Signaling mechanisms that direct cell fate specification and morphogenesis in human embryonic stem cells-based models of human gastrulation.

During mammalian gastrulation, a mass of pluripotent cells surrounded by extraembryonic tissues differentiates into germ layers, mesoderm, endoderm, and ectoderm. The three germ layers are then organized into a body plan with organ rudiments via morphogenetic gastrulation movements of emboly, epiboly, convergence, and extension. Emboly is the most conserved gastrulation movement, whereby mesodermal and endodermal progenitors undergo epithelial-to-mesenchymal transition (EMT) and move via a blastopore/primitive streak beneath the ectoderm. Decades of embryologic, genetic, and molecular studies in invertebrates and vertebrates, delineated a BMP > WNT > NODAL signaling cascade underlying mesoderm and endoderm specification. Advances have been made in the research animals in understanding the cellular and molecular mechanisms underlying gastrulation morphogenesis. In contrast, little is known about human gastrulation, which occurs in utero during the third week of gestation and its investigations face ethical and methodological limitations. This is changing with the unprecedented progress in modeling aspects of human development, using human pluripotent stem cells (hPSCs), including embryonic stem cells (hESC)-based embryo-like models (SCEMs). In one approach, hESCs of various pluripotency are aggregated to self-assemble into structures that resemble pre-implantation or post-implantation embryo-like structures that progress to early gastrulation, and some even reach segmentation and neurulation stages. Another approach entails coaxing hESCs with biochemical signals to generate germ layers and model aspects of gastrulation morphogenesis, such as EMT. Here, we review the recent advances in understanding signaling cascades that direct germ layers specification and the early stages of gastrulation morphogenesis in these models. We discuss outstanding questions, challenges, and opportunities for this promising area of developmental biology.

Deletion of taf1 and taf5 in zebrafish capitulate cardiac and craniofacial abnormalities associated with TAFopathies through perturbations in metabolism.

Intellectual disability is a neurodevelopmental disorder that affects 2-3% of the general population. Syndromic forms of intellectual disability frequently have a genetic basis and are often accompanied by additional developmental anomalies. Pathogenic variants in components of TATA-binding protein associated factors (TAFs) have recently been identified in a subset of patients with intellectual disability, craniofacial hypoplasia, and congenital heart disease. This syndrome has been termed as a TAFopathy and includes mutations in TATA binding protein (TBP), TAF1, TAF2, and TAF6. The underlying mechanism by which TAFopathies give rise to neurodevelopmental, craniofacial, and cardiac abnormalities remains to be defined. Through a forward genetic screen in zebrafish, we have recovered a recessive mutant phenotype characterized by craniofacial hypoplasia, ventricular hypoplasia, heart failure at 96 h post-fertilization and lethality, and show it is caused by a nonsense mutation in taf5. CRISPR/CAS9 mediated gene editing revealed that these defects where phenocopied by mutations in taf1 and taf5. Mechanistically, taf5-/- zebrafish displayed misregulation in metabolic gene expression and metabolism as evidenced by RNA sequencing, respiration assays, and metabolite studies. Collectively, these findings suggest that the TAF complex may contribute to neurologic, craniofacial, and cardiac development through regulation of metabolism.

Dissecting cell identity via network inference and in silico gene perturbation.

Cell identity is governed by the complex regulation of gene expression, represented as gene-regulatory networks1. Here we use gene-regulatory networks inferred from single-cell multi-omics data to perform in silico transcription factor perturbations, simulating the consequent changes in cell identity using only unperturbed wild-type data. We apply this machine-learning-based approach, CellOracle, to well-established paradigms-mouse and human haematopoiesis, and zebrafish embryogenesis-and we correctly model reported changes in phenotype that occur as a result of transcription factor perturbation. Through systematic in silico transcription factor perturbation in the developing zebrafish, we simulate and experimentally validate a previously unreported phenotype that results from the loss of noto, an established notochord regulator. Furthermore, we identify an axial mesoderm regulator, lhx1a. Together, these results show that CellOracle can be used to analyse the regulation of cell identity by transcription factors, and can provide mechanistic insights into development and differentiation.

VPS13B is localized at the cis-trans Golgi complex interface and is a functional partner of FAM177A1.

Mutations in VPS13B, a member of a protein family implicated in bulk lipid transport between adjacent membranes, cause Cohen syndrome. VPS13B is known to be concentrated in the Golgi complex, but its precise location within this organelle and thus the site(s) where it achieves lipid transport remains unclear. Here we show that VPS13B is localized at the interface between cis and trans Golgi sub-compartments and that Golgi complex re-formation after Brefeldin A (BFA) induced disruption is delayed in VPS13B KO cells. This delay is phenocopied by loss of FAM177A1, a Golgi complex protein of unknown function reported to be a VPS13B interactor and whose mutations also result in a developmental disorder. In zebrafish, the vps13b orthologue, not previously annotated in this organism, genetically interacts with fam177a1. Collectively, these findings raise the possibility that bulk lipid transport by VPS13B may play a role in expanding Golgi membranes and that VPS13B may be assisted in this function by FAM177A1.

CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish.

Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). Mutant KVs were smaller, contained fewer cells and displayed decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.

The zebrafish issue: 25†years on.

In the 1990s, labs on both sides of the Atlantic performed the largest genetic mutagenesis screen at that time using an emerging model organism: the zebrafish. Led by Christiane Nüsslein-Volhard in Tübingen, Germany, and Wolfgang Driever in Boston, USA, these colossal screens culminated in 1996 with the publication of 37 articles in a special issue of Development, which remains the journal's largest issue to this day. To celebrate the anniversary of the zebrafish issue and reflect on the 25 years since its publication, five zebrafish researchers share what the issue means to them, how it has contributed to their career and its impact on the zebrafish community.

20 years of Developmental Cell: Looking back.

In our 20th anniversary year, we reflect on how the cell and developmental biology fields have changed since the publication of Developmental Cell's first few issues. In this collection of Voices, authors who published in our early issues discuss the advances that helped shape their field over the past two decades.

Model organisms contribute to diagnosis and discovery in the undiagnosed diseases network: current state and a future vision.

Decreased sequencing costs have led to an explosion of genetic and genomic data. These data have revealed thousands of candidate human disease variants. Establishing which variants cause phenotypes and diseases, however, has remained challenging. Significant progress has been made, including advances by the National Institutes of Health (NIH)-funded Undiagnosed Diseases Network (UDN). However, 6000-13,000 additional disease genes remain to be identified. The continued discovery of rare diseases and their genetic underpinnings provides benefits to affected patients, of whom there are more than 400 million worldwide, and also advances understanding the mechanisms of more common diseases. Platforms employing model organisms enable discovery of novel gene-disease relationships, help establish variant pathogenicity, and often lead to the exploration of underlying mechanisms of pathophysiology that suggest new therapies. The Model Organism Screening Center (MOSC) of the UDN is a unique resource dedicated to utilizing informatics and functional studies in model organisms, including worm (Caenorhabditis elegans), fly (Drosophila melanogaster), and zebrafish (Danio rerio), to aid in diagnosis. The MOSC has directly contributed to the diagnosis of challenging cases, including multiple patients with complex, multi-organ phenotypes. In addition, the MOSC provides a framework for how basic scientists and clinicians can collaborate to drive diagnoses. Customized experimental plans take into account patient presentations, specific genes and variant(s), and appropriateness of each model organism for analysis. The MOSC also generates bioinformatic and experimental tools and reagents for the wider scientific community. Two elements of the MOSC that have been instrumental in its success are (1) multidisciplinary teams with expertise in variant bioinformatics and in human and model organism genetics, and (2) mechanisms for ongoing communication with clinical teams. Here we provide a position statement regarding the central role of model organisms for continued discovery of disease genes, and we advocate for the continuation and expansion of MOSC-type research entities as a Model Organisms Network (MON) to be funded through grant applications submitted to the NIH, family groups focused on specific rare diseases, other philanthropic organizations, industry partnerships, and other sources of support.

Gene expression dynamics underlying cell fate emergence in 2D micropatterned human embryonic stem cell gastruloids.

Human embryonic stem cells cultured in 2D micropatterns with BMP4 differentiate into a radial arrangement of germ layers and extraembryonic cells. Single-cell transcriptomes demonstrate generation of cell types transcriptionally similar to their in vivo counterparts in Carnegie stage 7 human gastrula. Time-course analyses indicate sequential differentiation, where the epiblast arises by 12 h between the prospective ectoderm in the center and the cells initiating differentiation toward extraembryonic fates at the edge. Extraembryonic and mesendoderm precursors arise from the epiblast by 24 h, while nascent mesoderm, endoderm, and primordial germ cell-like cells form by 44 h. Dynamic changes in transcripts encoding signaling components support a BMP, WNT, and Nodal hierarchy underlying germ-layer specification conserved across mammals, and FGF and HIPPO pathways being active throughout differentiation. This work also provides a resource for mining genes and pathways expressed in a stereotyped 2D gastruloid model, common with other species or unique to human gastrulation.

Deeply-supervised density regression for automatic cell counting in microscopy images.

Accurately counting the number of cells in microscopy images is required in many medical diagnosis and biological studies. This task is tedious, time-consuming, and prone to subjective errors. However, designing automatic counting methods remains challenging due to low image contrast, complex background, large variance in cell shapes and counts, and significant cell occlusions in two-dimensional microscopy images. In this study, we proposed a new density regression-based method for automatically counting cells in microscopy images. The proposed method processes two innovations compared to other state-of-the-art density regression-based methods. First, the density regression model (DRM) is designed as a concatenated fully convolutional regression network (C-FCRN) to employ multi-scale image features for the estimation of cell density maps from given images. Second, auxiliary convolutional neural networks (AuxCNNs) are employed to assist in the training of intermediate layers of the designed C-FCRN to improve the DRM performance on unseen datasets. Experimental studies evaluated on four datasets demonstrate the superior performance of the proposed method.

Postembryonic screen for mutations affecting spine development in zebrafish.

The spine gives structural support for the adult body, protects the spinal cord, and provides muscle attachment for moving through the environment. The development and maturation of the spine and its physiology involve the integration of multiple musculoskeletal tissues including bone, cartilage, and fibrocartilaginous joints, as well as innervation and control by the nervous system. One of the most common disorders of the spine in human is adolescent idiopathic scoliosis (AIS), which is characterized by the onset of an abnormal lateral curvature of the spine of <10° around adolescence, in otherwise healthy children. The genetic basis of AIS is largely unknown. Systematic genome-wide mutagenesis screens for embryonic phenotypes in zebrafish have been instrumental in the understanding of early patterning of embryonic tissues necessary to build and pattern the embryonic spine. However, the mechanisms required for postembryonic maturation and homeostasis of the spine remain poorly understood. Here we report the results from a small-scale forward genetic screen for adult-viable recessive and dominant zebrafish mutations, leading to overt morphological abnormalities of the adult spine. Germline mutations induced with N-ethyl N-nitrosourea (ENU) were transmitted and screened for dominant phenotypes in 1229 F1 animals, and subsequently bred to homozygosity in F3 families; from these, 314 haploid genomes were screened for adult-viable recessive phenotypes affecting general body shape. We cumulatively found 40 adult-viable (3 dominant and 37 recessive) mutations each leading to a defect in the morphogenesis of the spine. The largest phenotypic group displayed larval onset axial curvatures, leading to whole-body scoliosis without vertebral dysplasia in adult fish. Pairwise complementation testing of 16 mutant lines within this phenotypic group revealed at least 9 independent mutant loci. Using massively-parallel whole genome or whole exome sequencing and meiotic mapping we defined the molecular identity of several loci for larval onset whole-body scoliosis in zebrafish. We identified a new mutation in the skolios/kinesin family member 6 (kif6) gene, causing neurodevelopmental and ependymal cilia defects in mouse and zebrafish. We also report multiple recessive alleles of the scospondin and a disintegrin and metalloproteinase with thrombospondin motifs 9 (adamts9) genes, which all display defects in spine morphogenesis. Our results provide evidence of monogenic traits that are essential for normal spine development in zebrafish, that may help to establish new candidate risk loci for spine disorders in humans.

High-resolution transcriptional and morphogenetic profiling of cells from micropatterned human ESC gastruloid cultures.

During mammalian gastrulation, germ layers arise and are shaped into the body plan while extraembryonic layers sustain the embryo. Human embryonic stem cells, cultured with BMP4 on extracellular matrix micro-discs, reproducibly differentiate into gastruloids, expressing markers of germ layers and extraembryonic cells in radial arrangement. Using single-cell RNA sequencing and cross-species comparisons with mouse, cynomolgus monkey gastrulae, and post-implantation human embryos, we reveal that gastruloids contain cells transcriptionally similar to epiblast, ectoderm, mesoderm, endoderm, primordial germ cells, trophectoderm, and amnion. Upon gastruloid dissociation, single cells reseeded onto micro-discs were motile and aggregated with the same but segregated from distinct cell types. Ectodermal cells segregated from endodermal and extraembryonic but mixed with mesodermal cells. Our work demonstrates that the gastruloid system models primate-specific features of embryogenesis, and that gastruloid cells exhibit evolutionarily conserved sorting behaviors. This work generates a resource for transcriptomes of human extraembryonic and embryonic germ layers differentiated in a stereotyped arrangement.

MYH3-associated distal arthrogryposis zebrafish model is normalized with para-aminoblebbistatin.

Distal arthrogryposis (DA) is group of syndromes characterized by congenital joint contractures. Treatment development is hindered by the lack of vertebrate models. Here, we describe a zebrafish model in which a common MYH3 missense mutation (R672H) was introduced into the orthologous zebrafish gene smyhc1 (slow myosin heavy chain 1) (R673H). We simultaneously created a smyhc1 null allele (smyhc1- ), which allowed us to compare the effects of both mutant alleles on muscle and bone development, and model the closely related disorder, spondylocarpotarsal synostosis syndrome. Heterozygous smyhc1R673H/+ embryos developed notochord kinks that progressed to scoliosis with vertebral fusions; motor deficits accompanied the disorganized and shortened slow-twitch skeletal muscle myofibers. Increased dosage of the mutant allele in both homozygous smyhc1R673H/R673H and transheterozygous smyhc1R673H/- embryos exacerbated the notochord and muscle abnormalities, causing early lethality. Treatment of smyhc1R673H/R673H embryos with the myosin ATPase inhibitor, para-aminoblebbistatin, which decreases actin-myosin affinity, normalized the notochord phenotype. Our zebrafish model of MYH3-associated DA2A provides insight into pathogenic mechanisms and suggests a beneficial therapeutic role for myosin inhibitors in treating disabling contractures.

Maternal contributions to gastrulation in zebrafish.

Gastrulation is a critical early morphogenetic process of animal development, during which the three germ layers; mesoderm, endoderm and ectoderm, are rearranged by internalization movements. Concurrent epiboly movements spread and thin the germ layers while convergence and extension movements shape them into an anteroposteriorly elongated body with head, trunk, tail and organ rudiments. In zebrafish, gastrulation follows the proliferative and inductive events that establish the embryonic and extraembryonic tissues and the embryonic axis. Specification of these tissues and embryonic axes are controlled by the maternal gene products deposited in the egg. These early maternally controlled processes need to generate sufficient cell numbers and establish the embryonic polarity to ensure normal gastrulation. Subsequently, after activation of the zygotic genome, the zygotic gene products govern mesoderm and endoderm induction and germ layer patterning. Gastrulation is initiated during the maternal-to-zygotic transition, a process that entails both activation of the zygotic genome and downregulation of the maternal transcripts. Genomic studies indicate that gastrulation is largely controlled by the zygotic genome. Nonetheless, genetic studies that investigate the relative contributions of maternal and zygotic gene function by comparing zygotic, maternal and maternal zygotic mutant phenotypes, reveal significant contribution of maternal gene products, transcripts and/or proteins, that persist through gastrulation, to the control of gastrulation movements. Therefore, in zebrafish, the maternally expressed gene products not only set the stage for, but they also actively participate in gastrulation morphogenesis.

The Reissner Fiber Is Highly Dynamic In Vivo and Controls Morphogenesis of the Spine.

Cerebrospinal fluid (CSF) physiology is important for the development and homeostasis of the central nervous system, and its disruption has been linked to scoliosis in zebrafish [1, 2]. Suspended in the CSF is an extracellular structure called the Reissner fiber, which extends from the brain through the central canal of the spinal cord. Zebrafish scospondin-null mutants are unable to assemble a Reissner fiber and fail to form a straight body axis during embryonic development [3]. Here, we describe hypomorphic missense mutations of scospondin, which allow Reissner fiber assembly and extension of a straight axis. However, during larval development, these mutants display progressive Reissner fiber disassembly, which is concomitant with the emergence of axial curvatures and scoliosis in adult animals. Using a scospondin-GFP knockin zebrafish line, we demonstrate several dynamic properties of the Reissner fiber in vivo, including embryonic fiber assembly, the continuous rostral to caudal movement of the fiber within the brain and central canal, and subcommissural organ (SCO)-spondin-GFP protein secretion from the floor plate to merge with the fiber. Finally, we show that disassembly of the Reissner fiber is also associated with the progression of axial curvatures in distinct scoliosis mutant zebrafish models. Together, these data demonstrate a critical role for the Reissner fiber for the maintenance of a straight body axis and spine morphogenesis in adult zebrafish. Our study establishes a framework for future investigations to address the cellular effectors responsible for Reissner-fiber-dependent regulation of axial morphology. VIDEO ABSTRACT.

Nodal and planar cell polarity signaling cooperate to regulate zebrafish convergence and extension gastrulation movements.

During vertebrate gastrulation, convergence and extension (C and E) of the primary anteroposterior (AP) embryonic axis is driven by polarized mediolateral (ML) cell intercalations and is influenced by AP axial patterning. Nodal signaling is essential for patterning of the AP axis while planar cell polarity (PCP) signaling polarizes cells with respect to this axis, but how these two signaling systems interact during C and E is unclear. We find that the neuroectoderm of Nodal-deficient zebrafish gastrulae exhibits reduced C and E cell behaviors, which require Nodal signaling in both cell- and non-autonomous fashions. PCP signaling is partially active in Nodal-deficient embryos and its inhibition exacerbates their C and E defects. Within otherwise naïve zebrafish blastoderm explants, however, Nodal induces C and E in a largely PCP-dependent manner, arguing that Nodal acts both upstream of and in parallel with PCP during gastrulation to regulate embryonic axis extension cooperatively.

Derivation of trophoblast stem cells from naïve human pluripotent stem cells.

Naïve human pluripotent stem cells (hPSCs) provide a unique experimental platform of cell fate decisions during pre-implantation development, but their lineage potential remains incompletely characterized. As naïve hPSCs share transcriptional and epigenomic signatures with trophoblast cells, it has been proposed that the naïve state may have enhanced predisposition for differentiation along this extraembryonic lineage. Here we examined the trophoblast potential of isogenic naïve and primed hPSCs. We found that naïve hPSCs can directly give rise to human trophoblast stem cells (hTSCs) and undergo further differentiation into both extravillous and syncytiotrophoblast. In contrast, primed hPSCs do not support hTSC derivation, but give rise to non-self-renewing cytotrophoblasts in response to BMP4. Global transcriptome and chromatin accessibility analyses indicate that hTSCs derived from naïve hPSCs are similar to blastocyst-derived hTSCs and acquire features of post-implantation trophectoderm. The derivation of hTSCs from naïve hPSCs will enable elucidation of early mechanisms that govern normal human trophoblast development and associated pathologies.

The placenta is one of the most important human organs, but it is perhaps the least understood. The first decision the earliest human cells have to make, shortly after the egg is fertilized by a sperm, is whether to become part of the embryo or part of the placenta. This choice happens before a pregnancy even implants into the uterus. The cells that commit to becoming the embryo transform into 'naïve pluripotent' cells, capable of becoming any cell in the body. Those that commit to becoming the placenta transform into 'trophectoderm' cells, capable of becoming the two types of cell in the placenta. Placental cells either invade into the uterus to anchor the placenta or produce hormones to support the pregnancy. Once a pregnancy implants into the uterus, the naïve pluripotent cells in the embryo become 'primed'. This prevents them from becoming cells of the placenta, and it poses a problem for placental research. In 2018, scientists in Japan reported conditions for growing trophectoderm cells in the laboratory, where they are known as "trophoblast stem cells". These cells were capable of transforming into specialized placental cells, but needed first to be isolated from the human embryo or placenta itself. Dong et al. now show how to reprogram other pluripotent cells grown in the laboratory to produce trophoblast stem cells. The first step was to reset primed pluripotent cells to put them back into a naïve state. Then, Dong et al. exposed the cells to the same concoction of nutrients and chemicals used in the 2018 study. This fluid triggered a transformation in the naïve pluripotent cells; they started to look like trophoblast stem cells, and they switched on genes normally active in trophectoderm cells. To test whether these cells had the same properties as trophoblast stem cells, Dong et al. gave them chemical signals to see if they could mature into placental cells. The stem cells were able to transform into both types of placental cell, either invading through a three-dimensional gel that mimics the wall of the uterus or making pregnancy hormones. There is a real need for a renewable supply of placental cells in pregnancy research. Animal placentas are not the same as human ones, so it is not possible to learn everything about human pregnancy from animal models. A renewable supply of trophoblast stem cells could aid in studying how the placenta forms and why this process sometimes goes wrong. This could help researchers to better understand miscarriage, pre-eclampsia and other conditions that affect the growth of an unborn baby. In the future, it may even be possible to make custom trophoblast stem cells to study the specific fertility issues of an individual.

Cellular and molecular mechanisms of convergence and extension in zebrafish.

Gastrulation is the period of development when the three germ layers, mesoderm, endoderm and ectoderm, are not only formed, but also shaped into a rudimentary body plan. An elongated anteroposterior (AP) axis is a key feature of all vertebrate body plans, and it forms during gastrulation through the highly conserved morphogenetic mechanism of convergence & extension (C&E). As the name suggests, this process requires that cells within each germ layer converge toward the dorsal midline to narrow the tissue in the mediolateral (ML) dimension and concomitantly extend it in the AP dimension. In a number of vertebrate species, C&E is driven primarily by mediolateral intercalation behavior (MIB), during which cells elongate, align, and extend protrusions in the ML direction and interdigitate between their neighbors. MIB is only one of many complex cellular mechanisms that contributes to C&E in zebrafish embryos, however, where a combination of individual cell migration, collective migration, random walk, radial intercalation, epiboly movements, and MIB all act together to shape the nascent germ layers. Each of these diverse cell movements is driven by a distinct suite of dynamic cellular properties/activities, such as actin-rich protrusions, myosin contractility, and blebbing. Here, we discuss the spatiotemporal patterns of cellular behaviors underlying C&E gastrulation movements within each germ layer of zebrafish embryos. These behaviors must be coordinated with the embryonic axes, and we highlight the roles of Planar Cell Polarity (PCP) in orienting and BMP signaling in patterning C&E cell behaviors with respect to the AP and dorsoventral axes. Finally, we address the role of GPCR signaling, extracellular matrix, and mechanical signals in coordination of C&E movements between adjacent germ layers.

Spatio-temporal regulation of concurrent developmental processes by generic signaling downstream of chemokine receptors.

Chemokines are secreted proteins that regulate a range of processes in eukaryotic organisms. Interestingly, different chemokine receptors control distinct biological processes, and the same receptor can direct different cellular responses, but the basis for this phenomenon is not known. To understand this property of chemokine signaling, we examined the function of the chemokine receptors Cxcr4a, Cxcr4b, Ccr7, Ccr9 in the context of diverse processes in embryonic development in zebrafish. Our results reveal that the specific response to chemokine signaling is dictated by cell-type-specific chemokine receptor signal interpretation modules (CRIM) rather than by chemokine-receptor-specific signals. Thus, a generic signal provided by different receptors leads to discrete responses that depend on the specific identity of the cell that receives the signal. We present the implications of employing generic signals in different contexts such as gastrulation, axis specification and single-cell migration.