Fig. 4. Functional analysis reveals that fgf8 and fgf24 are together required for posterior mesodermal development. In situ hybridization and immunohistochemistry in 10-somite stage wild-type (A), fgf24^MO embryos (B), fgf8^- (C) and fgf8^-;fgf24^MO embryos (D). In A-D, pax2.1, krx20 and myod are stained purple, and Ntl protein is stained brown. At this stage in wild-type embryos, pax2.1 is expressed in the mid-hindbrain boundary (MHB), the otic placode and precursors of the pronephric ducts (black asterisks), krx20 in rhombomeres 3 and 5 (white asterisks), myod in adaxial cells (arrowhead) and a subset of cells in the forming somites (arrow), and Ntl protein in the developing notochord. At this stage, fgf24^MO embryos (B) are indistinguishable from wild type, while fgf8 mutants (C) have reduced expression of pax2.1 in the MHB, and a reduced number of cells expressing myod in the forming somites. By contrast, fgf8^-;fgf24^MO embryos (D) have significantly reduced numbers of myod- (arrow), pax2.1- (asterisks) and Ntl-expressing cells relative to wild-type, fgf24^MO and fgf8^-;fgf24^MO embryos. (E-H) Live wild-type and mutant embryos at 24 hpf. fgf24^MO embryos (F) are morphologically indistinguishable from wild-type embryos (E), while fgf8^- embryos (G) have a slightly shorter tail and a prominent MHB defect (arrowhead). fgf8^-;fgf24^MO embryos (H) have MHB defect (arrowhead), and produce significantly less posterior tissue than either fgf8 mutant or fgf24^MO embryos. Scale bars: in A, 50 µm for A-D; in E, 100 µm for E-H.