In this study, a significantly higher proportion of TDF-treated patients at week 48 achieved the primary end-point, compared with those treated with ADV (66% vs 12% in HBeAg-positive; and 71% vs 49% in HBeAg-negative; P < 0.001). At the end of treatment, 76% and 93% of the patients in the TDF group had HBV DNA levels of < 80 IU/mL,

compared with 13% and 63% of patients in the ADV group in both HBeAg-positive and HBeAg-negative patients, respectively (P < 0.001). Notably, 3% of HBeAg-positive patients treated with TDF lost HBsAg while no patients in the ADV-treated group encountered HBsAg loss. The drug resistance rate was 0% for TDF at weeks 48 and 72. The purpose of viral load measurement is Maraviroc very important during antiviral treatment. First, Dorsomorphin order it can measure the magnitude of viral load suppression, and second, it can detect viral breakthrough as early as possible.31 An on-treatment adjustment algorithm or the so-called ‘roadmap’ for NA therapy was proposed by several international experienced hepatologists in 2007 and was updated in 2008.32 Briefly, the serum HBV DNA

levels can be assessed at week 12 to check the initial antiviral response. If the serum HBV DNA levels declined less than 1 log10 IU/mL after antiviral agent therapy, it is called a ‘primary treatment failure,’ which is an indication to change treatment regimen at an early stage. The next early predictor of efficacy should be done at week 24 of therapy. This measurement is considered essential in the management of both HBeAg-positive and HBeAg-negative patients. This is because it was found to be the main predictor of subsequent treatment efficacy in terms of HBeAg seroconversion in HBeAg-positive patients, and of subsequent resistance. Notably, the

incidence of drug resistance in ETV or TDF therapy is too low to identify using any on-treatment predictors to date. At week 24, the declined serum HBV DNA levels should 上海皓元医药股份有限公司 further be categorized as complete (< 60 IU/mL), partial (60 to 2000 IU/mL), or inadequate (≧ 2000 IU/mL). In the face of suboptimal responses, further management strategies using LAM, Ldt or ADV are then based on the status of the virological response at week 12 and 24 as shown in Figure 1. Furthermore, periodical monitoring of HBV DNA levels should be done every 3–6 months to confirm adequate viral suppression and to detect viral breakthrough early. Once virological breakthrough has occurred, the recommendation is to use add-on therapy with a drug without cross-resistance. For patients with LAM resistance, ADV add-on therapy is highly effective at restoring viral suppression and preventing the emergence of resistance to ADV.33 Add-on therapy with TDF might be an even more attractive option for these patients.

All three patients (two viral and one alcoholic cirrhosis, who had nadolol stopped for impotence) remained abstinent during all follow-ups (range, 50-80 months), and none of them rebled. On the other hand, the two patients with dose reduction and loss of hemodynamic response rebled (at 13 and 17 months, respectively). Both patients in this

subgroup had mixed viral and alcohol cirrhosis and had resumed alcohol drinking prior to the rebleeding episode. Drug therapy in the remaining 43 patients was BGJ398 well tolerated, and all of them were kept during all follow-up on the same maximal tolerated doses they had at the moment of the second HVPG. To evaluate whether the differences between alcohol abstinents and nonabstinents in long-term response and outcomes may have been related to baseline differences or beta-blocker doses received compliance, a comparison of the most relevant among these parameters was performed (Table 4). Both subgroups were comparable. Cox multivariate analysis identified I-BET-762 in vivo loss of hemodynamic response (HR, 7.5; 95% CI, 2.1-27.0; P = 0.002) and history of previous variceal bleeding

(HR, 8.6; 95% CI, 2.1-34.5; P = 0.002) as risk factors for rebleeding, and viral etiology (HR, 4.1; 95% CI, 1.0-18.6; P = 0.05) and Child-Pugh score at 1 year (HR, 1.5; 95% CI, 1.1-2.1; P = 0.015) as main determinants of death/LT. It is currently accepted in clinical guidelines1, 2 that, in an HVPG-guided prophylactic strategy after a variceal hemorrhage, those patients meeting the accepted criteria

of hemodynamic response are reasonably 上海皓元医药股份有限公司 protected from rebleeding under drug therapy alone. This view is supported by longitudinal and randomized trials in which the responder status is based on HVPG measurements taken shortly (from 2 weeks to 3 months) after the index hemorrhage, with no subsequent reassessments during the usual 2-year follow up of these studies (range, 8-28 months).5, 6 Nevertheless, although it is assumed that these patients should be kept indefinitely on drug therapy only, there is no evidence that the initial hemodynamic response is maintained after this 2-year period. In this longitudinal observational study, we followed up for a median of 4 years a large cohort of hemodynamic responders treated with beta-blockers and nitrates after a variceal bleeding, and found that this response was lost in the long term in one out of three patients, an observation that was associated with a clear negative impact on their outcomes. This is the only available report in which sequential protocol HVPG measurements were performed in responders after a variceal bleeding to check their hemodynamic status. The previous study by Merkel et al.10 shared a similar approach and reported analogous results, but it was performed in patients with no previous history of bleeding, a setting with clearly lower baseline risk of progression of portal hypertension and incidence of related complications.

All three patients (two viral and one alcoholic cirrhosis, who had nadolol stopped for impotence) remained abstinent during all follow-ups (range, 50-80 months), and none of them rebled. On the other hand, the two patients with dose reduction and loss of hemodynamic response rebled (at 13 and 17 months, respectively). Both patients in this

subgroup had mixed viral and alcohol cirrhosis and had resumed alcohol drinking prior to the rebleeding episode. Drug therapy in the remaining 43 patients was isocitrate dehydrogenase inhibitor well tolerated, and all of them were kept during all follow-up on the same maximal tolerated doses they had at the moment of the second HVPG. To evaluate whether the differences between alcohol abstinents and nonabstinents in long-term response and outcomes may have been related to baseline differences or beta-blocker doses received compliance, a comparison of the most relevant among these parameters was performed (Table 4). Both subgroups were comparable. Cox multivariate analysis identified CHIR-99021 ic50 loss of hemodynamic response (HR, 7.5; 95% CI, 2.1-27.0; P = 0.002) and history of previous variceal bleeding

(HR, 8.6; 95% CI, 2.1-34.5; P = 0.002) as risk factors for rebleeding, and viral etiology (HR, 4.1; 95% CI, 1.0-18.6; P = 0.05) and Child-Pugh score at 1 year (HR, 1.5; 95% CI, 1.1-2.1; P = 0.015) as main determinants of death/LT. It is currently accepted in clinical guidelines1, 2 that, in an HVPG-guided prophylactic strategy after a variceal hemorrhage, those patients meeting the accepted criteria

of hemodynamic response are reasonably MCE protected from rebleeding under drug therapy alone. This view is supported by longitudinal and randomized trials in which the responder status is based on HVPG measurements taken shortly (from 2 weeks to 3 months) after the index hemorrhage, with no subsequent reassessments during the usual 2-year follow up of these studies (range, 8-28 months).5, 6 Nevertheless, although it is assumed that these patients should be kept indefinitely on drug therapy only, there is no evidence that the initial hemodynamic response is maintained after this 2-year period. In this longitudinal observational study, we followed up for a median of 4 years a large cohort of hemodynamic responders treated with beta-blockers and nitrates after a variceal bleeding, and found that this response was lost in the long term in one out of three patients, an observation that was associated with a clear negative impact on their outcomes. This is the only available report in which sequential protocol HVPG measurements were performed in responders after a variceal bleeding to check their hemodynamic status. The previous study by Merkel et al.10 shared a similar approach and reported analogous results, but it was performed in patients with no previous history of bleeding, a setting with clearly lower baseline risk of progression of portal hypertension and incidence of related complications.

Of the 10 protocol-defined failures identified in the study, postbaseline resistance testing was not performed in 5 patients because of low HCV RNA levels (<1,000 IU/mL by day 42 of the study). Of the remaining 5

selleck compound patients, 2 genotype 1b–infected patients (ANs 2957 and 3290) receiving placebo did not exhibit a greater than 2log10 decrease in HCV RNA during the dosing period (classified as “nonresponders”). RAVs were not detected in viruses from these patients by population sequencing (Table 4). The R155K variant was detected in viruses from 1 genotype 1a–infected patient (AN 3249), who exhibited a greater than 1log10 increase from nadir while receiving vaniprevir 800 mg QD (classified as a “breakthrough”) (Fig. 3). Two patients (1 infected with genotype 1a and 1 with genotype 1b) who received vaniprevir 300 mg BID exhibited a greater than 1log10 increase in HCV RNA from nadir after

completion of the 28-day vaniprevir dosing period (classified as “relapse after vaniprevir/placebo selleck products dosing”). RAVs R155K and D168V were detected by population sequencing in the genotype 1a–infected patient (AN 3242; Table 4). Clonal analysis revealed that these RAVs were not linked (data on file; Merck & Co., Inc., Whitehouse Station, NJ). RAVs D168V and D168T were identified in viruses from the genotype 1b–infected patient (AN 2966). In total, 70 patients provided consent medchemexpress for inclusion in the host genetic analysis, but 3 samples had insufficient template. The IL28B genotype analysis therefore compared genotype at loci rs12979860, rs12980275, and rs8103142 with RVR and SVR outcomes in 67 patients with samples available for testing from all treatment groups. IL28B genotype did not correlate significantly with SVR outcome (Supporting Table 3 and data not shown; P = 0.486 for rs12979860), in contrast to previous published work on response to Peg-IFN-α-2a/RBV treatment in a larger cohort of patients.19 IL28B genotype also did not associate

with the primary endpoint for this study, RVR (Supporting Table 4 and data not shown; P = 0.312 for rs12979860). In total, AEs were reported by 85 (90.4%) of the 94 treated patients across all treatment groups, with no notable between-group differences (Table 5). Among patients receiving vaniprevir, nausea (34.7%), headache (33.3%), influenza-like illness (22.7%), and fatigue (21.3%) were the most frequently reported AEs. These incidence rates were generally comparable with those among patients in the placebo group: nausea (26.3%), headache (36.8%), influenza-like illness (21.1%), and fatigue (36.8%). However, vomiting was reported by 40.0% (8 of 20) of the patients in the vaniprevir 600-mg BID group, compared to 0% (0 of 19) of the patients in the placebo group, and the difference of 40.0% (95% CI: 19.9%-61.

The other 28 patients who were followed up for at least 5 years postoperatively were enrolled in cohort B, which represented the period of the regional pilot study of the stool card screening program in Taiwan. Seventy-five BA patients were born between 2004 and 2005. After excluding one patient without Kasai operation, the 74 patients who were followed up for at least 3 years postoperatively were enrolled in cohort C, which represented the

nationwide screening data in Taiwan. Cohort B+C was the merged data of cohorts B and C and represented the era of the stool color card screening program. Statistical analyses were performed using Stata software (StataCorp LP, College Station, TX). A chi-square test was used to compare categorical variables, including age at Kasai operation <60 days, jaundice-free rates at 3 months after Kasai Roxadustat supplier operation, 3- Palbociclib and 5-year survival rates with native liver, 3- and 5-year jaundice-free survival rates with native liver, and 3-year and 5-year overall survival rates between different cohorts. Overall survival included those patients who survived with either their native liver

or a transplanted liver. Jaundice-free was defined as total serum bilirubin <2.0 mg/dL (34 μmol/L). Quality outcome was defined as jaundice-free survival with native liver. All survival time was calculated after the date of the Kasai operation. Relative odds ratios were computed using logistic regression models to compare the different factors affecting survival time. The Kaplan-Meier method and a log-rank test were also used to assess factors affecting survival. P < 0.05 was considered statistically significant. From 1990 until now, there was no systemic change in post-Kasai operation care except for the concept of prophylactic antibiotics use in Taiwan. Since 1997, most patients have been prescribed

medchemexpress with prophylactic antibiotics after operation. The data of the use of prophylactic antibiotics are collected by chart review, and this possible confounding factor is taken into consideration for analyses. The regimen of prophylactic antibiotics is trimethoprim-sulfamethoxazole (TMP-SMZ, 7 days per week) or neomycin (4 days per week). Our previous study reveals the superior effect of using prophylactic antibiotic versus not using it, and the equal effect for the prophylaxis of cholangitis between the two antibiotic regimens.9 There was no significant difference in the sex distribution between cohort A and cohort (B+C). The rates of Kasai operation performed before 60 days of age were 49.4% in cohort A and 65.7% in cohort B+C (P = 0.02). At 3 months after Kasai operation, the jaundice-free rate was significantly higher in cohort B+C than in cohort A (60.8% versus 34.8%; P < 0.001). The 3-year survival rates with native liver in cohort A and cohort B+C were 51.7% and 61.8%, respectively. The 3-year jaundice-free survival rate with native liver was significantly higher in cohort B+C than in cohort A (56.9% versus 31.

The other 28 patients who were followed up for at least 5 years postoperatively were enrolled in cohort B, which represented the period of the regional pilot study of the stool card screening program in Taiwan. Seventy-five BA patients were born between 2004 and 2005. After excluding one patient without Kasai operation, the 74 patients who were followed up for at least 3 years postoperatively were enrolled in cohort C, which represented the

nationwide screening data in Taiwan. Cohort B+C was the merged data of cohorts B and C and represented the era of the stool color card screening program. Statistical analyses were performed using Stata software (StataCorp LP, College Station, TX). A chi-square test was used to compare categorical variables, including age at Kasai operation <60 days, jaundice-free rates at 3 months after Kasai DNA-PK inhibitor operation, 3- JNK inhibitor and 5-year survival rates with native liver, 3- and 5-year jaundice-free survival rates with native liver, and 3-year and 5-year overall survival rates between different cohorts. Overall survival included those patients who survived with either their native liver

or a transplanted liver. Jaundice-free was defined as total serum bilirubin <2.0 mg/dL (34 μmol/L). Quality outcome was defined as jaundice-free survival with native liver. All survival time was calculated after the date of the Kasai operation. Relative odds ratios were computed using logistic regression models to compare the different factors affecting survival time. The Kaplan-Meier method and a log-rank test were also used to assess factors affecting survival. P < 0.05 was considered statistically significant. From 1990 until now, there was no systemic change in post-Kasai operation care except for the concept of prophylactic antibiotics use in Taiwan. Since 1997, most patients have been prescribed

medchemexpress with prophylactic antibiotics after operation. The data of the use of prophylactic antibiotics are collected by chart review, and this possible confounding factor is taken into consideration for analyses. The regimen of prophylactic antibiotics is trimethoprim-sulfamethoxazole (TMP-SMZ, 7 days per week) or neomycin (4 days per week). Our previous study reveals the superior effect of using prophylactic antibiotic versus not using it, and the equal effect for the prophylaxis of cholangitis between the two antibiotic regimens.9 There was no significant difference in the sex distribution between cohort A and cohort (B+C). The rates of Kasai operation performed before 60 days of age were 49.4% in cohort A and 65.7% in cohort B+C (P = 0.02). At 3 months after Kasai operation, the jaundice-free rate was significantly higher in cohort B+C than in cohort A (60.8% versus 34.8%; P < 0.001). The 3-year survival rates with native liver in cohort A and cohort B+C were 51.7% and 61.8%, respectively. The 3-year jaundice-free survival rate with native liver was significantly higher in cohort B+C than in cohort A (56.9% versus 31.

First, the authors mention that the relatively high misclassification of the “gold standard” (liver biopsy) makes it impossible for noninvasive tests to achieve high concordances. This is indeed true for noninvasive tests whose development was independent from liver histology (e.g., elastography). However, serum markers have been calibrated with direct reference to sets of liver biopsies. Therefore, the perfect serum marker would replicate even the misclassifications of the “golden” histological standard and could theoretically reach an AUROC (area under the receiver operating characteristic curve) of 1. Second,

the explosive development and overenthusiastic acceptance of noninvasive markers is not always supported by sufficient evidence and validation. In our meta-analysis on see more elastography, we exposed issues such as invalidated stiffness cutoffs for specific liver disease stages and low methodological quality in

the vast majority of published studies.2 In numerous studies, the maximum interval between elastography and liver biopsy was >3-6 months; in such cases, there is an erroneous assumption that fibrosis remains stable over these periods of time. Furthermore, from all publications, only six studies had both optimal histological and elastography measurements.2 Third, the authors correctly state that liver biopsy is more of a reference standard than a gold standard for assessing fibrosis. As we have pointed out before, histological “scores” of fibrosis are ordinal categories that incorporate

both architectural changes and fibrosis, 上海皓元医药股份有限公司 and Selleckchem CH5424802 have no quantitative relationship between them.3 Therefore, using them as continuous variables is inappropriate.3 Validation of noninvasive markers of “fibrosis” should ideally use quantitative histological measures. We have described such a measure, namely, collagen proportionate area, and correlated it with hepatic venous pressure gradient.4 More importantly, we evaluated its prognostic value with respect to patient outcome.5 Notably, collagen proportionate area performed better than Ishak staging and hepatic venous pressure gradient for predicting decompensation (AUROC = 0.97).5 In conclusion, the way forward involves carefully designed studies that validate noninvasive fibrosis markers against quantitative histological measures and/or clinical outcomes. Because we are ultimately treating patients, the clinical consequences of false positive and false negative classifications should be incorporated in the validation processes. Emmanuel A. Tsochatzis M.D.*, Giacomo Germani M.D.*, Andrew Hall M.D.†, Pinelopi Manousou M.D.*, Amar P. Dhillon M.D.†, Andrew K. Burroughs M.D., F.Med.Sci.*, * The Royal Free Sheila Sherlock Liver Centre and University Department of Surgery, Royal Free Hospital and University College London, London, UK, † Department of Cellular Pathology, UCL Medical School, Royal Free Campus, Rowland Hill Street, London, UK.

First, the authors mention that the relatively high misclassification of the “gold standard” (liver biopsy) makes it impossible for noninvasive tests to achieve high concordances. This is indeed true for noninvasive tests whose development was independent from liver histology (e.g., elastography). However, serum markers have been calibrated with direct reference to sets of liver biopsies. Therefore, the perfect serum marker would replicate even the misclassifications of the “golden” histological standard and could theoretically reach an AUROC (area under the receiver operating characteristic curve) of 1. Second,

the explosive development and overenthusiastic acceptance of noninvasive markers is not always supported by sufficient evidence and validation. In our meta-analysis on click here elastography, we exposed issues such as invalidated stiffness cutoffs for specific liver disease stages and low methodological quality in

the vast majority of published studies.2 In numerous studies, the maximum interval between elastography and liver biopsy was >3-6 months; in such cases, there is an erroneous assumption that fibrosis remains stable over these periods of time. Furthermore, from all publications, only six studies had both optimal histological and elastography measurements.2 Third, the authors correctly state that liver biopsy is more of a reference standard than a gold standard for assessing fibrosis. As we have pointed out before, histological “scores” of fibrosis are ordinal categories that incorporate

both architectural changes and fibrosis, 上海皓元医药股份有限公司 and check details have no quantitative relationship between them.3 Therefore, using them as continuous variables is inappropriate.3 Validation of noninvasive markers of “fibrosis” should ideally use quantitative histological measures. We have described such a measure, namely, collagen proportionate area, and correlated it with hepatic venous pressure gradient.4 More importantly, we evaluated its prognostic value with respect to patient outcome.5 Notably, collagen proportionate area performed better than Ishak staging and hepatic venous pressure gradient for predicting decompensation (AUROC = 0.97).5 In conclusion, the way forward involves carefully designed studies that validate noninvasive fibrosis markers against quantitative histological measures and/or clinical outcomes. Because we are ultimately treating patients, the clinical consequences of false positive and false negative classifications should be incorporated in the validation processes. Emmanuel A. Tsochatzis M.D.*, Giacomo Germani M.D.*, Andrew Hall M.D.†, Pinelopi Manousou M.D.*, Amar P. Dhillon M.D.†, Andrew K. Burroughs M.D., F.Med.Sci.*, * The Royal Free Sheila Sherlock Liver Centre and University Department of Surgery, Royal Free Hospital and University College London, London, UK, † Department of Cellular Pathology, UCL Medical School, Royal Free Campus, Rowland Hill Street, London, UK.

If antiviral therapy is not introduced due to concerns about tolerability, and ALT levels are abnormal, protective therapy (stronger neo-minophagen C; SNMC and/or ursodeoxycholic acid; UDCA) should be commenced.[1] Long-term low dose Peg-IFN (IFN) therapy is another option.[1] Recommendations Elderly patients are at high risk of hepatocellular carcinogenesis, and should commence antiviral therapy promptly. SMV + Peg-IFN + RBV triple therapy is the antiviral treatment of first choice in treatment-naïve elderly

patients. If antiviral therapy is not introduced and ALT levels are abnormal, protective therapy (SNMC, UDCA) should be commenced. Long-term low dose Peg-IFN (IFN) therapy is another option. Although the risk of hepatocellular carcinogenesis EPZ-6438 supplier is relatively low in non-elderly patients, the introduction of antiviral therapy is inevitably necessary in cases of advanced hepatic fibrosis, as in elderly patients. Akt cancer In general, SMV + Peg-IFN + RBV triple therapy should be administered to patients with advanced fibrosis. Also consider IFNβ + RBV combination therapy in patients with depressive symptoms.[1] The risk of carcinogenesis is considered lower in patients with mild fibrosis, so it may be reasonable to await the advent of newer agents with fewer adverse

reactions. Determination of IL28B SNP status may be of benefit when the decision whether to commence treatment is a difficult one. However, as mentioned above, clinical

trials of SMV + Peg-IFN + RBV triple therapy in treatment-naïve subjects reported SVR rates of approximately 80% in patients with IL28B minor alleles (Fig. 4). SMV-based triple therapy should therefore be considered in all patients who meet the criteria for antiviral therapy (ALT > 30 U/L or platelet count < 150 000/μL)[1] if treatment is likely to be tolerated, irrespective of IL28B SNP MCE公司 status. If antiviral therapy is not introduced, and ALT levels are abnormal, protective therapy should be commenced.[1] Recommendations Although the risk of hepatocellular carcinogenesis is relatively low in non-elderly patients, the introduction of antiviral therapy is inevitably necessary in cases of advanced hepatic fibrosis, as in elderly patients. Waiting for advent of newer agents with fewer adverse reactions is an option in patients with mild fibrosis. In general, SMV + Peg-IFN + RBV triple therapy should be administered to treatment-naïve non-elderly patients with advanced fibrosis. Although treatment may be delayed in non-elderly patients with mild fibrosis, SMV-based triple therapy should be considered in all patients who meet the criteria for antiviral therapy (ALT > 30 U/L or platelet count < 150 000/μL) if treatment is likely to be tolerated. If antiviral therapy is not introduced, and ALT levels are abnormal, protective therapy should be commenced.

A number of studies have shown that the limb-inducing signal originates in the axial mesoderm and is relayed from there to the LPM. In mouse, chick, and zebrafish, this signal is thought to be retinoic acid (RA), the bulk of which is synthesized by retinaldehyde dehydrogenase type2 (RALDH2) in early somites and the LPM.8–15 With respect to downstream effectors,

molecular studies have clearly shown that RA signaling from the zebrafish somitic mesoderm leads to the expression of the wnt2ba gene Crizotinib in vivo in the intermediate mesoderm, which then signals to the LPM and triggers tbx5 expression. Tbx5 is required for Fgf signaling in the fin bud that leads to prdm1 expression, which in turn triggers fgf10 and bmp2b expression.7, 16 In contrast, the identity of an initial hepatic inducer in vertebrates has yet to be validated genetically. In the first report to isolate a single gene regulating vertebrate liver Small molecule library specification, Ober et al.17 characterized an interesting zebrafish mutant called prometheus (prt). In prt embryos, the liver is absent or greatly reduced in size at 50 hours post-fertilization but may start to develop and “catch up” to normal size at a later stage. Positional cloning and further analysis revealed that the prt mutation

altered the wnt2bb gene (the second wnt2b gene) and that prt/wnt2bb was expressed in restricted bilateral domains in the LPM directly adjacent to the liver-forming endoderm. Subsequently, Shin et al.18 reported that Fgf and Bmp signaling pathways play important roles in zebrafish liver specification and raised the possibility that these molecules act downstream of Wnt2bb. However, the molecules that act upstream of Wnt2bb during liver specification MCE公司 remain to be identified. In this study, we carried

out a detailed characterization of our medaka hio mutants, whose signature phenotypes are a small liver and no pectoral fins. Our results define hio as a missense mutation of the raldh2 gene, the expression of which likely results in a nonfunctional RALDH2 protein that cannot support fin development. We also show that the hio mutation causes a retardation of liver budding that resembles that observed in zebrafish prt mutants, and that wnt2bb expression is undetectable in hio LPM. Our data suggest that the role of RA signaling in the specification of both liver and fins is to induce expression of wnt2b family genes. AP, anteroposterior; atRA, all-trans retinoic acid; ck19, cytokeratin19; cp, ceruloplasmin; E, embryonic day; hio, hiohgi; LPM, lateral plate mesoderm; MO, Morpholino; mRNA, messenger RNA; nls, neckless; nof, no-fin; PED6, N-([6-(2,4-dinitro-phenyl)amino]hexanoyl)-1-palmitoyl-2-BODIPY-FL-pentanoyl-sn-glycero-3-phosphoethanolamine; prt, prometheus; RA, retinoic acid; RALDH2, Retinaldehyde dehydrogenase type2. Medaka were raised and maintained under standard laboratory conditions at approximately 27°C.