PBPK-based dose finding for sildenafil in pregnant women for antenatal treatment of congenital diaphragmatic hernia.

Sildenafil is a potent vasodilator and phosphodiesterase type five inhibitor, commercially known as Revatio® and approved for the treatment of pulmonary arterial hypertension. Maternal administration of sildenafil during pregnancy is being evaluated for antenatal treatment of several conditions, including the prevention of pulmonary hypertension in fetuses with congenital diaphragmatic hernia. However, determination of a safe and effective maternal dose to achieve adequate fetal exposure to sildenafil remains challenging, as pregnancy almost always is an exclusion criterion in clinical studies. Physiologically-based pharmacokinetic (PBPK) modelling offers an attractive approach for dose finding in this specific population. The aim of this study is to exploit physiologically-based pharmacokinetic modelling to predict the required maternal dose to achieve therapeutic fetal exposure for the treatment congenital diaphragmatic hernia. A full-PBPK model was developed for sildenafil and N-desmethyl-sildenafil using the Simcyp simulator V21 platform, and verified in adult reference individuals, as well as in pregnant women, taking into account maternal and fetal physiology, along with factors known to determine hepatic disposition of sildenafil. Clinical pharmacokinetic data in mother and fetus were previously obtained in the RIDSTRESS study and were used for model verification purposes. Subsequent simulations were performed relying either on measured values for fetal fraction unbound (fu = 0.108) or on values predicted by the simulator (fu = 0.044). Adequate doses were predicted according to the efficacy target of 15 ng/mL (or 38 ng/mL) and safety target of 166 ng/mL (or 409 ng/mL), assuming measured (or predicted) fu values, respectively. Considering simulated median profiles for average steady state sildenafil concentrations, dosing regimens of 130 mg/day or 150 mg/day (administered as t.i.d.), were within the therapeutic window, assuming either measured or predicted fu values, respectively. For safety reasons, dosing should be initiated at 130 mg/day, under therapeutic drug monitoring. Additional experimental measurements should be performed to confirm accurate fetal (and maternal) values for fu. Additional characterization of pharmacodynamics in this specific population is required and may lead to further optimization of the dosing regimen.

Decreased fetal movements-the utility of ultrasound to identify infants at risk and prevent stillbirth is poor.

BACKGROUND: Despite a paucity of evidence, it is widely accepted that a perceived reduction in fetal movements is associated with an increased risk of stillbirth and poor obstetrical outcome. Consequently, many international guidelines recommend urgent ultrasound assessment of fetal well-being in women presenting with decreased fetal movements. OBJECTIVE: This study aimed to compare rates of abnormal ultrasound findings reflective of fetal compromise between women presenting with decreased fetal movements and gestation-matched controls in the third trimester. STUDY DESIGN: This was a retrospective cohort study performed at the Mater Mothers' Hospital in Brisbane between 2017 and 2020. We undertook propensity score matching analysis comparing abnormal ultrasound parameters in women with singleton, nonanomalous pregnancies presenting with decreased fetal movements after 28 weeks' gestation. The primary outcome was a composite of any abnormal scan parameter: umbilical artery pulsatility index >95th centile, middle cerebral artery pulsatility index <5th centile, cerebroplacental ratio <10th centile, estimated fetal weight <10th centile for gestation, middle cerebral artery peak systolic velocity >1.5 multiples of the median, or deepest vertical pocket of amniotic fluid <2 or >8 cm. RESULTS: After propensity score matching, the study cohort comprised 1466 cases and 2207 controls. The rate of the primary composite outcome was not significantly different between the 2 cohorts (20.2% vs 21.3%; P=.42). There were 30 new cases of small-for-gestational-age detected in the decreased fetal movements cohort, giving a number needed to scan of 48 in the decreased fetal movements group to detect 1 case of small-for-gestational-age. However, the frequency of the composite outcome was higher (13.0% vs 5.4%) at the final scan before birth in women with multiple decreased fetal movement presentations. Despite this, there was no significant difference in clinical outcomes between the 2 cohorts. CONCLUSION: Ultrasound abnormalities are not increased in women with decreased fetal movements compared with controls.

Phosphodiesterase-5 inhibitors in pregnancy: Systematic review and meta-analysis of maternal and perinatal safety and clinical outcomes.

BACKGROUND: The efficacy and safety profile of phosphodiesterase-5 inhibitors (PDE-5i) in pregnancy are unclear from the few relatively small diverse studies that have used them. OBJECTIVE: To assess the safety profile and clinical outcomes of PDE-5i use in pregnancy. SEARCH STRATEGY: We searched Embase, PubMed, CENTRAL, Prospero and Google Scholar to identify randomised controlled trials (RCTs) reporting the use of any PDE-5i in pregnancy up to September 2021. SELECTION CRITERIA: RCTs reporting obstetric or perinatal outcomes or maternal adverse outcomes in women taking PDE5i in pregnancy. DATA COLLECTION AND ANALYSIS: Risk ratios (RR), 95% confidence intervals (95% CI) and 95% prediction intervals were calculated and pooled for analysis. RESULTS: We identified 1324 citations, of which 10 studies including 1090 participants met the inclusion criteria. Only tadalafil and sildenafil were reported as used in pregnancy. Two studies using tadalafil and eight sildenafil. Nine of ten studies were assessed at having of low risk of bias. PDE-5i use was associated with an increased risk of headaches (RR 1.41, 95% CI 0.97-2.05), flushing (RR 2.59, 95% CI 0.69-9.90) and nasal bleeding (RR 10.53, 95% CI 1.36-81.3); an increase in vaginal birth when used for non-fetal growth restriction (FGR) indications (RR 1.24, 95% CI 1.00-1.55) and a reduction in risk of operative birth for intrapartum fetal compromise (RR 0.58, 95% CI 0.38-0.88). There was no evidence of any increase in risk of perinatal death (RR 0.89, 95% CI 0.56-1.43). However, use for the treatment of FGR increased the risk of persistent pulmonary hypertension of the newborn (PPHN) (RR 2.52, 95% CI 1.00-6.32). CONCLUSIONS: This meta-analysis suggests PDE-5i use in pregnancy is associated with mild maternal side effects and lower risk of operative birth for intrapartum fetal distress. Prolonged use for the treatment of FGR may increase the risk of PPHN. TWEETABLE ABSTRACT: PDE-5i use in pregnancy is associated with mild maternal side effects, lower operative birth for intrapartum fetal distress and a possible increase in persistent pulmonary hypertension of the newborn when used for the treatment of fetal growth restriction.

WITHDRAWN: Phosphodiesterase-5 inhibitors in Pregnancy: Systematic review and meta-analysis of maternal and perinatal safety and clinical outcomes.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause.

Evaluation of Pregnancy Outcomes Among Women With Decreased Fetal Movements.

Importance: Stillbirth is a devastating pregnancy outcome with far-reaching economic and psychosocial consequences, but despite significant investment, a screening tool for identifying those fetuses at risk for stillbirth remains elusive. Maternal reporting of decreased fetal movements (DFM) has been found to be associated with stillbirth and other adverse perinatal outcomes. Objective: To examine pregnancy outcomes of women presenting with DFM in the third trimester at a tertiary Australian center with a clear clinical management algorithm. Design, Setting, and Participants: This cohort study used data on all births meeting the inclusion criteria from 2009 through 2019 at Mater Mothers' Hospital in Brisbane, Australia. This is a tertiary center and Australia's largest maternity hospital. All singleton births without a known congenital anomaly after 28 weeks' gestation were included. Among 203 071 potential participants identified from the hospital database, 101 597 individuals met the eligibility criteria. Data analysis was performed from May through September 2020. Exposure: Presentation to hospital with DFM after 28 weeks gestation. Main Outcomes and Measures: The primary outcome of this study was the incidence of stillbirth. Multivariate analysis was undertaken to determine the association between DFM and stillbirth, obstetric intervention, and other adverse outcomes, including being born small for gestational age (SGA) and a composite adverse perinatal outcome (at least 1 of the following: neonatal intensive care unit admission, severe acidosis [ie, umbilical artery pH <7.0 or base excess -12.0 mmol/L or less], 5-minute Apgar score <4, or stillbirth or neonatal death). The hypothesis being tested was formulated prior to data collection. Results: Among 101 597 women with pregnancies that met the inclusion criteria, 8821 (8.7%) presented at least once with DFM and 92 776 women (91.3%) did not present with DFM (ie, the control population). Women presenting with DFM, compared with those presenting without DFM, were younger (mean [SD] age, 30.4 [5.4] years vs 31.5 [5.2] years; P < .001), more likely to be nulliparous (4845 women [54.9%] vs 42 210 women [45.5%]; P < .001) and have a previous stillbirth (189 women [2.1%] vs 1156 women [1.2%]; P < .001), and less likely to have a previous cesarean delivery (1199 women [13.6%] vs 17 444 women [18.8%]; P < .001). During the study period, the stillbirth rate was 2.0 per 1000 births after 28 weeks' gestation. Presenting with DFM was not associated with higher odds of stillbirth (9 women [0.1%] vs 185 women [0.2%]; adjusted odds ratio [aOR], 0.54; 95% CI, 0.23-1.26, P = .16). However, presenting with DFM was associated with higher odds of a fetus being born SGA (aOR, 1.14; 95% CI, 1.03-1.27; P = .01) and the composite adverse perinatal outcome (aOR, 1.14; 95% CI, 1.02-1.27; P = .02). Presenting with DFM was also associated with higher odds of planned early term birth (aOR, 1.26; 95% CI, 1.15-1.38; P < .001), induction of labor (aOR, 1.63; 95% CI, 1.53-1.74; P < .001), and emergency cesarean delivery (aOR, 1.18; 95% CI, 1.09-1.28; P < .001). Conclusions and Relevance: The presence of DFM is a marker associated with increased risk for a fetus. This study's findings of a nonsignificantly lower rate of stillbirth among women with DFM may be reflective of increased community awareness of timely presentation to their obstetric care clinician when concerned about fetal movements and the benefits of tertiary level care guided by a clear clinical management protocol. However, DFM was associated with increased odds of an infant being born SGA, obstetric intervention, early term birth, and a composite of adverse perinatal outcomes.

The Impact of Severe Maternal Morbidity on Perinatal Outcomes in High Income Countries: Systematic Review and Meta-Analysis.

While there is clear evidence that severe maternal morbidity (SMM) contributes significantly to poor maternal health outcomes, limited data exist on its impact on perinatal outcomes. We undertook a systematic review and meta-analysis to ascertain the association between SMM and adverse perinatal outcomes in high-income countries (HICs). We searched for full-text publications in PubMed, Embase, Cumulative Index of Nursing and Allied Health Literature (CINAHL), and Scopus databases. Studies that reported data on the association of SMM and adverse perinatal outcomes, either as a composite or individual outcome, were included. Two authors independently assessed study eligibility, extracted data, and performed quality assessment using the Newcastle-Ottawa Scale. We used random-effects modelling to calculate odds ratios (ORs) with 95% confidence intervals. We also assessed the risk of publication bias and statistical heterogeneity using funnel plots and Higgins I2, respectively. We defined sub-groups of SMM as hemorrhagic disorders, hypertensive disorders, cardiovascular disorders, hepatic disorders, renal disorders, and thromboembolic disorders. Adverse perinatal outcome was defined as preterm birth (before 37 weeks gestation), small for gestational age (SGA) (birth weight (BW) < 10th centile for gestation), low birthweight (LBW) (BW < 2.5 kg), Apgar score < 7 at 5 min, neonatal intensive care unit (NICU) admission, stillbirth and perinatal death (stillbirth and neonatal deaths up to 28 days). A total of 35 studies consisting of 38,909,426 women were included in the final analysis. SMMs associated with obstetric hemorrhage (OR 3.42, 95% CI: 2.55-4.58), severe hypertensive disorders (OR 6.79, 95% CI: 6.06-7.60), hepatic (OR 3.19, 95% CI: 2.46-4.13) and thromboembolic disorders (OR 2.40, 95% CI: 1.67-3.46) were significantly associated with preterm birth. SMMs from hypertensive disorders (OR 2.86, 95% CI: 2.51-3.25) or thromboembolic disorders (OR 1.48, 95% CI: 1.09-1.99) were associated with greater odds of having SGA infant. Women with severe hemorrhage had increased odds of LBW infant (OR 2.31, 95% CI: 1.57-3.40). SMMs from obstetric hemorrhage (OR 4.16, 95% CI: 2.54-6.81) or hypertensive disorders (OR 4.61, 95% CI: 1.17-18.20) were associated with an increased odds of low 5-min Apgar score and NICU admission (Severe obstetric hemorrhage: OR 3.34, 95% CI: 2.26-4.94 and hypertensive disorders: OR 3.63, 95% CI: 2.63-5.02, respectively). Overall, women with SMM were 4 times more likely to experience stillbirth (OR 3.98, 95%C 3.12-7.60) compared to those without SMM with cardiovascular disease (OR 15.2, 95% CI: 1.29-180.60) and thromboembolic disorders (OR 9.43, 95% CI: 4.38-20.29) conferring greatest risk of this complication. The odds of neonatal death were significantly higher in women with SMM (OR 3.98, 95% CI: 2.44-6.47), with those experiencing hemorrhagic (OR 7.33, 95% CI: 3.06-17.53) and hypertensive complications (OR 3.0, 95% CI: 1.78-5.07) at highest risk. Overall, SMM was also associated with higher odds of perinatal death (OR 4.74, 95% CI: 2.47-9.12) mainly driven by the increased risk in women experiencing severe obstetric hemorrhage (OR 6.18, 95% CI: 2.55-14.96). Our results highlight the importance of mitigating the impact of SMM not only to improve maternal health but also to ameliorate its consequences on perinatal outcomes.

Low First Trimester Pregnancy-Associated Plasma Protein-A Levels Are Not Associated with an Increased Risk of Intrapartum Fetal Compromise or Adverse Neonatal Outcomes: A Retrospective Cohort Study.

The aim of this study was to assess if women with a low first trimester maternal pregnancy-associated plasma protein-A (PAPP-A) level are at increased risk of emergency cesarean (EmCS) for intrapartum fetal compromise (IFC) and/or adverse neonatal outcomes. This was a retrospective cohort study performed at Mater Mother's Hospital, Brisbane, Australia, between 2016 and 2018. All women with a singleton, euploid, non-anomalous fetus with a documented PAPP-A level measured between 10 +0 and 13 +6 weeks gestation during the study period were included. Data were extracted from the institution's perinatal database and dichotomized according to PAPP-A level (≤0.4 Multiples of Medium (MoM) vs. >0.4 MoM). The primary outcomes were EmCS-IFC and a composite of severe adverse neonatal outcomes (SCNO). Nine thousand sixty-one pregnancies were included, 3.3% with a PAPP-A ≤ 0.4 MoM. Low maternal PAPP-A was not associated with an increased risk of EmCS-IFC (adjusted odds ratio (aOR) 0.77, 95% confidence interval (CI) 0.24-2.46, p = 0.66) or SCNO (aOR 0.65, 95% CI 0.39-1.07, p = 0.09). Low PAPP-A was associated with increased odds of pre-eclampsia, preterm birth and birthweight < 10th centile. In conclusion, low maternal PAPP-A level is not associated with an increased risk of EmCS IFC or adverse neonatal outcomes despite greater odds of low-birthweight infants and preterm birth.

The physiology of intrapartum fetal compromise at term.

Uterine contractions in labor result in a 60% reduction in uteroplacental perfusion, causing transient fetal and placental hypoxia. A healthy term fetus with a normally developed placenta is able to accommodate this transient hypoxia by activation of the peripheral chemoreflex, resulting in a reduction in oxygen consumption and a centralization of oxygenated blood to critical organs, namely the heart, brain, and adrenals. Providing there is adequate time for placental and fetal reperfusion between contractions, these fetuses will be able to withstand prolonged periods of intermittent hypoxia and avoid severe hypoxic injury. However, there exists a cohort of fetuses in whom abnormal placental development in the first half of pregnancy results in failure of endovascular invasion of the spiral arteries by the cytotrophoblastic cells and inadequate placental angiogenesis. This produces a high-resistance, low-flow circulation predisposing to hypoperfusion, hypoxia, reperfusion injury, and oxidative stress within the placenta. Furthermore, this renders the placenta susceptible to fluctuations and reduction in uteroplacental perfusion in response to external compression and stimuli (as occurs in labor), further reducing fetal capillary perfusion, placing the fetus at risk of inadequate gas/nutrient exchange. This placental dysfunction predisposes the fetus to intrapartum fetal compromise. In the absence of a rare catastrophic event, intrapartum fetal compromise occurs as a gradual process when there is an inability of the fetal heart to respond to the peripheral chemoreflex to maintain cardiac output. This may arise as a consequence of placental dysfunction reducing pre-labor myocardial glycogen stores necessary for anaerobic metabolism or due to an inadequate placental perfusion between contractions to restore fetal oxygen and nutrient exchange. If the hypoxic insult is severe enough and long enough, profound multiorgan injury and even death may occur. This review provides a detailed synopsis of the events that can result in placental dysfunction, how this may predispose to intrapartum fetal hypoxia, and what protective mechanisms are in place to avoid hypoxic injury.

Pathophysiological changes associated with sleep disordered breathing and supine sleep position in pregnancy.

Sleep is a complex and active physiological process that if disrupted, can result in adverse outcomes both within and outside of pregnancy. Sleep disordered breathing (SDB) occurs in 10-32% of pregnancies. Substantial physiological changes occur during pregnancy that impact on maternal sleep, which typically deteriorates with advancing gestation. Pregnancy challenges maternal homeostatic regulation of many systems which effect maternal sleep, including the respiratory, cardiovascular, endocrine, and immune systems. SDB can result from varying degrees of airway compromise and potentially cause systemic hypoxia. The hypoxia may be acute, intermittent or chronic in nature with complications dependant on the duration and the gestation at which the insult occurs. It is unlikely that this effect is mediated by a singular mechanistic pathway but results from a complex cascade of events across multiple maternal organ systems. Regardless of the etiology, both SDB and supine sleep position are associated with a variety of obstetric and perinatal complications including, pre-eclampsia/eclampsia, gestational diabetes mellitus, cardiomyopathy, heart failure, fetal growth restriction, poor neonatal condition at birth, stillbirth and neuro-psychiatric problems in offspring. Both maternal sleep position and sleep disordered breathing are potentially modifiable or treatable factors that if addressed have the potential to improve maternal and fetal outcomes. This narrative review summarizes the maternal and placental pathophysiological aberrations associated with sleep disordered breathing and supine sleep position in pregnancy.

A low fetal cerebroplacental ratio confers a greater risk of intrapartum fetal compromise and adverse neonatal outcomes in low risk multiparous women at term.

BACKGROUND: A low fetal cerebroplacental ratio (CPR) and nulliparity have independently been shown to be associated with adverse obstetric and perinatal outcomes. OBJECTIVES: To assess the effect of parity on the CPR and investigate the utility of a CPR threshold of <10th centile for predicting adverse outcomes. We hypothesised that nulliparous women would have a lower CPR than multiparous women, impacting the diagnostic performance of the <10th centile threshold. This is an important consideration for interpretation of a low CPR in clinical practice. STUDY DESIGN: This was a retrospective cohort study of low risk, singleton pregnancies delivering at term in Australia's largest maternity hospital. The primary outcome was emergency caesarean section for intrapartum fetal compromise (EmCS IFC). Data was dichotomised according to parity and further by CPR <10th centile. Multiple logistic regression was performed. RESULTS: 4737 women were included for analysis, 2333 were nulliparous and 2404 were multiparous. Overall the z-score (mean [SD])(CPR standardised for gestation) was lower in nulliparous compared to multiparous women (-0.16 [-1.73 - 1.42] vs 0.04 [-1.63 - 1.69], p < 0.001). Multiparous women had a non-significantly lower mean z-score for those who delivered by EmCS IFC than nulliparous women (-0.52 [-2.23 - 2.02] vs -0.45 [-2.22 - 1.1]). Nulliparous women had greater odds of having a CPR <10th centile compared to the multiparous cohort (OR 1.24, 95% CI 1.02-1.5 vs. OR 0.81, 95% CI 0.7-0.98, p < 0.001). A CPR thresholdd <10th centile in nulliparous women was associated with increased odds of intrapartum fetal compromise (IFC), EmCS IFC (aOR 1.72, 95CI 1.2-2.6, p < 0.05) and birthweight <10th centile. A low CPR in multiparous women was associated with increased odds of all adverse perinatal outcomes measured: IFC, meconium stained liquor, EmCS IFC (aOR 4.99, 95%CI 2.5-9.9, p < 0.001), birthweight <10th centile, acidosis, neonatal intensive care admission and severe composite neonatal outcome. These aORs were associated with specificities of >90% and false positive rates of <10% for all outcomes in multiparous women. CONCLUSIONS: A CPR <10th centile in multiparous women confers greater odds of adverse perinatal outcomes and as such of the influence of parity should be taken into account when decisions regarding clinical management are made because of a low CPR.

The intrapartum and perinatal risks of sleep-disordered breathing in pregnancy: a systematic review and metaanalysis.

OBJECTIVE DATA: Sleep-disordered breathing is an increasingly common condition in nonobstetric populations and is associated with significant morbidity. The incidence of sleep-disordered breathing in pregnancy is unknown, and it is likely that many cases go undiagnosed. STUDY: A systematic review and metaanalysis was undertaken to determine whether pregnant women who receive a diagnosis of sleep-disordered breathing are more likely to have adverse intrapartum and perinatal outcomes compared with control subjects. STUDY APPRAISAL AND SYNTHESIS METHODS: PubMed, Embase, and Cinahl databases were searched for full-text publications in English of sleep-disordered breathing and human pregnancy up to June 2017. Only studies that reported on sleep-disordered breathing in relation to gestational age or birthweight at delivery, preterm birth, mode of delivery, cord pH, Apgar score, nursery admission, stillbirth or perinatal death, meconium at delivery, or wound complications were included. RESULTS: A total of 1576 results were identified; 33 studies met inclusion criteria. Women with sleep-disordered breathing were older (mean difference, 1.66; 95% confidence interval, 1.04-2.28) and had a higher body mass index (mean difference, 3.31; 95% confidence interval, 2.30-4.32) than those who did not. Maternal sleep-disordered breathing was associated significantly with preterm birth (<37 weeks gestation; odds ratio, 1.86; 95% confidence interval, 1.50-2.31) and low birthweight (<2500 g; odds ratio, 1.67; 95% confidence interval, 1.00-2.78). These women were also less likely to have a vaginal delivery (odds ratio, 0.61; 95% confidence interval, 0.48-0.78) and to be at a higher risk of having an assisted vaginal delivery (odds ratio, 1.88; 95% confidence interval, 1.10-3.21) or a cesarean delivery (odds ratio, 1.81; 95% confidence interval, 1.55-2.11). The risk of both elective (odds ratio, 1.38; 95% confidence interval, 1.09 - 1.76) and emergency cesarean (odds ratio, 2.52; 95% confidence interval, 1.20-5.29) was increased. In addition, women with sleep-disordered breathing were at a higher risk of having an infant with a 5-minute Apgar score <7 (odds ratio, 2.14; 95% confidence interval, 1.24-3.71), stillbirth or perinatal death (odds ratio, 2.02; 95% confidence interval, 1.25-3.28), and neonatal nursery admission (odds ratio, 1.90; 95% confidence interval, 1.38-2.61). CONCLUSION: Maternal sleep-disordered breathing is associated with increased risks of adverse intrapartum and perinatal outcomes.

Pigment pattern evolution by differential deployment of neural crest and post-embryonic melanophore lineages in Danio fishes.

Latent precursors or stem cells of neural crest origin are present in a variety of post-embryonic tissues. Although these cells are of biomedical interest for roles in human health and disease, their potential evolutionary significance has been underappreciated. As a first step towards elucidating the contributions of such cells to the evolution of vertebrate form, we investigated the relative roles of neural crest cells and post-embryonic latent precursors during the evolutionary diversification of adult pigment patterns in Danio fishes. These pigment patterns result from the numbers and arrangements of embryonic melanophores that are derived from embryonic neural crest cells, as well as from post-embryonic metamorphic melanophores that are derived from latent precursors of presumptive neural crest origin. In the zebrafish D. rerio, a pattern of melanophore stripes arises during the larval-to-adult transformation by the recruitment of metamorphic melanophores from latent precursors. Using a comparative approach in the context of new phylogenetic data, we show that adult pigment patterns in five additional species also arise from metamorphic melanophores, identifying this as an ancestral mode of adult pigment pattern development. By contrast, superficially similar adult stripes of D. nigrofasciatus (a sister species to D. rerio) arise by the reorganization of melanophores that differentiated at embryonic stages, with a diminished contribution from metamorphic melanophores. Genetic mosaic and molecular marker analyses reveal evolutionary changes that are extrinsic to D. nigrofasciatus melanophore lineages, including a dramatic reduction of metamorphic melanophore precursors. Finally, interspecific complementation tests identify a candidate genetic pathway for contributing to the evolutionary reduction in metamorphic melanophores and the increased contribution of early larval melanophores to D. nigrofasciatus adult pigment pattern development. These results demonstrate an important role for latent precursors in the diversification of pigment patterns across danios. More generally, differences in the deployment of post-embryonic neural crest-derived stem cells or their specified progeny may contribute substantially to the evolutionary diversification of adult form in vertebrates, particularly in species that undergo a metamorphosis.

Essential role for puma in development of postembryonic neural crest-derived cell lineages in zebrafish.

Multipotent neural crest stem cells have been identified in late gestation amniote embryos. Yet, significant questions remain about the mechanisms by which these cells are generated, maintained, and recruited during postembryonic development. The zebrafish, Danio rerio, offers an opportunity to identify genes essential for these processes, by screening for mutants with defects in traits likely to depend on these cells during metamorphosis and adult life. One such trait is the pigment pattern formed by neural crest-derived pigment cells, or chromatophores, which include black melanophores, yellow xanthophores, and iridescent iridophores. Previous analyses have demonstrated that the adult zebrafish pigment pattern depends on the de novo differentiation of latent precursor cells during both early and late phases of pigment pattern metamorphosis. To better understand the development of these cells, in this study, we analyze the zebrafish puma mutant, which ablates most of the adult melanophores that differentiate during metamorphosis, but leaves intact early larval melanophores that differentiate during embryogenesis. We use epistasis analyses to show that puma promotes the development of both early-appearing metamorphic melanophores that depend on the kit receptor tyrosine kinase, as well as late-appearing metamorphic melanophores that depend on both the G-protein-coupled endothelin receptor b1 (ednrb1) and the kit-related fms receptor tyrosine kinase. We further demonstrate that, during pigment pattern metamorphosis, puma mutants have deficiencies in the numbers of cells expressing transcripts for kit, ednrb1, and fms, as well as the HMG domain transcription factor sox10. Because the puma mutant phenotype is temperature-sensitive, we use temperature-shift experiments to identify a critical period for puma activity during pigment pattern metamorphosis. Finally, we use cell transplantations to show that puma acts cell-autonomously to promote the expansion of pigment cell lineages during metamorphosis. These results suggest a model for the lineage diversification of neural crest stem cells during zebrafish postembryonic development.

Zebrafish puma mutant decouples pigment pattern and somatic metamorphosis.

The genetic and developmental bases for trait expression and variation in adults are largely unknown. One system in which genes and cell behaviors underlying adult traits can be elucidated is the larval-to-adult transformation of zebrafish, Danio rerio. Metamorphosis in this and many other teleost fishes resembles amphibian metamorphosis, as a variety of larval traits (e.g., fins, skin, digestive tract, sensory systems) are remodeled in a coordinated manner to generate the adult form. Among these traits is the pigment pattern, which comprises several neural crest-derived pigment cell classes, including black melanophores, yellow xanthophores, and iridescent iridophores. D. rerio embryos and early larvae exhibit a relatively simple pattern of melanophore stripes, but this pattern is transformed during metamorphosis into the more complex pattern of the adult, consisting of alternating dark (melanophore, iridophore) and light (xanthophore, iridophore) horizontal stripes. While it is clear that some pigment cells differentiate de novo during pigment pattern metamorphosis, the extent to which larval and adult pigment patterns are developmentally independent has not been known. In this study, we show that a subset of embryonic/early larval melanophores persists into adult stages in wild-type fish; thus, larval and adult pigment patterns are not completely independent in this species. We also analyze puma mutant zebrafish, derived from a forward genetic screen to isolate mutations affecting postembryonic development. In puma mutants, a wild-type embryonic/early larval pigment pattern forms, but supernumerary early larval melanophores persist in ectopic locations through juvenile and adult stages. We then show that, although puma mutants undergo a somatic metamorphosis at the same time as wild-type fish, metamorphic melanophores that normally appear during these stages are absent. The puma mutation thus decouples metamorphosis of the pigment pattern from the metamorphosis of many other traits. Nevertheless, puma mutants ultimately recover large numbers of melanophores and exhibit extensive pattern regulation during juvenile development, when the wild-type pigment pattern already would be completed. Finally, we demonstrate that the puma mutant is both temperature-sensitive and growth-sensitive: extremely severe pigment pattern defects result at a high temperature, a high growth rate, or both; whereas a wild-type pigment pattern can be rescued at a low temperature and a low growth rate. Taken together, these results provide new insights into zebrafish pigment pattern metamorphosis and the capacity for pattern regulation when normal patterning mechanisms go awry.

Temporal and cellular requirements for Fms signaling during zebrafish adult pigment pattern development.

Ectothermic vertebrates exhibit a diverse array of adult pigment patterns. A common element of these patterns is alternating dark and light stripes each comprising different classes of neural crest-derived pigment cells. In the zebrafish, Danio rerio, alternating horizontal stripes of black melanophores and yellow xanthophores are a prominent feature of the adult pigment pattern. In fms mutant zebrafish, however, xanthophores fail to develop and melanophore stripes are severely disrupted. fms encodes a type III receptor tyrosine kinase expressed by xanthophores and their precursors and is the closest known homologue of kit, which has long been studied for roles in pigment pattern development in amniotes. In this study we assess the cellular and temporal requirements for Fms activity in promoting adult pigment pattern development. By transplanting cells between fms mutants and either wild-type or nacre mutant zebrafish, we show that fms acts autonomously to the xanthophore lineage in promoting the striped arrangement of adult melanophores. To identify critical periods for fms activity, we isolated temperature sensitive alleles of fms and performed reciprocal temperature shift experiments at a range of stages from embryo to adult. These analyses demonstrate that Fms is essential for maintaining cells of the xanthophore lineage as well as maintaining the organization of melanophore stripes throughout development. Finally, we show that restoring Fms activity even at late larval stages allows essentially complete recovery of xanthophores and the development of a normal melanophore stripe pattern. Our findings suggest that fms is not required for establishing a population of precursor cells during embryogenesis but is required for recruiting pigment cell precursors to xanthophore fates, with concomitant effects on melanophore organization.