Regulation of spi-1l expression. (A-C) Lateral view of the yolk (YK) region at 36 hpf of cloche wild-type sibling, cloche, and injected cloche embryo, respectively. (A) Expression of spi-1l in a wild-type sibling from a cloche intercross. (B) Expression of spi-1l is absent in cloche mutant embryo. (C) Partial rescue of spi-1l expression in cloche mutant injected with pu.1 mRNA. (D-Q) Lateral view of embryos. Probes for genes, morpholino, and mRNA used are as indicated. Red and blue arrows indicate mRNA expression on the YK and in the CHT regions, respectively, of wild-type embryo. (D-I) Etsrp as an upstream component of spi-1l and pu.1. (D) There are 87 plus or minus 14 spi-1l cells and (E) 106 plus or minus 14 pu.1 cells expressed in control uninjected embryos at 28 hpf. Etsrp morphants lack spi-1l (F) and pu.1 (E) expression. Ectopic etsrp mRNA expression causes increase in the number of spi-1l cells to 114 plus or minus 27 (H), and pu.1 cells to 152 plus or minus 30 (I). (J-O) Lateral view of spi-1l and pu.1 morphants at 28 hpf. Wild-type expression of pu.1 (J) and spi-1l (M) in control uninjected embryos. (K) Expression of pu.1 is unaffected in spi-1l morphants. (L) As a control, expression of pu.1 is lost in pu.1 morphants. (N) As a control, expression of spi-1l is severely reduced in spi-1l morphants. (O) Expression of spi-1l is nearly absent in pu.1 morphants. (P) Wild-type expression of spi-1l at 22 hpf. (Q) Wild-type embryo injected with pu.1 mRNA has increased number of spi-1l–expressing cells. (R) Graph demonstrating the increase in the number of spi-1l–positive cells in pu.1-injected embryos compared with wild-type siblings. We determined that the number of spi-1l–positive cells was 59 plus or minus 9 in the injected embryos compared with 37 plus or minus 4 in uninjected siblings. A total of 30 embryos from 3 independent experiments were counted for each condition.