Fig. 4

Fig. 4 Morpholino knockdown of lrdr1 in all embryonic cells or specifically in DFCs perturbs LR patterning without affecting midline or KV development. (A-C) The majority of embryos injected with either 1 ng lrdr1-AUG MO (B) or 1.6 ng lrdr1E2/I2 MO (not shown) at the one- to four-cell stages (lrdr1 morphants) and embryos in which 1 ng lrdr1-AUG MO + 1.6 ng lrdr1E2/I2 MO was co-injected into the yolk at mid-blastula stages to target DFCs (DFC^lrdr1MO embryos) (C) appeared similar to uninjected controls (A) at 2 dpf. (D-F) ntl expression in the notochord was normal in lrdr1 morphants (E) and DFC^lrdr1MO embryos (F), but expression of the normally left-sided markers lft1 and lft2 (D) was altered, including right-sided (E) and bilateral (F) expression. (G-H) KV cilia were detected by anti-Tubulin antibodies (red) in both lrdr1 morphants (H) and DFC^lrdr1MO embryos (I), and resembled cilia in wild-type controls (G). (J) RT-PCR analysis of RNA extracted from embryos between 80-90% epiboly was used to determine the efficacy of lrdr1E2/I2 MO in DFCs. Primers in lrdr1 exons 2 and 4 amplified a fragment of the gene that includes exons 2-4 and introns 2-3 from genomic DNA (lane 2). The predominant cDNA fragment amplified in uninjected embryos (lane 3) and embryos injected with 1 ng of control MO at the one- to four-cell stages (lane 4) corresponds to spliced lrdr1 mRNA (exons 2-4). lrdr1E2/I2 MO (1.6 ng) injected at the one- to four-cell stages (lane 5) or at mid-blastula stage (lane 6) resulted in the accumulation of lrdr1 transcripts that retain intron 2. This was not observed in embryos injected with lrdr1E2/I2 MO between dome stage and 30% epiboly (lane 7) or with 1.5 ng lft1 MO at mid-blastula stages (lane 8). Negative control reactions that lacked reverse transcriptase (lane 9) amplified only a genomic fragment that probably reflects genomic DNA contamination in RNA extracts. DNA ladder was loaded in lane 1. Diagrams on the right represent the amplicons confirmed by direct sequencing of RT-PCR products.