The effect of nintedanib on health-related quality of life in Japanese patients with progressive fibrosing interstitial lung diseases: A subset analysis of the INBUILD trial.

BACKGROUND: In previous Japanese subgroup/subset analyses of the global INBUILD trial, nintedanib reduced the annual rate of forced vital capacity (FVC) decline and the risk of disease progression in patients with progressive fibrosing interstitial lung diseases (PF-ILDs). This exploratory subset analysis assessed the effect of nintedanib on symptoms and impacts of pulmonary fibrosis in Japanese patients with PF-ILDs, including those with usual interstitial pneumonia (UIP)-like fibrotic pattern on high-resolution computed tomography (HRCT). METHODS: This analysis included Japanese patients who received at least one dose of study treatment in the randomized, double-blind, placebo-controlled INBUILD trial. The Living with Pulmonary Fibrosis (L-PF) questionnaire was used to assess pulmonary fibrosis symptoms and impacts (higher scores indicated greater impairment) at baseline and weeks 12-52. RESULTS: In total, 108 Japanese patients (nintedanib: n = 52; placebo: n = 56) were included; 84 patients had UIP-like fibrotic pattern on HRCT. In the total Japanese subgroup and in those with UIP-like fibrotic pattern, numerically greater increases in L-PF total, symptoms total, symptoms fatigue domain, and impacts scores were observed in the placebo group than in the nintedanib group at all timepoints, starting from week 12. A numerically greater increase in the symptoms dyspnea domain score was observed with placebo versus nintedanib starting from week 36. Throughout the study, the symptoms cough domain score increased in the placebo group but decreased in the nintedanib group. CONCLUSIONS: Our findings demonstrate that nintedanib has the potential to reduce the worsening of symptoms and impacts of pulmonary fibrosis in Japanese patients with PF-ILDs.

Effects of nintedanib on symptoms in patients with progressive pulmonary fibrosis.

BACKGROUND: Dyspnoea and cough can have a profound impact on the lives of patients with pulmonary fibrosis. We investigated the effects of nintedanib on the symptoms and impact of pulmonary fibrosis in patients with progressive pulmonary fibrosis (PPF) in the INBUILD trial using the Living with Pulmonary Fibrosis (L-PF) questionnaire. METHODS: Patients had a fibrosing interstitial lung disease (ILD) (other than idiopathic pulmonary fibrosis) of >10% extent on high-resolution computed tomography (HRCT) and met criteria for ILD progression within the prior 24 months. Patients were randomised 1:1 to receive nintedanib or placebo. Changes in L-PF questionnaire scores from baseline to week 52 were assessed using mixed models for repeated measures. RESULTS: In total, 663 patients were treated. Compared with placebo, there were significantly smaller increases (worsenings) in adjusted mean L-PF questionnaire total (0.5 versus 5.1), symptoms (1.3 versus 5.3), dyspnoea (4.3 versus 7.8) and fatigue (0.7 versus 4.0) scores in the nintedanib group at week 52. L-PF questionnaire cough score decreased in the nintedanib group and increased in the placebo group (-1.8 versus 4.3). L-PF questionnaire impacts score decreased slightly in the nintedanib group and increased in the placebo group (-0.2 versus 4.6). Similar findings were observed in patients with a usual interstitial pneumonia-like fibrotic pattern on HRCT and in patients with other fibrotic patterns on HRCT. CONCLUSION: Based on changes in L-PF questionnaire scores, nintedanib reduced worsening of dyspnoea, fatigue and cough and the impacts of ILD over 52 weeks in patients with PPF.

Nintedanib in Asian patients with progressive fibrosing interstitial lung diseases: Results from the INBUILD trial.

BACKGROUND AND OBJECTIVE: In the INBUILD trial in patients with progressive fibrosing interstitial lung diseases (ILDs), nintedanib reduced the rate of decline in forced vital capacity (FVC) with an adverse event profile characterized mainly by gastrointestinal events. We analysed the effects of nintedanib in the subset of Asian subjects. METHODS: Subjects with fibrosing ILDs other than idiopathic pulmonary fibrosis who had shown progression of ILD at any time within the prior 24 months despite management deemed appropriate in clinical practice were randomized to receive nintedanib or placebo. We analysed the rate of decline in FVC (ml/year) over 52 weeks in all Asian subjects and in Asian subjects with a usual interstitial pneumonia (UIP)-like fibrotic pattern on high-resolution computed tomography (HRCT). RESULTS: One hundred sixty-four subjects in the INBUILD trial were of Asian race. The rate of decline in FVC (ml/year) over 52 weeks in this subgroup was -116.8 in the nintedanib group and -207.9 in the placebo group (difference: 91.0 [95% CI: 8.1, 173.9]; nominal p = 0.03). In Asian subjects with a UIP-like fibrotic pattern on HRCT, the rate of decline in FVC (ml/year) over 52 weeks was -130.1 in the nintedanib group and -224.2 in the placebo group (difference: 94.1 [5.5, 182.7]; nominal p = 0.04). Adverse events led to treatment discontinuation in 19.0% of the nintedanib group and 13.8% of the placebo group. CONCLUSION: In Asian patients with progressive fibrosing ILDs, nintedanib reduced the rate of decline in FVC with adverse events that were manageable for most patients.

Effects of nintedanib on disease progression and safety in Japanese patients with progressive fibrosing interstitial lung diseases: Further subset analysis from the whole INBUILD trial.

BACKGROUND: A previous subgroup analysis of data from the INBUILD trial showed that nintedanib reduced the annual rate of decline in forced vital capacity (FVC) in Japanese patients with progressive fibrosing interstitial lung diseases (PF-ILDs). The safety profile of nintedanib over 52 weeks in Japanese patients was similar to that of the overall population. METHODS: Using data from 108 Japanese patients with PF-ILDs who had received at least 1 dose of study medication in the INBUILD trial, we evaluated the effect of nintedanib on disease progression and assessed the safety profile over the whole trial period (i.e., a longer duration than the prior analysis) compared with placebo. ILD progression was defined as an absolute decline in FVC ≥10% predicted vs baseline. RESULTS: Over the whole trial, in Japanese patients with PF-ILDs, nintedanib numerically lowered the risk of progression of ILD or death (hazard ratio [HR], 0.66; 95% confidence intervals [CI]: 0.37, 1.16), acute exacerbation of ILD or death (HR, 0.28; 95% CI: 0.09, 0.83), and death (HR, 0.41; 95% CI: 0.11, 1.51). The most common adverse event over the whole trial in nintedanib-treated Japanese patients was diarrhea, which was manageable for most patients by dose reduction and interruption. The safety profile of nintedanib in this longer duration analysis was consistent with that previously reported. CONCLUSIONS: In this analysis of data from Japanese patients with PF-ILDs, nintedanib nominally reduced the risk of clinically meaningful outcomes reflecting disease progression, including death, over the whole trial, and no new safety concerns were observed. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT02999178.

Developing a conceptual model of symptoms and impacts in progressive fibrosing interstitial lung disease to evaluate patient-reported outcome measures.

Background: An understanding of the experience of patients with progressive fibrosing interstitial lung disease (PF-ILD) is needed to select appropriate patient-reported outcome measures (PROMs) to evaluate treatment effect in clinical trials. Methods: A systematic literature review was conducted to develop a preliminary conceptual model of the symptoms experienced by patients with PF-ILD and the impacts the disease has on them. An online survey and consensus meetings were then conducted with 12-14 stakeholders (patients, clinicians, regulatory and payer advisors) to refine the conceptual model and critically appraise how key concepts should be measured by PROMs. PROMs assessed included Living with Idiopathic Pulmonary Fibrosis, Living with Pulmonary Fibrosis, the King's Brief Interstitial Lung Disease questionnaire, Cough and Sputum Assessment Questionnaire, Evaluating Respiratory Symptoms, Leicester Cough Questionnaire, Functional Assessment of Chronic Illness Therapy (Dyspnoea/Fatigue) and St George's Respiratory Questionnaire for Idiopathic Pulmonary Fibrosis. Results: The literature review identified 36 signs/symptoms and 43 impacts directly or indirectly related to pulmonary aspects of PF-ILD. The most relevant symptoms identified by participants included shortness of breath on exertion, fatigue and cough; relevant impacts included effects on physical functioning, activities of daily living and emotional wellbeing. These are presented in a conceptual model. Consensus opinion was that existing PROMs need further modification and validation before use in clinical trials. Conclusions: The conceptual model improves understanding of the symptoms and impacts that living with PF-ILD has on patients' wellbeing. It can help to inform the choice of PROMs in clinical trials and highlight aspects to assess in the clinical care of patients with PF-ILD.

Meta-Analysis of Effect of Nintedanib on Reducing FVC Decline Across Interstitial Lung Diseases.

INTRODUCTION: The effect of nintedanib on slowing the rate of decline in forced vital capacity (FVC) has been investigated in randomized placebo-controlled trials in subjects with idiopathic pulmonary fibrosis (IPF), other progressive fibrosing interstitial lung diseases (ILDs), and ILD associated with systemic sclerosis (SSc-ILD). We assessed the consistency of the effect of nintedanib on the rate of decline in FVC over 52 weeks across four placebo-controlled phase III trials. METHODS: We used data on FVC decline from the INPULSIS-1 and INPULSIS-2 trials in subjects with IPF, the INBUILD trial in subjects with progressing fibrosing ILDs other than IPF, and the SENSCIS trial in subjects with SSc-ILD. In each trial, the primary endpoint was the annual rate of decline in FVC (mL/year) assessed over 52 weeks. We performed fixed effect and random effects meta-analyses based on the relative treatment effect of nintedanib versus placebo on the rate of decline in FVC (mL/year) over 52 weeks. Heterogeneity of the relative treatment effect of nintedanib across populations was assessed using the I2 statistic, τ2 and corresponding p value from a Q test for heterogeneity. RESULTS: The combined analysis comprised 1257 subjects treated with nintedanib and 1042 subjects who received placebo. Nintedanib reduced the rate of decline in FVC (mL/year) over 52 weeks by 51.0% (95% CI 39.1, 63.0) compared with placebo. The relative effect (95% CI) was the same using the fixed effect and random effects models. There was no evidence of heterogeneity in the relative treatment effect of nintedanib across the populations studied (I2 = 0%, τ2 = 0, p = 0.93). CONCLUSIONS: A meta-analysis of data from four placebo-controlled trials demonstrated that nintedanib approximately halved the rate of decline in FVC over 52 weeks across subjects with different forms of pulmonary fibrosis, with no evidence of heterogeneity in its relative treatment effect across patient populations.

Phase 3 Trial of BI 695502 Plus Chemotherapy Versus Bevacizumab Reference Product Plus Chemotherapy in Patients With Advanced Nonsquamous NSCLC.

INTRODUCTION: Biological therapies such as bevacizumab have improved survival in patients with NSCLC. This study was conducted to confirm the equivalent efficacy of the biosimilar candidate BI 695502 to the bevacizumab reference product (RP). METHODS: In this phase 3, multicenter, randomized, double-blind trial of adult patients with recurrent or metastatic NSCLC received up to 18 weeks of induction treatment with BI 695502 or bevacizumab RP 15 mg/kg plus paclitaxel and carboplatin. Subsequent maintenance therapy comprised BI 695502 or bevacizumab RP monotherapy until disease progression or unacceptable toxicity. The primary end point was the best overall response rate (ORR) per Response Evaluation Criteria in Solid Tumors version 1.1 assessed by central imaging review, until 18 weeks after the start of treatment. RESULTS: In total, 671 patients were randomized at one-to-one ratio to BI 695502 or bevacizumab RP, of whom 335 and 328, respectively, received treatment. Of these, 228 (68.1%) and 256 (78.0%), respectively, proceeded to maintenance monotherapy. A manufacturing issue led to a small number of patients treated with BI 695502 switching to bevacizumab RP late in the study. The primary end point, best ORR, was 54.0% in the BI 695502 group and 63.1% in the bevacizumab RP group. The 90% confidence interval for the between-group ratio of best ORR (0.770 to 0.951) was within the prespecified range for equivalence (0.736-1.359). Adverse events were class-related and similar between the two treatment arms. CONCLUSIONS: This study revealed equivalent ORR after 18 weeks of treatment with BI 695502 or bevacizumab RP, with similar adverse event profiles.

The psychometric properties of the King's Brief Interstitial Lung Disease questionnaire and thresholds for meaningful treatment response in patients with progressive fibrosing interstitial lung diseases.

BACKGROUND: There is a lack of fully validated patient-reported outcome measures for progressive fibrosing interstitial lung disease (ILD). We aimed to validate the King's Brief Interstitial Lung Disease (K-BILD) questionnaire for measuring health-related quality of life (HRQoL) in these patients. We also aimed to estimate the meaningful change threshold for interpreting stabilisation of HRQoL as a clinical end-point in progressive fibrosing ILD, where the current goal of treatment is disease stability and slowing progression. METHODS: This analysis evaluated data from 663 patients with progressive fibrosing ILD other than idiopathic pulmonary fibrosis from the INBUILD trial. Validation of the measurement properties was assessed for internal consistency, test-retest reliability, construct validity, known-groups validity and responsiveness. We calculated meaningful change thresholds for treatment response using anchor-based (within-patient) and distribution-based methods. RESULTS: K-BILD had strong internal consistency (Cronbach's α was 0.94 for total score, 0.88 for breathlessness and activities, 0.91 for psychological, and 0.79 for chest symptoms). The test-retest reliability intraclass correlation coefficient was 0.74 for K-BILD total score. K-BILD demonstrated weak correlations with forced vital capacity (FVC) percent predicted. Known-groups validity showed significant differences in K-BILD scores for patient groups with different disease severity based on use of supplemental oxygen or baseline FVC % pred (≤70% or >70%). We estimated a meaningful change threshold of ≥ -2 units for K-BILD total score for defining patients who remain stable/improved versus those with progressive deterioration. CONCLUSIONS: Our results validate K-BILD as a tool for assessing HRQoL in patients with progressive fibrosing ILD and set a meaningful change threshold of ≥ -2 units for K-BILD total score.

Using Data on Survival with Idiopathic Pulmonary Fibrosis to Estimate Survival with Other Types of Progressive Fibrosis Interstitial Lung Disease: A Bayesian Framework.

INTRODUCTION: Among the various types of progressive fibrosing interstitial lung diseases (PF-ILDs), substantial survival data exist for idiopathic pulmonary fibrosis (IPF) but not for other types. This hinders evidence-based decisions about treatment and management, as well as the economic modelling needed to justify research into new treatments and reimbursement approvals. Given the clinical similarities between IPF and other PF-ILDs, we reasoned that patient survival data from four major IPF trials could be used to estimate long-term survival in other PF-ILDs. METHODS: We used propensity score matching to match patients with IPF taking either nintedanib or placebo in the TOMORROW, INPULSIS-1, INPULSIS-2 and INPULSIS-ON trials to patients with PF-ILDs other than IPF in the INBUILD trial. Seven models were fitted to the survival data for the matched patients with IPF, and the three best-fitting models were used to generate informative priors in a Bayesian framework to extrapolate patient survival of the INBUILD population. RESULTS: After propensity score matching, the analysis included data from 1099 patients with IPF (640 nintedanib patients; 459 placebo patients) and 654 patients with other PF-ILDs (326 nintedanib patients; 328 placebo patients). Gamma, log-logistic and Weibull models best fit the survival of the matched patients with IPF. All three models led to consistent Bayesian estimates of survival for the matched patients with other PF-ILDs, with median rates of overall survival ranging from 6.34 to 6.50 years after starting nintedanib. The corresponding control group survival estimates were 3.42 to 3.76 years. CONCLUSION: We provide the first estimates of long-term overall survival for patients with PF-ILDs other than IPF, and our analysis suggests that nintedanib may prolong their survival. Our Bayesian approach to estimating survival of one disease based on clinical trial data from a similar disease may help inform economic modelling of rare, orphan and newly defined disorders.

The Living with Pulmonary Fibrosis questionnaire in progressive fibrosing interstitial lung disease.

The Living with Idiopathic Pulmonary Fibrosis (L-IPF) questionnaire was developed with substantial input from patients with idiopathic pulmonary fibrosis (IPF) to assess symptoms and health-related quality of life (HRQoL). Because IPF is the prototypical chronic fibrosing interstitial lung disease (ILD) with a progressive phenotype, we expanded applicability of the L-IPF by deleting the word "idiopathic", creating the L-PF (Living with Pulmonary Fibrosis) questionnaire, and then assessed its relevance among patients with progressive fibrosing ILDs in one-to-one interviews. Patients in the USA and Germany with any progressive fibrosing ILD other than IPF were asked about their disease and symptoms, completed the 44-item L-PF questionnaire (comprising two modules that assess symptoms and impacts of disease) and then answered a series of debriefing questions. Interviews were recorded, transcribed and coded for qualitative content analysis. 20 patients were interviewed, but time constraints meant not all were asked about all items. The most frequent diagnoses were rheumatoid arthritis-associated ILD (25%) and mixed connective tissue disease-associated ILD (20%). Almost all patients endorsed the symptoms assessed by the L-PF: shortness of breath (19 out of 20 patients), cough (19 out of 20) and fatigue (18 out of 20). Most patients endorsed impacts of progressive fibrosing ILD on activities of daily living, physical well-being, sleep, emotional well-being, and social aspects of their lives. Most patients had an overall positive impression of the Symptoms module and understood items as intended. All seven patients asked understood the items of the Impacts module. The L-PF contains concepts relevant and important to patients with progressive fibrosing ILD, and items are understood as intended.

Home spirometry in patients with idiopathic pulmonary fibrosis: data from the INMARK trial.

BACKGROUND: Data from the INMARK trial were used to investigate the feasibility and validity of home spirometry as a measure of lung function decline in patients with idiopathic pulmonary fibrosis (IPF). METHODS: Subjects with IPF and preserved forced vital capacity (FVC) were randomised to receive nintedanib or placebo for 12 weeks followed by open-label nintedanib for 40 weeks. Clinic spirometry was conducted at baseline and weeks 4, 8, 12, 16, 20, 24, 36 and 52. Subjects were asked to perform home spirometry at least once a week and ideally daily. Correlations between home- and clinic-measured FVC and rates of change in FVC were assessed using Pearson correlation coefficients. RESULTS: In total, 346 subjects were treated. Mean adherence to weekly home spirometry decreased over time but remained above 75% in every 4-week period. Over 52 weeks, mean adherence was 86%. Variability in change from baseline in FVC was greater when measured by home rather than clinic spirometry. Strong correlations were observed between home- and clinic-measured FVC at all time-points (r=0.72-0.84), but correlations between home- and clinic-measured rates of change in FVC were weak (r=0.26 for rate of decline in FVC over 52 weeks). CONCLUSION: Home spirometry was a feasible and valid measure of lung function in patients with IPF and preserved FVC, but estimates of the rate of FVC decline obtained using home spirometry were poorly correlated with those based on clinic spirometry.

Biomarkers of extracellular matrix turnover in patients with idiopathic pulmonary fibrosis given nintedanib (INMARK study): a randomised, placebo-controlled study.

BACKGROUND: A hallmark of idiopathic pulmonary fibrosis is the excess accumulation of extracellular matrix in the lungs. Degradation of extracellular matrix generates free-circulating protein fragments called neoepitopes. The aim of the INMARK trial was to investigate changes in neoepitopes as predictors of disease progression in patients with idiopathic pulmonary fibrosis and the effect of nintedanib on these biomarkers. METHODS: In this randomised, double-blind, placebo-controlled trial, patients with a diagnosis of idiopathic pulmonary fibrosis within the past 3 years and forced vital capacity (FVC) of 80% predicted or higher were eligible to participate. Patients were recruited from hospitals, private practices, clinical research units, and academic medical centres. Patients were randomly assigned (1:2) with the use of a pseudo-random number generator to receive oral nintedanib 150 mg twice a day or placebo for 12 weeks in a double-blind fashion, followed by open-label nintedanib for 40 weeks. The primary endpoint was the rate of change in C-reactive protein (CRP) degraded by matrix metalloproteinases 1 and 8 (CRPM) from baseline to week 12 in the intention-to-treat population. The trial has been completed and is registered with ClinicalTrials.gov, number NCT02788474, and with the European Clinical Trials Database, number 2015-003148-38. FINDINGS: Between June 27, 2016, and May 15, 2017, 347 patients were randomly assigned to the nintedanib group (n=116) or to the placebo group (n=231). One patient from the placebo group was not treated owing to a randomisation error. At baseline, mean FVC was 97·5% (SD 13·5) predicted. In the double-blind period, 116 patients received nintedanib and 230 patients received placebo. The rate of change in CRPM from baseline to week 12 was -2·57 × 10-3 ng/mL/month in the nintedanib group and -1·90 × 10-3 ng/mL/month in the placebo group (between-group difference -0·66 × 10-3 ng/mL/month [95% CI -6·21 × 10-3 to 4·88 × 10-3]; p=0·8146). The adjusted rate of change in FVC over 12 weeks was 5·9 mL in the nintedanib group and -70·2 mL in the placebo group (difference 76·1 mL/12 weeks [31·7 to 120·4]). In patients who received placebo for 12 weeks followed by open-label nintedanib, rising concentrations of CRPM over 12 weeks were associated with disease progression (absolute decline in FVC ≥10% predicted or death) over 52 weeks. In the double-blind period, serious adverse events were reported in eight (7%) patients given nintedanib and 18 (8%) patients given placebo. Grade 3 diarrhoea was reported in two (2%) patients in the nintedanib group and two (1%) patients in the placebo group. No patients had grade 4 diarrhoea. INTERPRETATION: In patients with idiopathic pulmonary fibrosis and preserved lung function, treatment with nintedanib versus placebo for 12 weeks did not affect the rate of change in CRPM but was associated with a reduced rate of decline in FVC. These results suggest that change in CRPM is not a marker of response to nintedanib in patients with idiopathic pulmonary fibrosis. FUNDING: Boehringer Ingelheim.

Early developmental specification of the thyroid gland depends on han-expressing surrounding tissue and on FGF signals.

The thyroid is an endocrine gland in all vertebrates that develops from the ventral floor of the anterior pharyngeal endoderm. Unravelling the molecular mechanisms of thyroid development helps to understand congenital hypothyroidism caused by the absence or reduction of this gland in newborn humans. Severely reduced or absent thyroid-specific developmental genes concomitant with the complete loss of the functional gland in the zebrafish hands off (han, hand2) mutant reveals the han gene as playing a novel, crucial role in thyroid development. han-expressing tissues surround the thyroid primordium throughout development. Fate mapping reveals that, even before the onset of thyroid-specific developmental gene expression, thyroid precursor cells are in close contact with han-expressing cardiac lateral plate mesoderm. Grafting experiments show that han is required in surrounding tissue, and not in a cell-autonomous manner, for thyroid development. Loss of han expression in the branchial arches and arch-associated cells after morpholino knock-down of upstream regulator genes does not impair thyroid development, indicating that other han-expressing structures, most probably cardiac mesoderm, are responsible for the thyroid defects in han mutants. The zebrafish ace (fgf8) mutant has similar thyroid defects as han mutants, and chemical suppression of fibroblast growth factor (FGF) signalling confirms that this pathway is required for thyroid development. FGF-soaked beads can restore thyroid development in han mutants, showing that FGFs act downstream of or in parallel to han. These data suggest that loss of FGF-expressing tissue in han mutants is responsible for the thyroid defects.

fgf1 is required for normal differentiation of erythrocytes in zebrafish primitive hematopoiesis.

Hematopoiesis in vertebrate development involves an embryonic, primitive wave and a later, definitive wave in which embryonic blood cells are replaced with adult blood cells. We here show that zebrafish fgf1 is involved in vivo in primitive hematopoiesis. Fibroblast growth factor-1 (FGF1) morpholino knockdown leads to abnormal accumulation of blood cells in the posterior intermediate cell mass at 32 hr postfertilization. Expression of the erythroid markers gata1 and ika, normally diminishing in differentiating erythrocytes at this stage, is maintained at abnormally high levels in primitive blood cells. The onset of erythrocyte differentiation as assessed by o-dianisidine staining is severely delayed. Most fgf1 morphants later recover to wild-type appearance, and primitive erythrocytes eventually differentiate. Zebrafish fgf1 is syntenic to human FGF1, which maps to a critically deleted region in human del(5q) syndrome posing an increased risk of leukemia to patients. As its knockdown in zebrafish changes expression of gata1, a gene involved in hematopoietic stem cell decisions, FGF1 should be considered to play a role in the pathogenesis of del(5q) syndrome.

Pituitary-interrenal interaction in zebrafish interrenal organ development.

To further elucidate pituitary adrenal interactions during development, we studied the organogenesis of the interrenal organ, the teleost homolog of the mammalian adrenal gland, in zebrafish. To this end we compared wild-type zebrafish interrenal development with that of mutants lacking pituitary cell types including corticotrophs. In addition, we studied the effects of ACTH receptor (Mc2r) knockdown and dexamethasone (dex) on interrenal development and pituitary feedback. Until 2 d post fertilization (2 dpf) interrenal development assessed by transcripts of key steroidogenic genes (cyp11a1, mc2r, star) is independent of proopiomelanocortin (Pomc) as demonstrated in aal/eya1and lia/fgf3 mutants. However, at 5 dpf lack of pituitary cells leads to reduced expression of steroidogenic genes at both the transcriptional and the protein level. Pituitary control of interrenal development resides in corticotrophs, because pit1 mutants lacking pituitary cells except corticotrophs have a phenotype similar to that of wild-type controls. Furthermore, development in mc2r knockdown morphants does not differ from aal/eya1 and lia/fgf3 mutants. Inhibition of steroidogenesis by mc2r knockdown induces up-regulation of pomc expression in the anterior domain of pituitary corticotrophs. Accordingly, dex suppresses pomc in the anterior domain only, leading to impaired expression of steroidogenic genes commencing at 3 dpf and interrenal hypoplasia via reduced interrenal proliferation. In contrast, negative feedback on pituitary corticotrophs by dex is evident at 2 dpf and precedes effects of Pomc on the interrenal primordium. These data demonstrate a gradual transition from early pituitary-independent interrenal organogenesis to developmental control by the anterior domain of pituitary corticotrophs acting via Mc2 receptors.

Arteries define the position of the thyroid gland during its developmental relocalisation.

During vertebrate development, the thyroid gland undergoes a unique relocalisation from its site of induction to a distant species-specific position in the cervical mesenchyme. We have analysed thyroid morphogenesis in wild-type and mutant zebrafish and mice, and find that localisation of growing thyroid tissue along the anteroposterior axis in zebrafish is linked to the development of the ventral aorta. In grafting experiments, ectopic vascular cells influence the localisation of thyroid tissue cell non-autonomously, showing that vessels provide guidance cues in zebrafish thyroid morphogenesis. In mouse thyroid development, the midline primordium bifurcates and two lobes relocalise cranially along the bilateral pair of carotid arteries. In hedgehog-deficient mice, thyroid tissue always develops along the ectopically and asymmetrically positioned carotid arteries, suggesting that, in mice (as in zebrafish), co-developing major arteries define the position of the thyroid. The similarity between zebrafish and mouse mutant phenotypes further indicates that thyroid relocalisation involves two morphogenetic phases, and that variation in the second phase accounts for species-specific differences in thyroid morphology. Moreover, the involvement of vessels in thyroid relocalisation sheds new light on the interpretation of congenital thyroid defects in humans.

Analysis of origin and growth of the thyroid gland in zebrafish.

The zebrafish thyroid gland shows a unique pattern of growth as a differentiated endocrine gland. Here, we analyze the onset of differentiation, the contribution of lineages, and the mode of growth of this gland. The expression of genes involved in hormone production and the establishment of epithelial polarity show that differentiation into a first thyroid follicle takes place early during embryonic development. Thyroid follicular tissue then grows along the pharyngeal midline, initially independently of thyroid stimulating hormone. Lineage analysis reveals that thyroid follicle cells are exclusively recruited from the pharyngeal endoderm. The ultimobranchial bodies that merge with the thyroid in mammals form separate glands in zebrafish as visualized by calcitonin precursor gene expression. Mosaic analysis suggests that the first thyroid follicle differentiating at 55 hours postfertilization corresponds later to the most anterior follicle and that new follicles are added caudally.

Anatomical and molecular reinvestigation of lamprey endostyle development provides new insight into thyroid gland evolution.

The thyroid gland of vertebrates is considered to be homologous to the endostyle of non-vertebrate chordates (cephalochordates, urochordates), a key character for understanding the origin and evolution of the chordate body plan. In lampreys, the larval endostyle transforms into an adult thyroid gland during metamorphosis, reflecting evolutionary changes that occurred in the vertebrate lineage. Focussing on thyroid-like cells in the endostyle, we here relate morphologically visible steps of lamprey (Lampetra fluviatilis) endostyle differentiation to embryonic stages and determine the onset of thyroid-like function. Analysing lamprey endostyle development using semi-thin histological sections, immunohistochemical detection of thyroid hormone, and the molecular marker thyroid transcription factor1 (Ttf1) refines our current view of the homology between endostyle and thyroid gland. In contrast to earlier literature, we find that a duct always persists to connect the endostyle lumen to the pharynx, a structure that resembles the thyroglossal duct in thyroid development and could further support the homology between endostyle and thyroid. Before the onset of thyroid-like function, Ttf1 expression becomes restricted to the ventral part of the endostyle, on the one hand showing that dorsal thyroid-like cells produce thyroid hormone in the absence of Ttf1, and on the other suggesting that Ttf1 was initially involved in specifying ventral fates in the endostyle.

Zebrafish hhex, nk2.1a, and pax2.1 regulate thyroid growth and differentiation downstream of Nodal-dependent transcription factors.

During zebrafish development, the thyroid primordium initiates expression of molecular markers such as hhex and nk2.1a in the endoderm prior to pharynx formation. As expected for an endodermally derived organ, initiation of thyroid development depends on Nodal signalling. We find that it also depends on three downstream effectors of Nodal activity, casanova (cas), bonnie and clyde (bon), and faust (fau)/gata5. Despite their early Nodal-dependent expression in the endoderm, both hhex and nk2.1a are only required relatively late during thyroid development. In hhex and nk2.1a loss-of-function phenotypes, thyroid development is initiated and arrests only after the primordium has evaginated from the pharyngeal epithelium. Thus, like pax2.1, both hhex and nk2.1a have similarly late roles in differentiation or growth of thyroid follicular cells, and here, we show that all three genes act in parallel rather than in a single pathway. Our functional analysis suggests that these genes have similar roles as in mammalian thyroid development, albeit in a different temporal mode of organogenesis.

Anterior and posterior waves of cyclic her1 gene expression are differentially regulated in the presomitic mesoderm of zebrafish.

Somite formation in vertebrates depends on a molecular oscillator in the presomitic mesoderm (PSM). In order to get a better insight into how oscillatory expression is achieved in the zebrafish Danio rerio, we have analysed the regulation of her1 and her7, two bHLH genes that are co-expressed in the PSM. Using specific morpholino oligonucleotide mediated inhibition and intron probe in situ hybridisation, we find that her7 is required for initiating the expression in the posterior PSM, while her1 is required to propagate the cyclic expression in the intermediate and anterior PSM. Reporter gene constructs with the her1 upstream sequence driving green fluorescent protein (GFP) expression show that separable regulatory regions can be identified that mediate expression in the posterior versus intermediate and anterior PSM. Our results indicate that the cyclic expression is generated at the transcriptional level and that the resulting mRNAs have a very short half-life. A specific degradation signal for her1 mRNA must be located in the 5'-UTR, as this region also destabilises the GFP mRNA such that it mimics the dynamic pattern of the endogenous her1 mRNA. In contrast to the mRNA, GFP protein is stable and we find that all somitic cells express the protein, proving that her1 mRNA is transiently expressed in all cells of the PSM.