Fically knocked down TTP protein expression, we designed a pair of non-overlapping MOs that target the second exon in the TTP pre-mRNA. The exonexclusion (EXC) MOs are complementary to either end of the second exon (Homatropine (methylbromide) site Figure 3A). These MOs interfere with the splicing and processing of the pre-mRNA resulting in the deletion of exon two from the mature product [16,17]. This alteration would result in a truncated protein product, if the aberrant mRNA were translated, due to a reading-frame shift caused by the exon exclusion and resulting in a pre-mature stop codon (Figure S1). The efficacy of splice inhibition by the EXC MOs was verified by RT-PCR amplification of a region spanning exon two and size verification by gel electrophoresis (Figure S2, primer locations shown as black arrows in Figure S1). The RT-PCR gel shows a complete loss of proper-size TTP mRNA in the EXC MO-treated embryos; instead the products are smaller 1326631 due to the exclusion of exon two from the final product. Additionally, embryos injected with the EXC MOs present with a significantly lower amount of TTP transcript (Figure S3), regardless of mRNA size (primers complimentary with regions not affected by the EXC MOs, orange arrows Figure S1). This loss of TTP mRNA is likely due to nonsense-mediated decay of the aberrant transcripts. Importantly, employing the EXC MOs compared with the TRN MO K162 biological activity yielded the same phenotype, namely abnormal head and eye formation, and a truncated tail. These results confirm that TTP knockdown using either MO targeting strategy disrupts the normal developmental processes. Non-specific p53 induction has been observed following injection with some MOs [18,19]. To confirm that the phenotype observed with TTP knockdown was not a result of off-target p53 induction, co-injections with a p53 knockdown MO were performed. The p53 MO co-injection did not affect the TTP phenotype (data not shown), and was not used in subsequent experiments.Disruption of TTP Expression using MorpholinosMOs were used to evaluate the requirement for TTP during zebrafish embryogenesis. Our experiments focused on a translational blocking MO (TRN), complementary to a region including the start codon of the mature TTP mRNA (Figure 3A). Embryos injected with the TRN showed significant developmental defects along the anterior/posterior axis at 1 dpf, including both cranial and tail malformations (p,0.0001 by ANOVA; p,0.001 TRN compared to CTR or NON, Tukey’s multiple comparison test, Figure 3C). These malformations were noted in .88 of TRN embryos by 1 dpf, compared with the embryos injected with the CTR (5.6 ) or non-injected (NON) embryos (1.7 , Figure 3B). It is important to note that these malformations occur in the same regions as the expression of TTP mRNA at 1 dpf (Figure 2). To determine the sequence of the observed malformations, embryos injected with TRN and CTR, or NON-controls were followed using time-lapse microscopy from ,6 hpf until ,24 hpf (Videos S1 and S2). Throughout blastula formation, epiboly and gastrulation (6?1 hpf), all embryos appeared to develop normally. At ,12 hpf, the nascent eye of embryos injected with TRN begin to display tissue darkening (Figure 4), indicating the initiation ofDiscussionThis study shows that expression of TTP is essential for early embryonic development in the zebrafish. The high degree of sequence similarity suggests a functional conservation between the human and zebrafish TTP orthologs. This conclusion is further supported by the.Fically knocked down TTP protein expression, we designed a pair of non-overlapping MOs that target the second exon in the TTP pre-mRNA. The exonexclusion (EXC) MOs are complementary to either end of the second exon (Figure 3A). These MOs interfere with the splicing and processing of the pre-mRNA resulting in the deletion of exon two from the mature product [16,17]. This alteration would result in a truncated protein product, if the aberrant mRNA were translated, due to a reading-frame shift caused by the exon exclusion and resulting in a pre-mature stop codon (Figure S1). The efficacy of splice inhibition by the EXC MOs was verified by RT-PCR amplification of a region spanning exon two and size verification by gel electrophoresis (Figure S2, primer locations shown as black arrows in Figure S1). The RT-PCR gel shows a complete loss of proper-size TTP mRNA in the EXC MO-treated embryos; instead the products are smaller 1326631 due to the exclusion of exon two from the final product. Additionally, embryos injected with the EXC MOs present with a significantly lower amount of TTP transcript (Figure S3), regardless of mRNA size (primers complimentary with regions not affected by the EXC MOs, orange arrows Figure S1). This loss of TTP mRNA is likely due to nonsense-mediated decay of the aberrant transcripts. Importantly, employing the EXC MOs compared with the TRN MO yielded the same phenotype, namely abnormal head and eye formation, and a truncated tail. These results confirm that TTP knockdown using either MO targeting strategy disrupts the normal developmental processes. Non-specific p53 induction has been observed following injection with some MOs [18,19]. To confirm that the phenotype observed with TTP knockdown was not a result of off-target p53 induction, co-injections with a p53 knockdown MO were performed. The p53 MO co-injection did not affect the TTP phenotype (data not shown), and was not used in subsequent experiments.Disruption of TTP Expression using MorpholinosMOs were used to evaluate the requirement for TTP during zebrafish embryogenesis. Our experiments focused on a translational blocking MO (TRN), complementary to a region including the start codon of the mature TTP mRNA (Figure 3A). Embryos injected with the TRN showed significant developmental defects along the anterior/posterior axis at 1 dpf, including both cranial and tail malformations (p,0.0001 by ANOVA; p,0.001 TRN compared to CTR or NON, Tukey’s multiple comparison test, Figure 3C). These malformations were noted in .88 of TRN embryos by 1 dpf, compared with the embryos injected with the CTR (5.6 ) or non-injected (NON) embryos (1.7 , Figure 3B). It is important to note that these malformations occur in the same regions as the expression of TTP mRNA at 1 dpf (Figure 2). To determine the sequence of the observed malformations, embryos injected with TRN and CTR, or NON-controls were followed using time-lapse microscopy from ,6 hpf until ,24 hpf (Videos S1 and S2). Throughout blastula formation, epiboly and gastrulation (6?1 hpf), all embryos appeared to develop normally. At ,12 hpf, the nascent eye of embryos injected with TRN begin to display tissue darkening (Figure 4), indicating the initiation ofDiscussionThis study shows that expression of TTP is essential for early embryonic development in the zebrafish. The high degree of sequence similarity suggests a functional conservation between the human and zebrafish TTP orthologs. This conclusion is further supported by the.