Fig. S1

(A,B) Expression of gata-2 in the zebrafish neural tube. At the 20-s stage, gata-2 is expressed in single cells of the ventral neural tube, as shown in (A) in dorsal view of trunk region. These cells are irregularly distributed along the AP axis, similar to tal2-positive cells (Fig. 1E) and located between MFP and motoneuronal domain (B). gata-2 is also expressed in cells located more dorsally within the neural tube (B). (C–J) Control experiment for nkx2.2a and nkx2.2b morpholino activity and specificity. To test the functionality of the nkx2.2a and nkx2.2b morpholino oligos, the target binding sequences of each morpholino were cloned in front of the GFP open reading frame in an RNA expression vector. Capped mRNA of nkx2.2a-GFP or nkx2.2b-GFP was injected into 2 to 4-cell stage embryos. This resulted in strong GFP expression in nkx2.2a-GFP RNA (A) and nkx2.2b-GFP RNA (B) injected embryos. In the same experiment, nkx2.2a or nkx2.2b morpholinos were injected into 1-cell stage embryos and the corresponding nkx2.2a-GFP RNA or nkx2.2b-GFP RNA was injected 20 to 30 minutes later into the same embryos, at the 2 to 4-cell stage. This resulted in a strong repression of GFP expression in nkx2.2a-GFP RNA and nkx2.2a morpholino coinjected embryos (E) as well as nkx2.2b-GFP RNA and nkx2.2b morpholino coinjected embryos (F). Embryos in the left bottom corner indicated with asterisk are injected with nkx2.2a-GFP RNA only (E) or nkx2.2b-GFP RNA only (F), to compare differences in GFP expression levels. These experiments show that the nkx2.2a and nkx2.2b morpholinos can both efficiently block translation of the target mRNA. To control the specificity of the nkx2.2a and nkx2.2b morpholinos, 5 mismatches were introduced into the target sequence of each morpholino before cloning them upstream of GFP in a RNA expression vector. Injection of RNA of MM nkx2.2a-GFP (G) and MM nkx2.2b-GFP (H) both resulted in strong expression of GFP. Injection of the nkx2.2a (I) or nkx2.2b morpholino (J) at the 1-cell stage and subsequent injection of mismatch GFP RNA in the same embryos at the 2 to 4-cell stage resulted in a similar strong expression of GFP. This shows that both morpholinos are not able to block translation of related sequences containing 5 mismatches or more. Therefore, both morpholinos are specific in binding of the RNA target sequence.