (al), blood island mesoderm (bi), headfold mesoderm (hfm), heart primordia (h), sinus venosus (sv), dorsal aorta (da), intersomitic vessels (iv), eye primordia (ey), ear primordia (er), nasal pits (np), caudal region of somites (cds) and ganglia (g). Scale bars: 50 µm in A-F; 400 µm in G-L.

View larger version (124K): [in a new window]   Fig. 2. Transient ERK signaling in intersomitic blood vessels. Low magnification view of Cy3-dpERK stained embryos at 9 dpc (A) and 9.5 dpc (B). (C) Higher magnification of region indicated in B. (D-F) Confocal composites of dp-ERK staining in somitic region at various stages of somite development. Dp-ERK staining is red. Nuclei (YOYO-1 staining) are shown in blue. Dp-ERK positive intersomitic vessels growing between newly formed somites are seen in D,E. In more mature blood vessels associated with more mature somites, dp-ERK staining is no longer associated with intersomitic vessels but rather with the caudal region of somites (B,F). da, dorsal aorta; iv, intersomitic vessels; cds, caudal portion of somite; psm, presomitic mesoderm. Scale bars: 500 µm.

View larger version (115K): [in a new window]   Fig. 3. ERK activation in peripheral nervous system. Dp-ERK stained 9.0 dpc (A) and 10.5 dpc (D) embryos with yellow arrowheads indicating neural crest and black arrowheads indicating nerve tracts of glossopharyngeal and vagus ganglia. Broken boxes approximate positions of the higher magnification panels indicated and broken lines in B and D approximate position of sections shown in C and E-H, respectively. White arrowhead (F) indicates dp-ERK positive dorsal root ganglia and white arrow (H) indicates dp-ERK staining in sensory nerve root found along the length of the neural tube at this stage. Scale bars: 600 µm.

View larger version (113K): [in a new window]   Fig. 4. FGFR-dependent ERK signaling in extra-embryonic ectoderm (exe), heart primordia (h), frontal nasal process (fnp), midbrain-hindbrain boundary (mhb), eye primordia (ey), branchial arches (ba) and limb buds (lb) at 6.5, 8.0 and 10.5 dpc. Prior to dp-ERK staining, embryos were cultured in the absence (A-C) or presence (D-F) of the FGFR inhibitor (40 µM SU5402) for 30-90 minutes. Note that culturing embryos alters dp-ERK patterns somewhat causing more diffuse boundaries of dp-ERK, some ectopic regions of dp-ERK and a loss of dp-ERK staining in some weaker regions of signaling. Dp-ERK staining in the ectoplacental cone (epc) and left heart ventricle (hv) were unaffected by the FGFR inhibitor. Scale bars: 50 µm in A,D; 100 µm in B,E; 700 µm in C,F.

View larger version (50K): [in a new window]   Fig. 5. Atlas of dp-ERK during mouse embryogenesis. Schematic overview of prominent ERK signaling domains during mouse development. Regions of FGFR signaling are colored red, non-FGFR signaling regions are blue and unclassified dp-ERK domains are purple. Note that regions of weak or transient ERK activation were not maintained in culture and thus no conclusion can be made about the effect of the FGFR inhibitor and hence the role of FGFR signaling in these domains. Diagrams of mouse embryos are adapted from http://genex.hgu.mrc.ac.uk

View larger version (122K): [in a new window]   Fig. 6. Subcellular localization of activated ERK in mouse embryos. (A,E,I,M) Whole-mount views of dp-ERK stained embryos at 10.5, 8.5, 9.0 and 6.5 dpc, respectively. High magnification confocal and deconvolution sections of these dp-ERK stained embryos reveal cytoplasmic subcellular localization in endogeneous signaling domains such as limb bud mesenchyme (B-D) and forebrain neural ectoderm (F-H), but cytoplasmic and nuclear staining in mitotic cells (N-P) and regions of injury (J-L). (B-D,F-H,J-L) Confocal sections of dp-ERK/YOYO1 stained embryos in the regions indicated in A,E,I, respectively. (N-P) Deconvolution images of a dp-ERK/Hoechst-labeled embryo sectioned transversely at the position indicated in M. Arrowheads indicate mitotic dp-ERK positive cells. Arrows indicate non-mitotic cells that exhibit both cytoplasmic and nuclear dp-ERK staining in the region of torn somites. Scale bars: 700 µm in A; 250 µm in E; 400 µm in I; 75 µm in M.

View larger version (46K): [in a new window]   Fig. 7. FGFR-ERK signaling in limb buds. Dp-ERK staining in forelimb region at 9.0 dpc (A), 9.5 dpc (B,C) and 10.5 dpc (D). Initially, dp-ERK is detected in surface ectoderm (se) overlying lateral plate mesoderm (A). As limb bud outgrowth continues (B-D), a gradient of dp-ERK is seen in mesenchyme directly beneath the apical ectodermal ridge (AER). Transverse sections through hindlimbs at 9.5 dpc (E), 10.0 dpc (F) and 10.5 dpc (G). (H,I) Domains of FGF-dependent dp-ERK in developing limbs and proposed directions of FGF signaling shown with arrows. Fgfr1 expression is shown in yellow, Fgfr2 expression in brown and dp-ERK regions in red. Blue arrows indicate FGF signaling in the proximodistal directions, which is proposed by current models of limb development (Martin, 1998). Dp-ERK staining in the dorsal and ventral surface ectoderm suggest the possibility of mesenchyme to surface ectoderm FGF signaling or autocrine signaling within the surface ectoderm, as indicated by green arrows. Gradients of dp-ERK in mesenchyme beneath the surface ectoderm suggests FGF signaling from the surface ectoderm to mesenchyme (purple arrows). Scale bars: 50 µm.

View larger version (97K): [in a new window]   Fig. 8. FGFR-ERK signaling in extra-embryonic ectoderm. Confocal images (400x) of dp-ERK stained embryos superimposed on DIC images at (A) 5.5 dpc, (B) 6.0 dpc, (C) 7.0 dpc and (D-E) 7.5 dpc. The entire extra-embryonic region is shown in A,B whereas the EPC has been removed in C-E. White brackets indicate extent of dp-ERK staining in extra-embryonic ectoderm. (E) Sagittal section of D showing dp-ERK in both the internal and peripheral layers of the extra-embryonic ectoderm. (F-H) Schematic diagram depicting the Fgfr2-expressing extra-embryonic ectoderm in yellow, the domains of dp-ERK in the extra-embryonic ectoderm in red and the location of the proposed FGF signaling sources in blue. Boxes in F-H demarcate the region of embryo shown in confocal images (A,B,E). Initially, a gradient of dp-ERK can be seen throughout the entire extra-embryonic ectoderm at 5.5 dpc (A,F). This gradient becomes reduced and spans a distance of six to nine cell diameters by 6.0 (B,G). The upper boundary sharpens and the band of dp-ERK is reduced to four to six cell-diameters by 7.0 (C) and to two to four cell-diameters by 7.5 dpc (D,H). In situs show expression pattern of Fgfr2 throughout the extra-embryonic ectoderm at 6.5 (I) and 7.5 dpc (J). In situs of Eomes at 5.5 (K), 6.5 (L) and 7.5 dpc (M) correspond closely to regions of ERK activation. (I-M) Adapted, with permission, from Ciruna and Rossant (Ciruna and Rossant, 1999).