Antenatal detection of large-for-gestational-age fetuses following implementation of the Growth Assessment Protocol: secondary analysis of a randomised control trial.

OBJECTIVE: To determine whether the Growth Assessment Protocol (GAP) affects the antenatal detection of large for gestational age (LGA) or maternal and perinatal outcomes amongst LGA babies. DESIGN: Secondary analysis of a pragmatic open randomised cluster control trial comparing the GAP with standard care. SETTING: Eleven UK maternity units. POPULATION: Pregnant women and their LGA babies born at ≥36+0  weeks of gestation. METHODS: Clusters were randomly allocated to GAP implementation or standard care. Data were collected from electronic patient records. Trial arms were compared using summary statistics, with unadjusted and adjusted (two-stage cluster summary approach) differences. MAIN OUTCOME MEASURES: Rate of detection of LGA (estimated fetal weight on ultrasound scan above the 90th centile after 34+0  weeks of gestation, defined by either population or customised growth charts), maternal and perinatal outcomes (e.g. mode of birth, postpartum haemorrhage, severe perineal tears, birthweight and gestational age, neonatal unit admission, perinatal mortality, and neonatal morbidity and mortality). RESULTS: A total of 506 LGA babies were exposed to GAP and 618 babies received standard care. There were no significant differences in the rate of LGA detection (GAP 38.0% vs standard care 48.0%; adjusted effect size -4.9%; 95% CI -20.5, 10.7; p = 0.54), nor in any of the maternal or perinatal outcomes. CONCLUSIONS: The use of GAP did not change the rate of antenatal ultrasound detection of LGA when compared with standard care.

Effect of the Growth Assessment Protocol on the DEtection of Small for GestatioNal age fetus: process evaluation from the DESiGN cluster randomised trial.

BACKGROUND: Reducing the rate of stillbirth is an international priority. At least half of babies stillborn in high-income countries are small for gestational-age (SGA). The Growth Assessment Protocol (GAP), a complex antenatal intervention that aims to increase the rate of antenatal detection of SGA, was evaluated in the DESiGN type 2 hybrid effectiveness-implementation cluster randomised trial (n = 13 clusters). In this paper, we present the trial process evaluation. METHODS: A mixed-methods process evaluation was conducted. Clinical leads and frontline healthcare professionals were interviewed to inform understanding of context (implementing and standard care sites) and GAP implementation (implementing sites). Thematic analysis of interview text used the context and implementation of complex interventions framework to understand acceptability, feasibility, and the impact of context. A review of implementing cluster clinical guidelines, training and maternity records was conducted to assess fidelity, dose and reach. RESULTS: Interviews were conducted with 28 clinical leads and 27 frontline healthcare professionals across 11 sites. Staff at implementing sites generally found GAP to be acceptable but raised issues of feasibility, caused by conflicting demands on resource, and variable beliefs among clinical leaders regarding the intervention value. GAP was implemented with variable fidelity (concordance of local guidelines to GAP was high at two sites, moderate at two and low at one site), all sites achieved the target to train > 75% staff using face-to-face methods, but only one site trained > 75% staff using e-learning methods; a median of 84% (range 78-87%) of women were correctly risk stratified at the five implementing sites. Most sites achieved high scores for reach (median 94%, range 62-98% of women had a customised growth chart), but generally, low scores for dose (median 31%, range 8-53% of low-risk women and median 5%, range 0-17% of high-risk women) were monitored for SGA as recommended. CONCLUSIONS: Implementation of GAP was generally acceptable to staff but with issues of feasibility that are likely to have contributed to variation in implementation strength. Leadership and resourcing are fundamental to effective implementation of clinical service changes, even when such changes are well aligned to policy mandated service-change priorities. TRIAL REGISTRATION: Primary registry and trial identifying number: ISRCTN 67698474. Registered 02/11/16. https://doi.org/10.1186/ISRCTN67698474 .

Is there a role for carbetocin in the prophylaxis of postpartum obstetric haemorrhage?

Postpartum haemorrhage is a common complication of pregnancy, most commonly due to uterine atony. Uterotonics have a vital role in preventing postpartum haemorrhage but the choice of the most effective agent with the fewest adverse effects is a subject of debate. Carbetocin, a synthetic analogue of oxytocin has been available in the UK since 2007 but is not currently widely used. It has a longer duration of action than oxytocin, which avoids the need for an infusion and as it is heat-stable it can be stored at room temperature. Current UK clinical guidelines, based on the results of older meta-analyses, do not recommend carbetocin as a first-line agent. A Cochrane review, published in 2018, ranked carbetocin in the top three drug regimens for preventing postpartum haemorrhage and an international consensus statement on uterotonic use for caesarean birth concluded that carbetocin may become the preferred drug for caesarean birth, by reducing the need for additional uterotonics. The higher cost of carbetocin when compared with oxytocin is a limiting factor, but the significant healthcare costs of a postpartum haemorrhage and the physiological impact of this event suggests it a reasonable alternative to consider, especially if ergometrine is contraindicated or in those who are undergoing a caesarean birth or are at high risk of bleeding.

Evaluation of the Growth Assessment Protocol (GAP) for antenatal detection of small for gestational age: The DESiGN cluster randomised trial.

BACKGROUND: Antenatal detection and management of small for gestational age (SGA) is a strategy to reduce stillbirth. Large observational studies provide conflicting results on the effect of the Growth Assessment Protocol (GAP) in relation to detection of SGA and reduction of stillbirth; to the best of our knowledge, there are no reported randomised control trials. Our aim was to determine if GAP improves antenatal detection of SGA compared to standard care. METHODS AND FINDINGS: This was a pragmatic, superiority, 2-arm, parallel group, open, cluster randomised control trial. Maternity units in England were eligible to participate in the study, except if they had already implemented GAP. All women who gave birth in participating clusters (maternity units) during the year prior to randomisation and during the trial (November 2016 to February 2019) were included. Multiple pregnancies, fetal abnormalities or births before 24+1 weeks were excluded. Clusters were randomised to immediate implementation of GAP, an antenatal care package aimed at improving detection of SGA as a means to reduce the rate of stillbirth, or to standard care. Randomisation by random permutation was stratified by time of study inclusion and cluster size. Data were obtained from hospital electronic records for 12 months prerandomisation, the washout period (interval between randomisation and data collection of outcomes), and the outcome period (last 6 months of the study). The primary outcome was ultrasound detection of SGA (estimated fetal weight <10th centile using customised centiles (intervention) or Hadlock centiles (standard care)) confirmed at birth (birthweight <10th centile by both customised and population centiles). Secondary outcomes were maternal and neonatal outcomes, including induction of labour, gestational age at delivery, mode of birth, neonatal morbidity, and stillbirth/perinatal mortality. A 2-stage cluster-summary statistical approach calculated the absolute difference (intervention minus standard care arm) adjusted using the prerandomisation estimate, maternal age, ethnicity, parity, and randomisation strata. Intervention arm clusters that made no attempt to implement GAP were excluded in modified intention to treat (mITT) analysis; full ITT was also reported. Process evaluation assessed implementation fidelity, reach, dose, acceptability, and feasibility. Seven clusters were randomised to GAP and 6 to standard care. Following exclusions, there were 11,096 births exposed to the intervention (5 clusters) and 13,810 exposed to standard care (6 clusters) during the outcome period (mITT analysis). Age, height, and weight were broadly similar between arms, but there were fewer women: of white ethnicity (56.2% versus 62.7%), and in the least deprived quintile of the Index of Multiple Deprivation (7.5% versus 16.5%) in the intervention arm during the outcome period. Antenatal detection of SGA was 25.9% in the intervention and 27.7% in the standard care arm (adjusted difference 2.2%, 95% confidence interval (CI) -6.4% to 10.7%; p = 0.62). Findings were consistent in full ITT analysis. Fidelity and dose of GAP implementation were variable, while a high proportion (88.7%) of women were reached. Use of routinely collected data is both a strength (cost-efficient) and a limitation (occurrence of missing data); the modest number of clusters limits our ability to study small effect sizes. CONCLUSIONS: In this study, we observed no effect of GAP on antenatal detection of SGA compared to standard care. Given variable implementation observed, future studies should incorporate standardised implementation outcomes such as those reported here to determine generalisability of our findings. TRIAL REGISTRATION: This trial is registered with the ISRCTN registry, ISRCTN67698474.

Pregnancy in women known to be living with a single kidney.

There is a paucity of data on pregnancy outcome in women living with a single kidney from all causes. Current thinking is extrapolated from living kidney donors, a group biased by strict selection criteria. We present a cohort of 26 women with a solitary functioning kidney; 11 women had an acquired single kidney of whom only 1 was a living donor and 15 had a congenital single kidney. Median time living with a single kidney was 28 years. None booked with hypertension or proteinuria. Urinary tract infection complicated 50% of pregnancies. Worryingly, 35% developed pre-eclampsia, gestational proteinuria or gestational hypertension. We propose pre-conceptual counselling, education on how to protect their single kidney, pre eclampsia prophylaxis with low-dose aspirin and close monitoring for urinary tract infection, hypertension and proteinuria with lower thresholds for pharmaceutical management. We have devised a Patient Information leaflet - 'Living with a single kidney, pregnancy and beyond'.

The DESiGN trial (DEtection of Small for Gestational age Neonate), evaluating the effect of the Growth Assessment Protocol (GAP): study protocol for a randomised controlled trial.

BACKGROUND: Stillbirth rates in the United Kingdom (UK) are amongst the highest of all developed nations. The association between small-for-gestational-age (SGA) foetuses and stillbirth is well established, and observational studies suggest that improved antenatal detection of SGA babies may halve the stillbirth rate. The Growth Assessment Protocol (GAP) describes a complex intervention that includes risk assessment for SGA and screening using customised fundal-height growth charts. Increased detection of SGA from the use of GAP has been implicated in the reduction of stillbirth rates by 22%, in observational studies of UK regions where GAP uptake was high. This study will be the first randomised controlled trial examining the clinical efficacy, health economics and implementation of the GAP programme in the antenatal detection of SGA. METHODS/DESIGN: In this randomised controlled trial, clusters comprising a maternity unit (or National Health Service Trust) were randomised to either implementation of the GAP programme, or standard care. The primary outcome is the rate of antenatal ultrasound detection of SGA in infants found to be SGA at birth by both population and customised standards, as this is recognised as being the group with highest risk for perinatal morbidity and mortality. Secondary outcomes include antenatal detection of SGA by population centiles, antenatal detection of SGA by customised centiles, short-term maternal and neonatal outcomes, resource use and economic consequences, and a process evaluation of GAP implementation. Qualitative interviews will be performed to assess facilitators and barriers to implementation of GAP. DISCUSSION: This study will be the first to provide data and outcomes from a randomised controlled trial investigating the potential difference between the GAP programme compared to standard care for antenatal ultrasound detection of SGA infants. Accurate information on the performance and service provision requirements of the GAP protocol has the potential to inform national policy decisions on methods to reduce the rate of stillbirth. TRIAL REGISTRATION: Primary registry and trial identifying number: ISRCTN 67698474 . Registered on 2 November 2016.

Siphonariid development: Quintessential euthyneuran larva with a mantle fold innovation (Gastropoda; Panpulmonata).

Recent phylogenetic revisions of euthyneuran gastropods ("opisthobranchs" and "pulmonates") suggest that clades with a planktotrophic larva, the ancestral life history for euthyneurans, are more widely distributed along the trunk of the euthyneuran tree than previously realized. There is some indication that the planktotrophic larva of euthyneurans has distinctive features, but information to date has come mainly from traditional "opisthobranch" groups. Much less is known about planktotrophic "pulmonate" larvae. If planktotrophic larvae of "pulmonates" share unique traits with those of "opisthobranchs," then a distinctive euthyneuran larval-type has been the developmental starting template for a spectacular amount of evolved morphological and ecological disparity among adult euthyneurans. We studied development of a siphonariid by preparing sections of larval and postmetamorphic stages for histological and ultrastructural analysis, together with 3D reconstructions and data from immunolabeling of the larval apical sensory organ. We also sought a developmental explanation for the unusual arrangement of shell-attached, dorso-ventral muscles relative to the mantle cavity of adult siphonariids. Adult siphonariids ("false limpets") have a patelliform shell but their C-shaped shell muscle partially embraces a central mantle cavity, which is different from the arrangement of these components in patellogastropods ("true limpets"). It is not obvious how shell muscles extending into the foot become placed anterior to the mantle cavity during siphonariid development from a veliger larva. We found that planktotrophic larvae of Siphonaria denticulata are extremely similar to previously described, planktotrophic "opisthobranch" larvae. To emphasize this point, we update a list of distinctive characteristics of planktotrophic euthyneuran larvae, which can anchor future studies on the impressive evolvability of this larval-type. We also describe how premetamorphic and postmetamorphic morphogenesis of larval mantle fold tissue creates the unusual arrangement of shell-muscles and mantle cavity in siphonariids. This result adds to the known postmetamorphic evolutionary innovations involving mantle fold tissue among euthyneurans.

Muscle and nerve net organization in stalked jellyfish (Medusozoa: Staurozoa).

Staurozoan cnidarians display an unusual combination of polyp and medusa characteristics and their morphology may be informative about the evolutionary origin of medusae. We studied neuromuscular morphology of two staurozoans, Haliclystus 'sanjuanensis' and Manania handi, using whole mount immunohistochemistry with antibodies against FMRFamide and α-tubulin to label neurons and phalloidin to label muscles. All muscles appeared to lack striations. Longitudinal interradial muscles are probable homologues of stalk muscles in scyphopolyps, but in adult staurozoans they are elaborated to inwardly flex marginal lobes of the calyx during prey capture; these muscles are pennate in M. handi. Manubrial perradial muscles, like the manubrium itself, are an innovation shared with pelagic medusae and manubrial interradial muscles are shared with scyphozoan ephyra. Marginal muscles of M. handi displayed occasional synchronous contraction reminiscent of a medusa swim pulse, but contractions were not repetitive. The nerve net in both species showed regional variation in density and orientation of neurons. Some areas labeled predominantly by α-tubulin antibodies (exumbrellar epidermis), other areas labeled exclusively by FMRFamide antibodies (dense plexus of neurites surrounding the base of secondary tentacles, neuronal concentration at the base of transformed primary tentacles; gastrodermal nerve net), but most areas showed a mix of neurons labeled by these two antibodies and frequent co-labeling of neurons. Transformed primary tentacles had a concentration of FMRFamide-immunoreactive neurons at their base that was associated with a pigment spot in M. handi; this is consistent with their homology with rhopalia of medusae, which are also derived from primary tentacles. The muscular system of these staurozoans embodies characteristics of both scyphopolyps and pelagic medusae. However, their nerve net is more polyp-like, although marginal concentrations of the net associated with primary and secondary tentacles may facilitate the richer behavioral repertoire of staurozoans relative to polyps of other medusozoans. J. Morphol. 278:29-49, 2017. ©© 2016 Wiley Periodicals,Inc.

The other gastropod larvae: larval morphogenesis in a marine neritimorph.

Two of the three major gastropod clades with feeding larvae are sister groups and larval morphogenesis for members of these clades, the Caenogastropoda and Heterobranchia, has been well studied. The third clade, the Neritimorpha, has an unstable phylogenetic position and little is known about development of their planktotrophic larvae. Information about larval morphology of neritimorphs and resolution of their controversial phylogenetic placement is critically important for understanding evolution of larval feeding within the Gastropoda. We describe larval morphogenesis to metamorphic competence for laboratory-reared larvae of Nerita melanotragus (Smith, 1884) (Neritimorpha: Neritidae). Preliminary observations suggest that prehatch larvae are capable of delayed hatching, possibly by entering a diapause state. Our description of larval morphogenesis, as based on tissue sections for light and transmission electron microscopy, scanning electron microscopy, three-dimensional-reconstructions of sectioned tissue, and labeling of muscles with fluorphore-tagged phalloidin, revealed four features that are unprecedented among both feeding and nonfeeding gastropod larvae. Larvae of N. melanotragus have muscles on the left and right side that both meet current criteria of a larval retractor muscle; shell-anchored muscles with oblique striations that project inside the visceral nerve loop to insert mainly on the velar lobes. They also have left and right digestive glands of similar size and a left and right hypobranchial gland. A larval "heart" is absent, but water circulation through the mantle cavity may be facilitated by large circular orifices, lined by patches of motile cilia, leading in and out of the mantle cavity. Comparison of larval traits among all three groups of gastropods with feeding larvae indicates that larvae of N. melanotragus have many unique characteristics, but they show more similarities to caenogastropod than to heterobranch larvae. These results are a significant step toward the goal of identifying primitive versus derived larval traits among feeding gastropod larvae.

Developmental modularity and phenotypic novelty within a biphasic life cycle: morphogenesis of a cone snail venom gland.

The venom gland of predatory cone snails (Conus spp.), which secretes neurotoxic peptides that rapidly immobilize prey, is a proposed key innovation for facilitating the extraordinary feeding behaviour of these gastropod molluscs. Nevertheless, the unusual morphology of this gland has generated controversy about its evolutionary origin and possible homologues in other gastropods. I cultured feeding larvae of Conus lividus and cut serial histological sections through the developing foregut during larval and metamorphic stages to examine the development of the venom gland. Results support the hypothesis of homology between the venom gland and the mid-oesophageal gland of other gastropods. They also suggest that the mid-region of the gastropod foregut, like the anterior region, is divisible into dorsal and ventral developmental modules that have different morphological, functional and ontogenetic fates. In larvae of C. lividus, the ventral module of the middle foregut transformed into the anatomically novel venom gland of the post-metamorphic stage by rapidly pinching-off from the main dorsal channel of the mid-oesophagus, an epithelial remodelling process that may be similar to other cases where epithelial tubes and vesicles arise from a pre-existing epithelial sheet. The developmental remodelling mechanism could have facilitated an abrupt evolutionary transition to the derived morphology of this important gastropod feeding innovation.

Metamorphic remodeling of a planktotrophic larva to produce the predatory feeding system of a cone snail (Mollusca, Neogastropoda).

I used histological sections and 3D reconstructions to document development through metamorphosis of the foregut and proboscis in the conoidean neogastropod Conus lividus. A goal was to determine how highly derived features of the post-metamorphic feeding system of this gastropod predator develop without interfering with larval structures for microherbivory. A second goal was to compare foregut development in this conoidean with previous observations on foregut development in the buccinoidean neogastropod Nassarius mendicus. These two neogastropods both have a feeding larval stage, but they show major differences in post-metamorphic foregut morphology. Basic events in development of the proboscis and proboscis sheath in C. lividus and N. mendicus were similar. However, the elongate buccal tube of C. lividus forms during metamorphosis as a composite of apical epidermal tissue that grows inward and ventral foregut tissue that extends outward. The larval mouth is not carried through metamorphosis. Comparative observations on foregut development in caenogastropods, which now include data on C. lividus, suggest that the foregut incorporates dorsal and ventral modules having different ontogenetic and functional fates. This developmental modularity may have facilitated evolutionary diversification of the post-metamorphic foregut. Foregut diversification in predatory gastropods may have been further fast-tracked by developmental uncoupling of larval and post-metamorphic mouths.

Embryonic toxin expression in the cone snail Conus victoriae: primed to kill or divergent function?

Predatory marine cone snails (genus Conus) utilize complex venoms mainly composed of small peptide toxins that target voltage- and ligand-gated ion channels in their prey. Although the venoms of a number of cone snail species have been intensively profiled and functionally characterized, nothing is known about the initiation of venom expression at an early developmental stage. Here, we report on the expression of venom mRNA in embryos of Conus victoriae and the identification of novel α- and O-conotoxin sequences. Embryonic toxin mRNA expression is initiated well before differentiation of the venom gland, the organ of venom biosynthesis. Structural and functional studies revealed that the embryonic α-conotoxins exhibit the same basic three-dimensional structure as the most abundant adult toxin but significantly differ in their neurological targets. Based on these findings, we postulate that the venom repertoire of cone snails undergoes ontogenetic changes most likely reflecting differences in the biotic interactions of these animals with their prey, predators, or competitors. To our knowledge, this is the first study to show toxin mRNA transcripts in embryos, a finding that extends our understanding of the early onset of venom expression in animals and may suggest alternative functions of peptide toxins during development.

Molluscan larvae: Pelagic juveniles or slowly metamorphosing larvae?

Asking the right questions about evolution of development, larval morphology, and life history requires knowledge of ancestral state. Two hypotheses dominate current opinion about the ancestral life cycle of bilaterians: the "larva-first" and the "intercalation" hypotheses. Until recently, the larva-first hypothesis was preeminent. This proposes that the original indirect life cycle of bilaterians included a planktotrophic larva followed by a benthic adult. Phylogenetic evidence suggests that a planktotrophic larva is plesiomorphic for echinoderms. A preponderance of developmental studies on echinoderms may have fostered a tendency to extrapolate conclusions about echinoderm development to other clades, particularly the concept that larval and juvenile/adult bodies are mostly separate entities. However, some of the recent reconstructions of bilaterian phylogeny suggest that nonfeeding larvae may have been ancestral for bilaterians, and these may have been intercalated into a life cycle that was originally direct. I review comparative data on molluscan development that suggests the trochophore-like stage is little more than a gastrula with transient structures (prototroch and apical sensory organ) to allow a temporary planktonic phase during development. Most lineage founder cells of molluscan embryos generate progeny that develop through the veliger stage into structures of the juvenile, which becomes benthic when the prototroch and apical sensory organ are lost. In light of this, the model of separate larval and juvenile bodies with the latter developing from nests of multipotent cells within the larva is inappropriate for molluscs. The intercalation hypothesis may be a better model for interpreting development of molluscs and other lophotrochozoans.

Shrinking to fit: fluid jettison from a haemocoelic hydrostatic skeleton during defensive withdrawals of a gastropod larva.

Although most of the basic animal body plans are supported by hydrostatic skeletons consisting of fluid maintained at constant volume, studies on how animals have solved biomechanical scaling dilemmas during evolution of large body size have emphasized cases where skeletons are formed by rigid solids. Larvae of gastropod molluscs swim using ciliated velar lobes supported by a constant volume hydrostatic skeleton. Defensive behaviour involves rapid withdrawal of the velar lobes and foot into a protective biomineralized shell. Some gastropod larvae grow to giant size and the velar lobes enlarge allometrically, but the lobes and foot of many can still withdraw completely into the mineral-stiffened shell. I dyed internal fluid of a large gastropod larva with fluorescein to show that fluid supporting the extended velar lobes is expelled from discrete release sites during defensive withdrawals. Scanning electron microscopy suggested that release sites are distinctive papillae on the upper velar epidermis. Ultrathin sections revealed that branched tracks of microvilli-free membrane on the surface of these papillae were formed by very thin epithelial cells, which may rupture and re-anneal during and after defensive withdrawals. Behaviours facilitated by fluid discharge from a haemocoelic (non-coelomic) body compartment have been rarely reported among aquatic invertebrates, but may be more widespread than currently recognized.

Diagnosis of abnormal uterine bleeding.

Abnormal uterine bleeding is an extremely common indication for referral to a gynaecologist. This chapter examines the modes of presentation and the causes of such symptoms, which range from physiological variations to more sinister underlying pathology. A thorough understanding of these causes is required to direct investigation in an appropriate manner. The full range of possible investigations is discussed with emphasis on how to choose the most appropriate tests for a particular patient. This is fundamental to ensure that tests are pertinent and streamlined, and to prevent unnecessary anxiety and delay. Once the underlying causes have been clarified, a suitable management plan can be made.

Early differentiating neuron in larval abalone (Haliotis kamtschatkana) reveals the relationship between ontogenetic torsion and crossing of the pleurovisceral nerve cords.

Crossing of the pleurovisceral nerve cords in gastropods has supported the view that gastropods evolved by 180 degrees rotation between the ventral and dorsal body regions. Indeed, a rotation of this type occurs as a dramatic morphogenetic movement ("ontogenetic torsion") during the development of basal gastropods. According to a long-standing hypothesis, ontogenetic torsion in basal gastropods preserves an ancient developmental aberration that generated the contorted gastropod body plan. It follows from this reasoning that crossing of the pleurovisceral nerve cords during gastropod development should be mechanically coupled to ontogenetic torsion. The predicted mechanical coupling can now be examined because of the discovery of an early differentiating neuron in Haliotis kamtschatkana (Vetigastropoda) that expresses 5-hydroxytryptamine-like immunoreactivity. The neuron appeared to delineate the trajectory of the pleurovisceral nerve cords beginning before ontogenetic torsion. Before torsion, the neuronal soma is embedded in mantle epithelium at the ventral midline and two neurites extend anteriorly toward the apical sensory organ. Contrary to expectation, the two neurites of this cell did not cross-over during ontogenetic torsion because the soma of this mantle neuron shifted in the same direction as the rotating head and foot. Full crossing of the pleurovisceral nerve cords occurred gradually during later development as the mantle cavity deepened and expanded leftward. These results are consistent with a generalization emerging from comparative studies indicating a conserved developmental stage for gastropods in which the mantle cavity is localized to one side, despite a fully "post-torsional" orientation for other body components. Developmental morphology before this stage is much more variable among different gastropod clades.

A role for GnRH in early brain regionalization and eye development in zebrafish.

Gonadotropin-releasing hormone (GnRH) is a highly conserved peptide that is expressed early in brain development in vertebrates. In zebrafish, we detected GnRH mRNA within 2h post fertilization by RT-PCR. To determine if GnRH is involved in development, we used gene knockdown techniques to block translation of gnrh2 or gnrh3 mRNA after which the expression patterns for gene markers were examined at 24h post fertilization with in situ hybridization. First, loss of either GnRH2 or GnRH3 affected regionalization of the brain as shown by a change in expression of fgf8 or pax2.1 genes in the midbrain-hindbrain boundary or diencephalon-midbrain boundary. Second, lack of GnRH2 and/or GnRH3 altered gene markers expressed in the formation of the eye cup (pax2.1, pax6.1, mab21l2 and meis1.1) or eye stalk (fgf8 and pax2.1). Third, knockdown of GnRH2 affected the size and shape of the midbrain and expression of gene markers therein. Results from assays with the TUNEL method and caspase-3 and -9 activity showed the brain and eye changes were unlikely to result from secondary apoptotic cell death before 24h post fertilization. These experiments suggest that GnRH loss-of-function affects early brain and eye formation during development.