The FAM13A Long Isoform Regulates Cilia Movement and Co-ordination in Airway Mucociliary Transport.

SNPs in the FAM13A locus are amongst the most commonly reported risk alleles associated with chronic obstructive pulmonary disease (COPD) and other respiratory diseases, however the physiological role of FAM13A is unclear. In humans, two major protein isoforms are expressed at the FAM13A locus: 'long' and 'short', but their functions remain unknown, partly due to a lack of isoform conservation in mice. We performed in-depth characterisation of organotypic primary human airway epithelial cell subsets and show that multiciliated cells predominantly express the FAM13A long isoform containing a putative N-terminal Rho GTPase activating protein (RhoGAP) domain. Using purified proteins, we directly demonstrate RhoGAP activity of this domain. In Xenopus laevis, which conserve the long isoform, Fam13a-deficiency impaired cilia-dependent embryo motility. In human primary epithelial cells, long isoform deficiency did not affect multiciliogenesis but reduced cilia co-ordination in mucociliary transport assays. This is the first demonstration that FAM13A isoforms are differentially expressed within the airway epithelium, with implications for the assessment and interpretation of SNP effects on FAM13A expression levels. We also show that the long FAM13A isoform co-ordinates cilia-driven movement, suggesting that FAM13A risk alleles may affect susceptibility to respiratory diseases through deficiencies in mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

Ventricular, atrial, and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak.

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.

Placentation defects are highly prevalent in embryonic lethal mouse mutants.

Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.

Behavioral and neural responses to high-strength magnetic fields are reduced in otolith mutant mice.

Static high magnetic fields (MFs) interact with the vestibular system of humans and rodents. In rats and mice, exposure to MFs causes perturbations such as head movements, circular locomotion, suppressed rearing, nystagmus, and conditioned taste aversion acquisition. To test the role of otoconia, two mutant mouse models were examined, head-tilt Nox3het (het) and tilted Otop1 (tlt), with mutations, respectively, in Nox3, encoding the NADPH oxidase 3 enzyme, and Otop1, encoding the otopetrin 1 proton channel, which are normally expressed in the otolith organs, and are critical for otoconia formation. Consequently, both mutants show a near complete loss of otoconia in the utricle and saccule, and are nonresponsive to linear acceleration. Mice were exposed to a 14.1 Tesla MF for 30 min. After exposure, locomotor activity, conditioned taste aversion and c-Fos (in het) were assessed. Wild-type mice exposed to the MF showed suppressed rearing, increased latency to rear, locomotor circling, and c-Fos in brainstem nuclei related to vestibular processing (prepositus, spinal vestibular, and supragenual nuclei). Mutant het mice showed no response to the magnet and were similar to sham animals in all assays. Unlike het, tlt mutants exposed to the MF showed significant locomotor circling and suppressed rearing compared with sham controls, although they failed to acquire a taste aversion. The residual responsiveness of tlt versus het mice might reflect a greater semicircular deficit in het mice. These results demonstrate the necessity of the otoconia for the full effect of exposure to high MFs, but also suggest a semicircular contribution.

PAWS1 controls cytoskeletal dynamics and cell migration through association with the SH3 adaptor CD2AP.

Our previous studies of PAWS1 (protein associated with SMAD1; also known as FAM83G) have suggested that this molecule has roles beyond BMP signalling. To investigate these roles, we have used CRISPR/Cas9 to generate PAWS1-knockout U2OS osteosarcoma cells. Here, we show that PAWS1 plays a role in the regulation of the cytoskeletal machinery, including actin and focal adhesion dynamics, and cell migration. Confocal microscopy and live cell imaging of actin in U2OS cells indicate that PAWS1 is also involved in cytoskeletal dynamics and organization. Loss of PAWS1 causes severe defects in F-actin organization and distribution as well as in lamellipodial organization, resulting in impaired cell migration. PAWS1 interacts in a dynamic fashion with the actin/cytoskeletal regulator CD2AP at lamellae, suggesting that its association with CD2AP controls actin organization and cellular migration. Genetic ablation of CD2AP from U2OS cells instigates actin and cell migration defects reminiscent of those seen in PAWS1-knockout cells.This article has an associated First Person interview with the first authors of the paper.

PAWS1 controls Wnt signalling through association with casein kinase 1α.

The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co-localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.

Transcriptomics of dorso-ventral axis determination in Xenopus tropicalis.

Amphibian embryos provide a powerful system to study early cell fate determination because their eggs are externally fertilised, large, and easy to manipulate. Ultraviolet (UV) or lithium chloride (LiCl) treatment are classic embryonic manipulations frequently used to perturb specification of the dorso-ventral (DV) axis by affecting the stability of the maternal Wnt mediator ß-catenin. Such treatments result in the formation of so-called ventralised or dorsalised embryos. Although these phenotypes have been well described with respect to their morphology and some aspects of gene expression, their whole transcriptomes have never been systematically characterised and compared. Here we show that at the early gastrula stage UV-treated embryos are transcriptionally more closely related to untreated embryos than to LiCl-treated embryos. Transcriptional comparisons with dissected ventral and dorsal regions of unperturbed gastrula embryos indicate that UV and LiCl treatments indeed enrich for ventral and dorsal cells, respectively. However, these treatments also affect the balance of neural induction in the ectodermal germ layer, with LiCl stimulating pro-neural BMP inhibition and UV preferentially generating epidermis because of elevated BMP levels. Thus the transcriptomes of UV- and LiCl-treated embryos can best be described as ventro-epidermalised and dorso-neuralised. These descriptions notwithstanding, our profiling reveals several hitherto uncharacterized genes with differential expression along the DV axis. At least one of these genes, a RNF220-like ubiquitin ligase, is activated dorsally by ß-catenin. Our analysis of UV/LiCl-mediated axis perturbation will enhance the mechanistic understanding of DV axis determination in vertebrates.

Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells.

The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments.

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq).

Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq.

New insights into the maternal to zygotic transition.

The initial phases of embryonic development occur in the absence of de novo transcription and are instead controlled by maternally inherited mRNAs and proteins. During this initial period, cell cycles are synchronous and lack gap phases. Following this period of transcriptional silence, zygotic transcription begins, the maternal influence on development starts to decrease, and dramatic changes to the cell cycle take place. Here, we discuss recent work that is shedding light on the maternal to zygotic transition and the interrelated but distinct mechanisms regulating the onset of zygotic transcription and changes to the cell cycle during early embryonic development.

High-resolution analysis of gene activity during the Xenopus mid-blastula transition.

The Xenopus mid-blastula transition (MBT) marks the onset of large-scale zygotic transcription, as well as an increase in cell cycle length and a loss of synchronous cell divisions. Little is known about what triggers the activation of transcription or how newly expressed genes interact with each other. Here, we use high-resolution expression profiling to identify three waves of gene activity: a post-fertilisation wave involving polyadenylation of maternal transcripts; a broad wave of zygotic transcription detectable as early as the seventh cleavage and extending beyond the MBT at the twelfth cleavage; and a shorter post-MBT wave of transcription that becomes apparent as development proceeds. Our studies have also allowed us to define a set of maternal mRNAs that are deadenylated shortly after fertilisation, and are likely to be degraded thereafter. Experimental analysis indicates that the polyadenylation of maternal transcripts is necessary for the establishment of proper levels of zygotic transcription at the MBT, and that genes activated in the second wave of expression, including Brachyury and Mixer, contribute to the regulation of genes expressed in the third. Together, our high-resolution time series and experimental studies have yielded a deeper understanding of the temporal organisation of gene regulatory networks in the early Xenopus embryo.

Identification of the zebrafish maternal and paternal transcriptomes.

Transcription is an essential component of basic cellular and developmental processes. However, early embryonic development occurs in the absence of transcription and instead relies upon maternal mRNAs and proteins deposited in the egg during oocyte maturation. Although the early zebrafish embryo is competent to transcribe exogenous DNA, factors present in the embryo maintain genomic DNA in a state that is incompatible with transcription. The cell cycles of the early embryo titrate out these factors, leading to zygotic transcription initiation, presumably in response to a change in genomic DNA chromatin structure to a state that supports transcription. To understand the molecular mechanisms controlling this maternal to zygotic transition, it is important to distinguish between the maternal and zygotic transcriptomes during this period. Here we use exome sequencing and RNA-seq to achieve such discrimination and in doing so have identified the first zygotic genes to be expressed in the embryo. Our work revealed different profiles of maternal mRNA post-transcriptional regulation prior to zygotic transcription initiation. Finally, we demonstrate that maternal mRNAs are required for different modes of zygotic transcription initiation, which is not simply dependent on the titration of factors that maintain genomic DNA in a transcriptionally incompetent state.

Patterns of ingestion of rats during chronic oral administration of lithium chloride.

Chronic lithium administration to rodents is used to explore the potential neural mechanisms of mood stabilization, as well as to model the side effects of chronic lithium on multiple organ systems. Oral administration of lithium in the maintenance diet or drinking water is convenient, but lithium can acutely affect intake and it can mediate acquisition of conditioned taste aversions (CTA). We compared ad libitum food and fluid intake by male rats with LiCl or NaCl solutions as their sole source of fluid across 20 days, with a commonly used dosage of LiCl (24 mM: 1 g / L LiCl). To quantify the pattern of intake, rats were housed in cages equipped with lickometers to detect licks and infrared photobeams to detect food access with 6-s resolution. To determine if rats formed a CTA to LiCl, they were subsequently tested with access to NaCl. Rats showed an immediate avoidance of the LiCl solution, as seen on the first day of access by an increased latency to initiate drinking and a decreased size of drinking bouts. Rats showed a differential response to LiCl vs. NaCl after as few as 5 licks. Chronic consumption of LiCl solution led to significantly decreased food and fluid intake compared to baseline, with concomitant weight loss. The decreased intake was realized by marked changes in the pattern of drinking and feeding bouts: a decrease in per-lick volume and a decrease in licks per drinking bout, and an increase in feeding bout duration resulting in an overall decrease in eating rate. Conversely, chronic NaCl access led to an increase in drinking bout number and licks/bout. The avoidance of LiCl was likely a combination of toxic effects of ingested LiCl and rapid acquisition of a learned aversion to the taste of LiCl, as shown by an extinguishable generalized aversion to NaCl solution during subsequent NaCl test days. The marked effect of chronic oral LiCl on ingestion may impact the oral dosing of lithium as well as the rat's metabolic status.

no tail integrates two modes of mesoderm induction.

During early zebrafish development the nodal signalling pathway patterns the embryo into three germ layers, in part by inducing the expression of no tail (ntl), which is essential for correct mesoderm formation. When nodal signalling is inhibited ntl fails to be expressed in the dorsal margin, but ventral ntl expression is unaffected. These observations indicate that ntl transcription is under both nodal-dependent and nodal-independent regulation. Consistent with these observations and with a role for ntl in mesoderm formation, some somites form within the tail region of embryos lacking nodal signalling. In an effort to understand how ntl is regulated and thus how mesoderm forms, we have mapped the elements responsible for nodal-dependent and nodal-independent expression of ntl in the margin of the embryo. Our work demonstrates that expression of ntl in the margin is the consequence of two separate enhancers, which act to mediate different mechanisms of mesoderm formation. One of these enhancers responds to nodal signalling, and the other to Wnt and BMP signalling. We demonstrate that the nodal-independent regulation of ntl is essential for tail formation. Misexpression of Wnt and BMP ligands can induce the formation of an ectopic tail, which contains somites, in embryos devoid of nodal signalling, and this tail formation is dependent on ntl function. Similarly, nodal-independent tail somite formation requires ntl. At later stages in development ntl is required for notochord formation, and our analysis has also led to the identification of the enhancer required for ntl expression in the developing notochord.

Orientation within a high magnetic field determines swimming direction and laterality of c-Fos induction in mice.

High-strength static magnetic fields (>7 tesla) perturb the vestibular system causing dizziness, nystagmus, and nausea in humans; and head motion, locomotor circling, conditioned taste aversion, and c-Fos induction in brain stem vestibular nuclei in rodents. To determine the role of head orientation, mice were exposed for 15 min within a 14.1-tesla magnet at six different angles (mice oriented parallel to the field with the head toward B+ at 0°; or pitched rostrally down at 45°, 90°, 90° sideways, 135°, and 180°), followed by a 2-min swimming test. Additional mice were exposed at 0°, 90°, and 180° and processed for c-Fos immunohistochemistry. Magnetic field exposure induced circular swimming that was maximal at 0° and 180° but attenuated at 45° and 135°. Mice exposed at 0° and 45° swam counterclockwise, whereas mice exposed at 135° and 180° swam clockwise. Mice exposed at 90° (with their rostral-caudal axis perpendicular to the magnetic field) did not swim differently than controls. In parallel, exposure at 0° and 180° induced c-Fos in vestibular nuclei with left-right asymmetries that were reversed at 0° vs. 180°. No significant c-Fos was induced after 90° exposure. Thus, the optimal orientation for magnetic field effects is the rostral-caudal axis parallel to the field, such that the horizontal canal and utricle are also parallel to the field. These results have mechanistic implications for modeling magnetic field interactions with the vestibular apparatus of the inner ear (e.g., the model of Roberts et al. of an induced Lorenz force causing horizontal canal cupula deflection).

Climate Change and Health: Local Government Capacity for Health Protection in Australia.

Climate change is the greatest global health threat of the 21st century, with numerous direct and indirect human health consequences. Local governments play a critical role in communities' response to climate change, both through strategies to reduce emissions and adaption plans to respond to changing climate and extreme weather events. Australian local government environmental health officers (EHOs) have the relevant skills and expertise to inform and develop adaptation plans for health protection in the context of climate change. This study used an online survey followed by phone interviews of local government management to determine the extent to which EHOs are involved in adaptation planning in health protection climate change plans. Questions were also asked to determine whether local councils are aware of EHOs' capability to contribute and to gauge the willingness of management to provide EHOs with the workload capacity to do so. The findings demonstrated that although climate adaptation and mitigation planning is occurring in local government, it is not including or considering the public health impacts on the community. Primarily, it was found that this oversight was due to a lack of awareness of the health impacts of climate change outside of a disaster or emergency scenario. Currently, EHOs are an untapped source of knowledge and skills that can contribute to climate change adaption planning. To support this, a framework of local environmental health practice was developed to assist the reconceptualization of the scope of practice required for the planning and response to climate change.

Climate Change and Health: Challenges to the Local Government Environmental Health Workforce in South Australia.

Climate change is the most urgent and significant public health risk facing the globe. In Australia, it has been identified that Environmental Health Officers/Practitioners (EHOs/EHPs, hereafter EHOs) are a currently underutilized source of knowledge and skills that can contribute to climate change adaptation planning at the local government level. The ability of local government EHOs to utilize their local knowledge and skills in human health risk assessment during a public health emergency was demonstrated through their role in the response to COVID-19. This study used a survey and follow up interviews to examine the roles and responsibilities of EHOs during the COVID-19 pandemic and used the results to examine the potential of the workforce to tackle climate change and health related issues. What worked well, what regulatory tools were helpful, how interagency collaboration worked and what barriers or hindering factors existed were also explored. A workforce review of EHOs in South Australia was also undertaken to identify current and future challenges facing EHOs and their capacity to assist in climate change preparedness. The findings demonstrated that the workforce was used in the response to COVID-19 for varying roles by councils, including in education and communication (both internally and externally) as well as monitoring and reporting compliance with directions. Notably, half the workforce believed they could have been better utilized, and the other half thought they were well utilized. The South Australian Local Government Functional Support Group (LGFSG) was praised by the workforce for a successful approach in coordinating multiagency responses and communicating directions in a timely fashion. These lessons learnt from the COVID-19 pandemic should be incorporated into climate change adaptation planning. To ensure consistent messaging and a consolidated information repository, a centralized group should be used to coordinate local government climate change adaptation plans in relation to environmental health and be included in all future emergency management response plans. The surveyed EHOs identified environmental health issues associated with climate change as the most significant future challenge; however, concerningly, participants believe that a lack of adequate resourcing, leading to workforce shortages, increasing workloads and a lack of support, is negatively impacting the workforce's preparedness to deal with these emerging issues. It was suggested that the misperception of environmental health and a failure to recognize its value has resulted in a unique dilemma where EHOs and their councils find themselves caught between managing current workload demands and issues, and endeavouring to prepare, as a priority, for emerging environmental health issues associated with climate change and insufficient resources.

In vitro cellular reprogramming to model gonad development and its disorders.

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro-derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9-mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

Generation of left ventricle-like cardiomyocytes with improved structural, functional, and metabolic maturity from human pluripotent stem cells.

Decreased left ventricle (LV) function caused by genetic mutations or injury often leads to debilitating and fatal cardiovascular disease. LV cardiomyocytes are, therefore, a potentially valuable therapeutical target. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are neither homogeneous nor functionally mature, which reduces their utility. Here, we exploit cardiac development knowledge to instruct differentiation of hPSCs specifically toward LV cardiomyocytes. Correct mesoderm patterning and retinoic acid pathway blocking are essential to generate near-homogenous LV-specific hPSC-CMs (hPSC-LV-CMs). These cells transit via first heart field progenitors and display typical ventricular action potentials. Importantly, hPSC-LV-CMs exhibit increased metabolism, reduced proliferation, and improved cytoarchitecture and functional maturity compared with age-matched cardiomyocytes generated using the standard WNT-ON/WNT-OFF protocol. Similarly, engineered heart tissues made from hPSC-LV-CMs are better organized, produce higher force, and beat more slowly but can be paced to physiological levels. Together, we show that functionally matured hPSC-LV-CMs can be obtained rapidly without exposure to current maturation regimes.

Rab5-mediated endocytosis of activin is not required for gene activation or long-range signalling in Xenopus.

Morphogen gradients provide positional cues for cell fate specification and tissue patterning during embryonic development. One important aspect of morphogen function, the mechanism by which long-range signalling occurs, is still poorly understood. In Xenopus, members of the TGF-beta family such as the nodal-related proteins and activin act as morphogens to induce mesoderm and endoderm. In an effort to understand the mechanisms and dynamics of morphogen gradient formation, we have used fluorescently labelled activin to study ligand distribution and Smad2/Smad4 bimolecular fluorescence complementation (BiFC) to analyse, in a quantitative manner, the cellular response to induction. Our results indicate that labelled activin travels exclusively through the extracellular space and that its range is influenced by numbers of type II activin receptors on responding cells. Inhibition of endocytosis, by means of a dominant-negative form of Rab5, blocks internalisation of labelled activin, but does not affect the ability of cells to respond to activin and does not significantly influence signalling range. Together, our data indicate that long-range signalling in the early Xenopus embryo, in contrast to some other developmental systems, occurs through extracellular movement of ligand. Signalling range is not regulated by endocytosis, but is influenced by numbers of cognate receptors on the surfaces of responding cells.