Expression of enosf1b during development. A: enosf1b expression measured by RT-PCR. Primers for full length enosf1b (Table 1) were used to test for the presence or absence of enosf1b in staged embryo lysate. Bp = base pairs. B: Whole mount in situ hybridization (WISH) with sense and antisense DIG-labelled riboprobe during early embryonic development. Negative control for WISH, DIG-labelled sense riboprobe, is free of WISH colored precipitate. C: WISH for enosf1b during later embryonic development. Except for panel C, original magnification for all photomicrographs is in the lower right hand corner of each picture.

In vivo expression of enosf1b-EGFP compared to EGFP. A-C: Lateral views of representative embryos injected with in vitro transcribed mRNA encoding enosf1b-EGFP or EGFP. Original magnification for all photomicrographs is in the lower right hand corner of each picture. D: Comparison of the effect of injecting equal doses of enosf1b-EGFP or EGFP mRNA on time embryos reach 24 hpf or 48 hpf developmental stage. Data is average of three independent experiments; "n" values are total of the three experiments. Error bars are standard deviation. Original magnification for all photomicrographs is in the lower right hand corner of each picture.

Morpholino (MO) antisense oligonucleotide knockdown of enosf1b expression. A: MO phenotypes. Photomicrograph of uninjected, standard control (Std Ctrl), e10i10, or ATG2 MO-injected embryos at 48 hpf. Original magnification is in the lower right hand corner. B (top panel): Effect of injecting three morpholinos at two different doses compared to uninjected embryos. B (bottom panel): Morpholino phenotypes are not dependent on p53 status. Data in B is average of three independent experiments; "n" values reported are total of the three experiments. Embryos were scored for MO phenotypes at 48 hpf. C: RT-PCR of uninjected, standard control, and e10i10 injected single embryos. Morpholinos were both injected at a final concentration of 7 ng/embryo. Single embryos were collected at 48 hpf and processed through RT-PCR as described in Methods. Bp = base pairs. D (left panel): Rescue of e10i10 phenotype by coinjection of e10i10 morpholino and enosf1b-EGFP mRNA. D (right panel): Titration of ATG2 phenotype by coinjecting ATG2 morpholino and enosf1b-EGFP mRNA. See Methods for MO and RNA doses. Data is average of three independent experiments; "n" values are total of the three experiments. Error bars are standard deviation. Embryos were scored at 48 hpf. Unpaired Student′s t test was used to evaluate statistical significance of observed differences.

Characterizing the enosf1b knockdown phenotype. A: WISH for no tail and pax2a on 36 hpf morpholino-injected embryos. Red, grey, blue, and green arrows indicate pax2a staining of the pronephros, midbrain-hindbrain boundary, prospective cranial nerves, and thyroid primordium respectively. B (left panel): TUNEL staining on uninjected, std ctrl, e10i10, and ATG2 injected 48 hpf embryos. B (right panel): Mitotic index measured in uninjected, std ctrl, e10i10, and ATG2 injected embryos by antibody staining for phospho-histone H3. C: Morpholino-injected embryos have increased TUNEL staining but unchanged pH3 staining. Data is average of 3 tail counts per condition. Error bars are standard deviation. YSE = yolk sac extension. Original magnification for all photomicrographs is in the lower right hand corner of each picture.