Harnessing the PRECISE network as a platform to strengthen global capacity for maternal and child health research in sub-Saharan Africa.

It is widely acknowledged across the global health sector that research programmes need to be designed and implemented in a way that maximise opportunities for strengthening local capacity. This paper examines how the United Kingdom Research and Innovation (UKRI) Grand Challenges Research Fund (GCRF) funded PRECISE (PREgnancy Care Integrating translational Science, Everywhere) Network has been established as a platform to strengthen global capacity for research focused on the improvement of maternal, fetal and newborn health in sub-Saharan Africa.Best practice principles outlined in an ESSENCE on Health Research report have been considered in relation to the PRECISE Network capacity-building activities described in this paper. These activities are described at the individual, programmatic and institutional levels, and successes, challenges and recommendations for future work are outlined.The paper concludes that the PRECISE leadership have an opportunity to review and refresh activity plans for capacity building at this stage in the project to build on achievements to date.

The contribution of qualitative research within the PRECISE study in sub-Saharan Africa.

The PRECISE Network is a cohort study established to investigate hypertension, fetal growth restriction and stillbirth (described as "placental disorders") in Kenya, Mozambique and The Gambia. Several pregnancy or birth cohorts have been set up in low- and middle-income countries, focussed on maternal and child health. Qualitative research methods are sometimes used alongside quantitative data collection from these cohorts. Researchers affiliated with PRECISE are also planning to use qualitative methods, from the perspective of multiple subject areas. This paper provides an overview of the different ways in which qualitative research methods can contribute to achieving PRECISE's objectives, and discusses the combination of qualitative methods with quantitative cohort studies more generally.We present planned qualitative work in six subject areas (health systems, health geography, mental health, community engagement, the implementation of the TraCer tool, and respectful maternity care). Based on these plans, with reference to other cohort studies on maternal and child health, and in the context of the methodological literature on mixed methods approaches, we find that qualitative work may have several different functions in relation to cohort studies, including informing the quantitative data collection or interpretation. Researchers may also conduct qualitative work in pursuit of a complementary research agenda. The degree to which integration between qualitative and quantitative methods will be sought and achieved within PRECISE remains to be seen. Overall, we conclude that the synergies resulting from the combination of cohort studies with qualitative research are an asset to the field of maternal and child health.

PRECISE pregnancy cohort: challenges and strategies in setting up a biorepository in sub-Saharan Africa.

BACKGROUND AND OBJECTIVE: PRECISE is a population-based, prospective pregnancy cohort study designed for deep phenotyping of pregnancies in women with placenta-related disorders, and in healthy controls. The PRECISE Network is recruiting ~ 10,000 pregnant women in three countries (The Gambia, Kenya, and Mozambique) representing sub-Saharan Africa. The principal aim is to improve our understanding of pre-eclampsia, fetal growth restriction and stillbirth. This involves the creation of a highly curated biorepository for state of the art discovery science and a rich database of antenatal variables and maternal and neonatal outcomes. Our overarching aim is to provide large sample numbers with adequate power to address key scientific questions. Here we describe our experience of establishing a biorepository in the PRECISE Network and review the issues and challenges surrounding set-up, management and scientific use. METHODS: The feasibility of collecting and processing each sample type was assessed in each setting and plans made for establishing the necessary infrastructure. Quality control (QC) protocols were established to ensure that biological samples are 'fit-for-purpose'. The management structures required for standardised sample collection and processing were developed. This included the need for transport of samples between participating countries and to external academic/commercial institutions. RESULTS: Numerous practical challenges were encountered in setting up the infrastructure including facilities, staffing, training, cultural barriers, procurement, shipping and sample storage. Whilst delaying the project, these were overcome by establishing good communication with the sites, training workshops and constant engagement with the necessary commercial suppliers. A Project Executive Committee and Biology Working Group together defined the biospecimens required to answer the research questions paying particular attention to harmonisation of protocols with other cohorts so as to enable cross-biorepository collaboration. Governance structures implemented include a Data and Sample Committee to ensure biospecimens and data will be used according to consent, and prioritisation by scientific excellence. A coordinated sample and data transfer agreement will prevent delay in sample sharing. DISCUSSION: With adequate training and infrastructure, it is possible to establish high quality sample collections to facilitate research programmes such as the PRECISE Network in sub-Saharan Africa. These preparations are pre-requisites for effective execution of a biomarker-based approach to better understand the complexities of placental disease in these settings, and others.

The PRECISE (PREgnancy Care Integrating translational Science, Everywhere) Network's first protocol: deep phenotyping in three sub-Saharan African countries.

BACKGROUND: The PRECISE (PREgnancy Care Integrating translational Science, Everywhere) Network is a new and broadly-based group of research scientists and health advocates based in the UK, Africa and North America. METHODS: This paper describes the protocol that underpins the clinical research activity of the Network, so that the investigators, and broader global health community, can have access to 'deep phenotyping' (social determinants of health, demographic and clinical parameters, placental biology and agnostic discovery biology) of women as they advance through pregnancy to the end of the puerperium, whether those pregnancies have normal outcomes or are complicated by one/more of the placental disorders of pregnancy (pregnancy hypertension, fetal growth restriction and stillbirth). Our clinical sites are in The Gambia (Farafenni), Kenya (Kilifi County), and Mozambique (Maputo Province). In each country, 50 non-pregnant women of reproductive age will be recruited each month for 1 year, to provide a final national sample size of 600; these women will provide culturally-, ethnically-, seasonally- and spatially-relevant control data with which to compare women with normal and complicated pregnancies. Between the three countries we will recruit ≈10,000 unselected pregnant women over 2 years. An estimated 1500 women will experience one/more placental complications over the same epoch. Importantly, as we will have accurate gestational age dating using the TraCer device, we will be able to discriminate between fetal growth restriction and preterm birth. Recruitment and follow-up will be primarily facility-based and will include women booking for antenatal care, subsequent visits in the third trimester, at time-of-disease, when relevant, during/immediately after birth and 6 weeks after birth. CONCLUSIONS: To accelerate progress towards the women's and children's health-relevant Sustainable Development Goals, we need to understand how a variety of social, chronic disease, biomarker and pregnancy-specific determinants health interact to result in either a resilient or a compromised pregnancy for either mother or fetus/newborn, or both. This protocol has been designed to create such a depth of understanding. We are seeking funding to maintain the cohort to better understand the implications of pregnancy complications for both maternal and child health.

Development of a cost-effective automated platform to produce human liver spheroids for basic and applied research.

Liver disease represents an increasing cause of global morbidity and mortality. Currently, liver transplant is the only treatment curative for end-stage liver disease. Donor organs cannot meet the demand and therefore scalable treatments and new disease models are required to improve clinical intervention. Pluripotent stem cells represent a renewable source of human tissue. Recent advances in three-dimensional cell culture have provided the field with more complex systems that better mimic liver physiology and function. Despite these improvements, current cell-based models are variable in performance and expensive to manufacture at scale. This is due, in part, to the use of poorly defined or cross-species materials within the process, severely affecting technology translation. To address this issue, we have developed an automated and economical platform to produce liver tissue at scale for modelling disease and small molecule screening. Stem cell derived liver spheres were formed by combining hepatic progenitors with endothelial cells and stellate cells, in the ratios found within the liver. The resulting tissue permitted the study of human liver biology 'in the dish' and could be scaled for screening. In summary, we have developed an automated differentiation system that permits reliable self-assembly of human liver tissue for biomedical application. Going forward we believe that this technology will not only serve as anin vitroresource, and may have an important role to play in supporting failing liver function in humans.

Studies on the pharmacokinetics and metabolism of a gamma-secretase inhibitor BMS-299897, and exploratory investigation of CYP enzyme induction.

BMS-299897 is a gamma-secretase inhibitor that was effective in reducing amyloid beta-peptide (A beta) in transgenic mice and guinea pigs. Therefore, pharmacokinetic and drug metabolism studies were conducted in animals to support its clinical development. The compound appeared to have low to intermediate total body clearance and was orally bioavailable (24-100%). The oral absorption of BMS-299897 from solid dosage forms appeared to be dissolution rate-limited. BMS-299897 was distributed into extravascular space (V(ss) >or= 1.3 l kg(-1)), including brain (brain-to-plasma ratio = 0.13-0.50). BMS-299897 appeared to be a P-glycoprotein (P-gp) substrate as the brain-to-plasma ratio was two-fold higher in the mdr1a knockout mouse as compared with the wild-type. Apparent autoinduction by BMS-299897 was observed in murine and rat efficacy and toxicity studies. In vitro, BMS-299897 was a weaker inducer of cytochrome P450 3A4 (CYP3A4) and a weaker transactivator of human pregnane X receptor (hPXR) as compared with rifampicin. Induction of human UGT1A and UGT2B was evaluated in primary human hepatocytes, but the results were inconclusive. A low potential for autoinduction in humans was predicted at a clinical dose of 250 mg and the prediction was consistent with the findings from a clinical multiple-dose study with BMS-299897 in probable Alzheimer's patients.

Workshop overview: scientific and regulatory challenges for the reduction, refinement, and replacement of animals in toxicity testing.

Public concern for animal welfare has been expressed through legislative control of animal use for experimental purposes since the first legislation was introduced in 1876 in the United Kingdom. Legislative control of animal use has been introduced in virtually every developed country, with major initiatives in Europe (1986) and the United States (1966 and 1985). Advances in scientific thinking resulted in the development of the concept of the three Rs--refinement, reduction, and replacement--by Russell and Burch in 1959. The field has expanded substantially since, with specialist scientific journals dedicated to alternatives, World Congresses organized to discuss the scientific and philosophical issues, and European and U.S. validation organizations being launched. Current scientific attention is focused on validation of alternative methods. The underlying scientific principles of chemical toxicity are complicated and insufficiently understood for alternative methods for all toxicity endpoints of importance in protecting human health to be available. Important lessons have been learned about how to validate methods, including the need to have prediction models available before the validation is undertaken, the need to understand the variability of the animal-based data which is to be used as the validation standard, and the need to have well-managed validation programs. Future progress will depend on the development of novel methods, which can now be validated through international collaborative efforts.

In vitro tests for teratogens: desirable endpoints, test batteries and current status of the micromass teratogen test.

Information from in vitro tests can be usefully used as a component of the risk/hazard assessment process. In vivo studies will be required to confirm the in vitro data. If the in vitro test system is designed around endpoints that reflect changes following in vivo toxic insult then it may be possible to modify the in vitro system to account for some of the discrepancies observed between in vivo and in vitro outcomes. When the discrepancy can be accounted for by low bioavailability in vivo, pharmacokinetic studies may be required to determine the relevance of the in vitro toxic concentrations. Reproductive hazard, especially teratogenicity, has been the subject of intensive in vitro test development. The observation of teratogenicity may affect the development of new products more significantly than any other type or category of reproductive toxicity. The micromass test, involving culture of differentiating rat embryo limb and midbrain cells exposed to test agents, may be useful as part of a battery of in vitro tests for teratogens. The most recent protocol for the micromass test is described, followed by a summary of validation and mechanistic studies confirming its usefulness. The test is robust in its transfer to new laboratories. Interlaboratory variability is small.

In vitro studies of the toxicity of nucleoside analogues used in the treatment of HIV infection.

The nucleoside analogues, 3'-azido-3'-deoxythymidine (AZT, zidovudine), 2',3'-dideoxyinosine (ddI, didanosine) and 2',3'-dideoxycytidine (ddC, zalcitabine), used in the treatment of human immunodeficiency virus (HIV) infection, have been associated with a number of dose-limiting toxicities in clinical studies. These include myelotoxicity (AZT), myopathy (AZT), peripheral neuropathy (ddC, ddI) and pancreatitis (ddI). Myopathy, peripheral neuropathy and pancreatitis are also observed in HIV-infected patients who have not been treated with the nucleoside analogues. Thus, nucleoside analogue toxicity can be confused with the adverse effects of HIV infection. Animal models exist for some, but not all, aspects of nucleoside analogue-related toxicity. In vitro studies have been used extensively to elucidate the mechanisms of nucleoside analogue toxicity. Cellular purine and pyrimidine metabolizing enzymes phosphorylate the analogues, which can then interact with DNA polymerases. Inhibition of one of these, HIV reverse transcriptase, is responsible for the antiviral activity of the nucleoside analogues. Toxicity is caused by inhibition of nuclear or mitochondrial DNA polymerases (or both) and by chain termination of replicating DNA at the point of insertion of the nucleoside analogue. The different toxicities observed in the case of each nucleoside analogue are most likely explained by different affinities for each of the cellular DNA polymerases.

Predicting In vivo Toxicity.

The objective of toxicity testing is to generate information that will help establish the safety of new products. Successful new in vitro tests will identify those endpoints and cell systems which provide evidence of pathologic change that can be used in determining safety. Chronic in vivo toxicity studies usually document adverse changes that are indicative of cell death in a target organ. For example, leakage of tissue specific enzymes is indicative of membrane damage and correlated with microscopic observation of cell death. Tissues, like the liver, can mount an effective regenerative response to moderate cell loss, but cell proliferation may be accompanied by the possibility of carcinogenesis. The balance between cell death and cell proliferation can make the difference between recovery, organ failure or tumour induction. Consequently, in vitro studies of toxicity have often focused on cell death as an endpoint. Molecular events preceding the irreversible commitment of a cell to die or proliferate are currently being investigated as additional potentially useful endpoints for in vitro toxicity studies. As molecular mechanisms become better understood, the number of potential endpoints for in vitro toxicity testing has greatly increased. However, at the earliest stages of interaction with the cell, it is difficult to predict whether changes induced by the xenobiotic are reversible (pharmacologic) or irreversible (pathologic) change. Changes in second messenger systems, binding to receptors, or early gene induction may explain the induction of toxicity by agents previously studied in vivo, but are potentially reversible changes that may not be useful for predicting the safety of untested products. Later changes that can be more easily identified as irreversible may provide more useful endpoints. One example is the activation of caspases in the cell as a precursor to apoptosis.

Localization and identification of cytochrome P-450 in differentiating rat embryo cells in vitro and in foetal tissue in vivo by immunocytochemistry.

The presence of the isoenzymes b, e and c of cytochrome P-450 in foetal rat limb-bud and mid-brain tissue has been investigated in vivo and in micromass cell cultures of limb-bud and mid-brain cells derived from rat embryos by a sensitive immunocytochemical technique. The cytochromes could not be detected by antibody staining at the start of the culture nor in 13-day-old embryos from which cultures were prepared. Two different antibodies directed against cytochrome P-450 revealed the ontogenic profile of the phenobarbitone-inducible b and e forms, which appeared at an earlier stage of development, day 1 of culture (equivalent to day 14 of gestation), than did the 3-methylcholanthrene-inducible c form, which appeared on day 3 of culture (equivalent to day 16 of gestation). These isoenzymes were not tissue specific. Comparison of the localization and intensity of staining of cells cultured in vitro for 5 days with tissue from the equivalent foetal developmental stage (day 18) in vivo revealed the presence of cytochrome P-450 in corresponding areas. In day 18 limb sections, cytochrome P-450 was localized in the perichondrial and myogenic tissue, which corresponded to the cells in the periphery of the chondrogenic foci in vitro. In mid-brain whole tissue, the enzyme was located in connective tissue and neurofibrils, corresponding to cells in the periphery of the foci of neurones in vitro. The correlation between in vitro and in vivo observations from time course, location and quantitative aspects, illustrated that the micromass culture technique is a valid model for metabolism studies with these specific isoenzymes.

Inducibility and functionality of rat embryonic/foetal cytochrome P-450: A study of differentiating limb-bud and mid-brain cells in vitro.

The presence of constitutive levels of cytochrome P-450 isoenzymes in cultures derived from rat embryo limb-bud (LB) and mid-brain (CNS) cells was demonstrated immunocytochemically by staining with specific monoclonal and polyclonal antibodies of cytochrome P-450. The b and e forms of cytochrome P-450 were found to be non-inducible by either in vitro co-incubation for 5 days or by transplacental maternal induction with phenobarbitone (PB), 3-methylcholanthrene (3MC) or beta-naphthoflavone (betaNF) in either cell type. Consistent with this lack of response was the observation that both in vitro and in vivo inducer treatment did not alter the toxicity of the teratogens diphenylhydantoin (DPH) or cyclophosphamide (CPA). In contrast, 3MC induction was achieved by both in vitro and transplacental regimens as gauged by the increased intensity of peroxidase staining using a monoclonal antibody to cytochrome t-450 c, in both cell types. There was also a concomitant increase in DPH toxicity (>20% gauged by a decrease in IC(50) values) in LB cells by both induction regimens but the CNS cells were refractory. betaNF induction of cytochrome P-450 was observed following in vitro and in vivo exposures in both cell types. There was no modulation of DPH or CPA toxicity after in vitro exposure to the inducers, but in vivo induction caused a strong staining reaction in both cell types, commensurate with a 30% increase in DPH toxicity in LB cells and activation of the pro-teratogen CPA. The b and e forms of cytochrome P-450 were non-inducible but it is highly likely that the c form was both inducible (by 3MC and betaNF) and functional, the latter being assessed by modulation of DPH toxicity and CPA activation. It may be possible to induce cytochrome P-450 in cells derived from embryos. The system used may be suitable for detailed investigations of the types of metabolizing systems involved in the mechanisms underlying toxicity/teratogenicity.

Characterization of rabbit primary proximal tubule kidney cell cultures grown on Millicell-HA membrane filters.

With the aim of developing a kidney cell culture system that can be used to assess renal toxicity in vivo, freshly isolated rabbit proximal tubules were plated on Millipore cellulose filters mounted in plastic inserts (Millicell-HA). DNA synthesis peaked on day 6 of culture and cells reached confluency by days 12-14. The integrity of the monolayer was confirmed by exclusion of [(14)C]inulin and cell viability demonstrated by linearity of protein synthesis over a 24-hr period. In confluent cultures, the organic anion, [(14)C]p-aminohippuric acid (PAH) and cation [(14)C]tetraethylammonium bromide (TEA) were shown to be transported from the basolateral to the apical side at a rate 5-6 times greater than that from the apical to basolateral side during the first 60 min of exposure. Probenecid decreased PAH transport by 60% and N-methylnicotinamide and quinine inhibited TEA transport by 40 and 56%, respectively. Uptake of [(14)C]alpha-methylglucopyranoside into the cells was three times greater when label was added to the apical side than when label was added to the basolateral side. Apical uptake of glucose was sodium dependent and inhibited by more than 90% with phloridzin. Thus, kidney proximal tubule cells in the filter insert culture system display functional polarity which appears to mimic function in vivo and may be useful for examining mechanisms of nephrotoxicity.

Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community--academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.

An in vitro test for teratogens: its practical application.

An in vitro test for teratogens has been used successfully for more than 3 years. The method involves exposing undifferentiated rat embryo midbrain and limb cells to test compounds and observing the effect on subsequent cell differentiation. Experience of using the test has confirmed the accuracy of prediction (greater than 90%) suggested by a blind trial. The test has been used at the early stages of pharmacological evaluation in the selection of non-teratogenic pharmaceuticals and 250 compounds are tested on average each year. Maternal metabolism is modelled by the inclusion of Aroclor 1254-induced rat liver homogenate plus cofactors (S-9 mix). The concentrations of S-9 mix (50-100 microliters/ml culture medium) conventionally used in the Ames bacterial mutagenicity test are toxic to rat embryo cells, but greatly reduced concentrations (3-5 microliters/ml) are not toxic but are still able to activate pro-teratogens such as cyclophosphamide. However, most potentially teratogenic compounds tested are toxic in the absence of active preparations of drug-metabolizing enzymes. The conclusion that most teratogens are direct acting may be premature, since evidence has been found for drug-metabolizing activity in the embryo cells themselves.

In vitro metabolism of teratogens by differentiating rat embryo cells.

Rapid and accurate prediction of teratogenic hazard had been achieved using cultures of differentiating limb mesenchyme (LB) and midbrain (CNS) cells from 13-day-old rat embryos. In this study we have used these cultures to examine the role of metabolism in the in vitro teratogenic activity of diphenylhydantoin (DPH) and cyclophosphamide (CPA). Two approaches were used. The first involved modulation of cytochrome P-450 activity by co-incubation in vitro with a variety of inhibitors at concentrations that were non-cytotoxic to the cells. This enhanced the toxicity of DPH by 13-82% in LB and by 3-52% in CNS cells. Benzimidazole and ellipticine caused the greatest enhancement and SKF 525A the least. DPH appears to be the proximate teratogen and there appear to be embryo-tissue cytochrome P-450s that assist in its detoxification. Following prior transplacental induction, CPA was toxic in vitro to LB cells from beta-naphthoflavone-pretreated mothers. CPA was non-toxic in cells of control, phenobarbitone- or 3-methylcholanthrene-treated embryos. Thus there appear to be inducible levels of cytochrome P-448 in embryo cells. In the second approach, positive immunocytochemical staining of the cells with both monoclonal and polyclonal P-450 antibodies identified phenobarbitone, beta-naphthoflavone- and 3-methylcholanthrene-inducible cytochrome P-450s at a constitutive level. Cytochromes P-448 (beta-naphthoflavone type) and P-450 (phenobarbitone type, PB3 fraction) were inducible, confirming that cytochrome P-450s are in fact present in the embryo cells.

In vitro toxicology: a commercial proposition?

1. The commercial value of any test system must be equated with its scientific value. In vitro toxicity tests only have scientific value when they are accurate models of toxicity in vivo. 2. Toxicity is a complex event so that the simple tests of cell viability are unlikely to be useful models as these tests often use cell lines which are functionally remote from cells in tissues of the whole animal. 3. Primary cultures retain their differentiated function for hours (hepatocytes) or days (nerve cells) and may acquire differentiated functions in vitro (embryo cells) and are the models of choice for predicting toxic hazard. 4. When the in vitro test is a satisfactory predictive model of toxicity there are sound commercial reasons for its use. It saves time and effort and can be used at an early stage in compound development to sort out the toxic from the non-toxic candidate compounds. Large numbers of structurally similar compounds can be tested (QSAR studies) and a greater understanding of the mechanism of toxic action can often be achieved than in vivo because of the numbers of animals required and the interplay of different organ systems in the toxic events that make interpretation difficult. Thus the in vitro test can not only save animals but can also save compounds.

A micromass culture method for rat embryonic neural cells.

A method of culturing early (13-day) rat embryo neural cells is described. Undifferentiated neural epithelium is disaggregated and cultured in small discrete islands. Cells that destined to differentiate as neurons actively segregate from the other cells in the island and aggregate together into small clumps. Other cells flatten and attach to the substrate and resemble typical fibroblasts throughout the culture period. The clumps of preneuron cells spread out forming large irregular foci. Spreading is mediated by active cell movements. Cells in the foci differentiate as a pure population of neurons identifiable by specific inhibition of 3H-labelled gamma-amino butyric acid incorporation or by labelling with a monoclonal antibody to GQ-ganglioside. The ganglioside is not found on the cell surface at the start of culture after trypsinization, but emerges during the 5 days of culture. The antigen is similarly not present in the embryonic mesencephalon in vivo at 13 days post coitum, only emerging later in the differentiated midbrain. There is thus an apparent de novo synthesis, which is paralleled in vivo and in vitro.

Cell behaviour and cleft palate in the mutant mouse, amputated.

Cleft palate with a genetic origin normally arises because of a failure of the palatal shelves to elevate or fuse. Until now attention in studies of palatal development development has been focused on two critical phases, those of elevation and fusion. In the mutant mouse, amputated, however, cleft palate arise because of a failure of the palatal shelves to make any significant outgrowth between the 12th day after conception when the palatal shelves are first observed and the 14th day when elevation and fusion normally occur. When cell proliferation (mitotic index) was measured in the palatal shelves on days 12, 13 and 14 no difference was found between mutant and normal. The failure of the mutant palate to grow cannot be accounted for on grounds of reduced cell proliferation. For this reason the palatal mesenchyme in 12.5-day and 14.5-day normal and amputated mice has been studied wit the scanning electron microscope. This work shows that the mesenchymal cells in the mutant palate are clumped together and have much greater areas of cell contact than are observed in the normal palate. The abnormal cell behaviour described in mutant palatal mesenchyme is typical of amputated embryonic mesenchyme in general, and in other cases has been shown to cause abnormal morphogenesis. We propose that aberrant cell behaviour causing an aggregation through increased cell adhesion inhibits palatal outgrowth in the mutant, and for this reason the palatal shelves subsequently fail to elevate and fuse.