Embryonic expression of spi-1l mRNA. (A) Spi-1l expression analysis by RT-PCR. −cDNA lane is a negative control in which cDNA was not added to the PCR. Lanes indicated with hours after fertilization (HPF) and days after fertilization (DPF) represent different developmental stages. K indicates adult kidney. −RT is a control reaction without the reverse-transcriptase enzyme added to the RT-PCR. β actin is used as a loading control. A dashed black line between the DPF and K lanes has been inserted to indicate a repositioned gel lane. (B-K) Whole-mount in situ hybridization analysis of spi-1l and pu.1 expression. The probe used is indicated to the left of the panels and the embryo's age marked by hours after fertilization (hpf). The embryos are oriented with anterior to the left and dorsal up. Red arrows point to the A-LPM region and blue arrows point to the P-LPM or PBI/CHT regions. All panels (B-K) except panels D and H show lateral views of embryos. (D,H) Posterior view of the same embryo as in panels C and G, respectively. Expression of spi-1l (B,C,E) and pu.1 (F,G,I) in the A-LPM. (D) spi-1l expression is absent in the P-LPM in contrast with pu.1, which is expressed in this region (H). (J) Expression of spi-1l throughout the yolk and in the posterior blood island (PBI). (K) Expression of spi-1l in the vessels of the eye and in the caudal hematopoietic tissue (CHT). (L) Single confocal section image at 28 hpf of fluorescent double in situ hybridization of spi-1l (green) and pu.1 (red). Right panel shows the merged image. Approximate region of the embryo scanned for the fluorescent double in situ hybridization is represented by the box in panel J. Hatched white circles indicate cells coexpressing spi-1l and pu.1 and yellow arrows indicate cells expressing only spi-1.