Supplemental Figure S1
-
Fig. S1. Morpholino control data. All morpholinos were used at a concentration of 150 µM in 0.1M KCl with 1% rhodamine. (A-C) Early prism stage larva from the same batch. (A) After injection with fluorescein-containing GeneTools standard control oligo (control MASO), imaged with a GFP filter set. (B) After injection with Lox1-MASO and imaged with a rhodamine filter set. Note the straight gut when compared with the control embryo imaged in A. (C) After injection with Lox2-MASO. Note that the phenotype is less severe than that shown in B. (D,E) Early pluteus larvae from the same batch, bright-field images. (D) After injection with GeneTools control MASO, larvae show no phenotype when compared with uninjected larva (not shown). (E) Larvae injected with Lox1-MASO show normal skeletogenesis and pigmentation patterns, but have an abnormal gut. (F) Injection with a GCM-MASO results in albino larvae that have otherwise normal morphology, including a fully differentiated gut. (G-I) Early pluteus larvae from the same batch imaged with DIC optics to highlight gut morphology. (G) Eggs injected with GeneTools control MASO develop a tripartite gut, with a well-developed posterior constriction (white arrows). (H) Eggs injected with Lox1-MASO develop into larvae with slightly smaller guts that lack the constriction marking the position where the posterior sphincter will form in normal embryos. (I) Introducing five mismatched nucleotides (see Materials and methods) into the Lox1-MASO sequence blocks the phenotypic effect, resulting in differentiation of the tripartite gut with a posterior constriction. (J) In vitro translation assay. In the absence of MASOs, a 42 kDa protein is translated (lane 1). Addition of 0.5 µM Lox1-MASO completely blocks this translation (lane 2). Neither the mismatched Lox1-MASO (mLox1), nor the GeneTools control morpholino (cont) at the same concentration affects the translation of SpLox. The luciferase translation control is shown in lane 5.
