Mesoderm spreading in mutant embryos. (A-F) Ectodermal cell fate and mesoderm morphogenesis. Cross sections of embryos showing the development of the mesoderm in embryos with altered ectodermal cell fates. The embryos have been stained to reveal the di-phosphorylated form of the MAP kinase ERK. Each set of panels show an early and late timepoint during the flattening of the mesoderm. (A,B) Sections of brk, sog mutant embryos. (C,D) Sections of Egfr (top^18A) mutant embryos. (E,F) Sections of brk, sog; Egfr (top^18A) triple mutant embryos. Arrowheads indicate mesodermal cells in which high levels of the di-phosphorylated form of ERK are present. (G,H) Sections of embryos stained with antibodies directed against Twist to reveal mesodermal cells. (G) A section of a late dof, stg double mutant embryo. (H) Mesoderm development in a late htl mutant embryo expressing an activated form of Htl throughout the mesoderm.

Activation of MAPK and rescue of Eve-positive cells in the mesoderm of htl mutants. Embryos were stained with antibodies directed against dp-ERK (left), or Even-skipped (right). (A,H) Whole-mounts of wild-type embryos; (B-G,I-N) enlarged views of the trunk regions of htl mutant embryos. (B,I) The homozygous htl mutant phenotype. (C-G,J-N) The effect of expressing transgenes in the mesoderm of htl mutant embryos. (C,J) Htl; (D,K) an activated form of Htl; (E,L) an activated form of Btl; (F,M) an activated form of EGFR; (G,N) an activated form of PVR.

Rescue of MAPK activation and spreading at early stages of mesoderm morphogenesis. Sections of mutants expressing transgenes in the mesoderm stained to reveal activated MAPK. (A) Expression of Htl in the mesoderm of an htl mutant embryo completely rescues mesoderm spreading. (B) An activated form of the EGF receptor expressed in an htl mutant fails to promote interaction of the mesoderm with the ectoderm and does not stimulate activation of MAPK within the mesoderm at this developmental stage. (C) An activated form of Raf does not induce mesoderm spreading or the activation of MAPK in a dof mutant embryo. Note that we chose a slightly older embryo, showing that even at a stage when the mesoderm collapses onto the ectoderm no MAPK activation is detectable. (D-I) Sections from a young embryo are shown on the left of each row, with older embryos towards the right. (D-F) Expression of an activated form of Htl rescues both early contact of the mesoderm with the ectoderm and the activation of MAPK, notably at early stages this is only evident in the mesodermal cells that make contact with the ectoderm. (G-I) Expression of an activated form of Btl also rescues mesoderm spreading, but the onset of spreading occurs slightly later than in wild-type embryos. Activated MAPK is only observed in mesodermal cells that are in contact with the ectoderm.

The role of Rho-family GTPases in mesoderm spreading. (A) A section through an embryo that lacks the function of Rac2. The mesoderm flattens down onto the ectoderm as in wild type, but does not adhere to the ectoderm properly. (B) The effect of reducing the maternal dose of RhoA. The interaction of the mesoderm with the ectoderm is not affected, but the shape of the cells in the mesoderm is abnormal. (C,D) Embryos derived from mothers with reduced dose of (C) Rac1 and Rac2, or (D) Rac1, Rac2 and Mtl. In these mutants, contact between the mesoderm and the ectoderm fails to be established properly. (E) Embryos that lack the materal and zygotic function of the Rac effector Pak show no effect upon the initation of mesodermal spreading. (F) A section of a homozygous pebble mutant embryo. Contact of the mesoderm with the ectoderm does not occur, and MAPK activation is not observed within the mesoderm at this stage of development. Embryos shown in A-E were stained with Twist and the embryo in Fwas stained with anti-dp-ERK.

Mesoderm spreading is initiated in the absence of MAPK activation. Cross sections of embryos stained for Twist to reveal the development of the mesoderm (brown). (A) A wild-type embryo. (B) A homozygous dof¹ mutant embryo. (C-F) Mesoderm development in embryos derived from germline mutant clones lacking both the maternal and zygotic function of (C) Dsor1^LH110, (D) Ras85D^c40B, (E) csw^E(sev)1A-eOP and (F) dShc^13G.