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Positive feedback driven by transcriptional regulation has long been considered a key mechanism underlying cell lineage segregation during embryogenesis. Using the developing spinal cord as a paradigm, we found that canonical, transcription‐driven feedback cannot explain robust lineage segregation of motor neuron subtypes marked by two cardinal factors, Hoxa5 and Hoxc8. We propose a feedback mechanism involving elementary microRNA–mRNA reaction circuits that differ from known feedback loop‐like structures. Strikingly, we show that a wide range of biologically plausible post‐transcriptional regulatory parameters are sufficient to generate bistable switches, a hallmark of positive feedback. Through mathematical analysis, we explain intuitively the hidden source of this feedback. Using embryonic stem cell differentiation and mouse genetics, we corroborate that microRNA–mRNA circuits govern tissue boundaries and hysteresis upon motor neuron differentiation with respect to transient morphogen signals. Our findings reveal a previously underappreciated feedback mechanism that may have widespread functions in cell fate decisions and tissue patterning. SYNOPSIS Robust cell fate decision and precise tissue boundary formation are critical for development. This study reports a feedback mechanism involving mRNA‐microRNA interactions during cell lineage segregation in mouse spinal cord development. Robust lineage segregation of mouse Hoxa5 + and Hoxc8 + motor neurons does not require canonical transcriptional feedback loops. Mathematical modeling derives a wide range of biologically plausible parameters that allow bistability to arise from post‐transcriptional networks. An intuitive interpretation of the mathematical analysis reveals a hidden feedback mechanism involving mRNA‐microRNA interactions. In vitro and in vivo experiments validate the critical roles of two microRNAs in lineage segregation and tissue boundary formation. Graphical Abstract Robust cell fate decision and precise tissue boundary formation are critical for development. This study reports a feedback mechanism involving mRNA‐microRNA interactions during cell lineage segregation in mouse spinal cord development.

Key Points Marginal zone (MZ) B cells are strategically located at the interface between the circulation and the white pulp of the spleen, where they provide a first line of defence by rapidly producing IgM and class-switched IgG antibodies in response to infections by blood-borne viruses and encapsulated bacteria. Mouse MZ B cells primarily express a non-mutated B cell receptor (BCR) and enter the circulation only to shuttle antigens to the follicle, whereas human MZ B cells express a mutated BCR and extensively recirculate. MZ B cells may also produce IgM and class-switched IgG and IgA antibodies in response to commensal antigens that physiologically translocate from the intestinal mucosa to the general circulation in the absence of infection. This innate-like humoral response may generate a ready-to-use pre-immune antibody repertoire that provides a rapid systemic line of defence not only against pathogens, but also against commensal bacteria that breach the mucosal barrier. In mice, MZ B cells originate from splenic transitional stage 2 B cells via a pathway involving the BCR, the B cell-activating factor (BAFF) receptor and the receptor NOTCH2. In humans, some MZ B cells may arise from immature or transitional B cells located in the liver and mesenteric lymph nodes via a fetal pathway involving NOTCH2, whereas other MZ B cells may emerge from transitional or germinal centre B cells located in the spleen through a postnatal pathway involving the BCR, Toll-like receptors (TLRs) and CD40. MZ B cells have a lower activation threshold than follicular B cells, which permits the rapid initiation of IgM production and of IgG- and IgA-inducing class-switch recombination (CSR) in the absence of CD40-dependent help from T follicular helper (T FH ) cells. This T cell-independent pathway requires dual BCR and TLR engagement by conserved microbial antigens together with co-stimulatory signals from dendritic cells, macrophages and neutrophils via various cytokines, including BAFF, a proliferation-inducing ligand (APRIL), interleukin-6 (IL-6), IL-10, IL-21, interferon-α (IFNα), IFNβ and CXC-chemokine ligand 10 (CXCL10). MZ B cells can also undergo T cell-dependent antibody production by following either a follicular pathway involving CD40-dependent help from germinal centre T FH cells or an extrafollicular pathway involving CD40-dependent help from extrafollicular T FH cells or invariant natural killer T (iNKT) cells. In addition to expressing CD40 ligand, T FH and iNKT cells secrete various CSR- and antibody-inducing cytokines, including IL-4, IL-21 and IFNγ. In humans, MZ B cells probably have a heterogeneous ontogeny and can undergo somatic hypermutation both before and after birth through either an extrafollicular T cell-independent or a follicular T cell-dependent pathway. The extrafollicular T cell-independent pathway diversifies MZ B cells mainly during fetal life or after postnatal exposure to conserved commensal antigens or native polysaccharides, whereas the follicular T cell-dependent pathway may diversify MZ B cells predominantly after postnatal exposure to proteins or protein-conjugated polysaccharides. Marginal zone B cells are strategically positioned at the interface between the circulation and the white pulp of the spleen, where they can mediate a rapid, innate-like humoral response against both pathogens and commensals that breach the mucosal barrier. Understanding the interplay of these cells with other immune components and the species-specific differences between mice and humans is of clinical importance. Protective responses to microorganisms involve the nonspecific but rapid defence mechanisms of the innate immune system, followed by the specific but slow defence mechanisms of the adaptive immune system. Located as sentinels at the interface between the circulation and lymphoid tissue, splenic marginal zone B cells rapidly respond to blood-borne antigens by adopting 'crossover' defensive strategies that blur the conventional boundaries of innate and adaptive immunity. This Review discusses how marginal zone B cells function as innate-like lymphocytes that mount rapid antibody responses to both T cell-dependent and T cell-independent antigens. These responses require the integration of activation signals from germline-encoded and somatically recombined receptors for microorganisms with helper signals from effector cells of the innate and adaptive immune systems.

Pluripotent embryonic stem cells (ESCs) can differentiate into any adult cell type; however, aggregates of these cells do not mimic embryonic architecture when grown in culture. To see whether mouse ESCs and their extraembryonic counterparts, trophoblast stem cells (TSCs), can recapitulate normal development, Harrison et al. combined ESCs and TSCs in an extracellular matrix culture (see the Perspective by Pera). The resultant “ETS-embryos” displayed considerable resemblance to normal embryos, even specifying mesoderm and primordial germ cells at the boundary between embryonic and extraembryonic compartments. These ETS-embryos are a genetically tractable tool for studying mammalian embryogenesis. Science , this issue p. eaal1810 ; see also p. 137 Embryonic and trophoblast stem cells self-assemble to generate a structure resembling a natural mouse embryo. Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos—ETS-embryos—depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos.

PREMISE OF THE STUDY: The flip‐leaved podocarp Retrophyllum has a disjunct extant distribution in South American and Australasian tropical rainforests and a Gondwanic fossil record since the Eocene. Evolutionary, biogeographic, and paleoecological insights from previously described fossils are limited because they preserve little foliar variation and no reproductive structures. METHODS: We investigated new Retrophyllum material from the terminal Cretaceous Lefipán, the early Eocene Laguna del Hunco, and the early/middle Eocene Río Pichileufú floras of Patagonian Argentina. We also reviewed type material of historical Eocene fossils from southern Chile. KEY RESULTS: Cretaceous Retrophyllum superstes sp. nov. is described from a leafy twig, while Eocene R. spiralifolium sp. nov. includes several foliage forms and a peduncle with 13 pollen cones. Both species preserve extensive damage from sap‐feeding insects associated with foliar transfusion tissue. The Eocene species exhibits a suite of characters linking it to both Neotropical and West Pacific Retrophyllum, along with several novel features. Retrophyllum araucoensis (Berry) comb. nov. stabilizes the nomenclature for the Chilean fossils. CONCLUSIONS: Retrophyllum is considerably older than previously thought and is a survivor of the end‐Cretaceous extinction. Much of the characteristic foliar variation and pollen‐cone morphology of the genus evolved by the early Eocene. The mixed biogeographic signal of R. spiralifolium supports vicariance and represents a rare Neotropical connection for terminal‐Gondwanan Patagonia, which is predominantly linked to extant Australasian floras due to South American extinctions. The leaf morphology of the fossils suggests significant drought vulnerability as in living Retrophyllum, indicating humid paleoenvironments.

The inner ear contains distinct sensory organs, produced sequentially by segregation from a large sensory-competent domain in the developing otic vesicle. To understand the mechanistic basis of this process, we investigated the changes in prosensory cell patterning, proliferation, and character during the segregation of some of the vestibular organs in the mouse and chicken otic vesicle. We discovered a specialised boundary domain, located at the interface of segregating organs. It is composed of prosensory cells that gradually enlarge, elongate, and are ultimately diverted from a prosensory fate. Strikingly, the boundary cells align their apical borders and constrict basally at the interface of cells expressing or not the Lmx1a transcription factor, an orthologue of Drosophila Apterous. The boundary domain is absent in Lmx1a -deficient mice, which exhibit defects in sensory organ segregation and is disrupted by the inhibition of ROCK-dependent actomyosin contractility. Altogether, our results suggest that actomyosin-dependent tissue boundaries ensure the proper separation of inner ear sensory organs and uncover striking homologies between this process and the compartmentalisation of the Drosophila wing disc by lineage-restricted boundaries.

Morphogen gradients encode positional information during development. How high patterning precision is achieved despite natural variation in both the morphogen gradients and in the readout process, is still largely elusive. Here, we show that the positional error of gradients in the mouse neural tube has previously been overestimated, and that the reported accuracy of the central progenitor domain boundaries in the mouse neural tube can be achieved with a single gradient, rather than requiring the simultaneous readout of opposing gradients. Consistently and independently, numerical simulations based on measured molecular noise levels likewise result in lower gradient variabilities than reported. Finally, we show that the patterning mechanism yields progenitor cell numbers with even greater precision than boundary positions, as gradient amplitude changes do not affect interior progenitor domain sizes. We conclude that single gradients can yield the observed developmental precision, which provides prospects for tissue engineering. Morphogen gradients encode positional information during development. Here the authors use theory and simulations to suggest a positional accuracy of single gradients that directly explains the observed precision of progenitor domain boundaries.

The remarkable regeneration capability of plant tissues or organs under culture conditions has underlain an extensive practice for decades. The initial step in plant in vitro regeneration often involves the induction of a pluripotent cell mass termed callus, which is driven by the phytohormone auxin and occurs via a root development pathway. However, the key molecules governing callus formation remain unknown. Here we demonstrate that Arabidopsis LATERAL ORGAN BOUNDARIES DOMAIN （LBD）/ASYMMETRIC LEAVES2-LIKE （ASL） transcription factors are involved in the control of callus formation program. The four LBD genes downstream of AUXIN RESPONSE FACTORs （ARFs）, LBD16, LBD17, LBD18 and LBD29, are rapidly and dramatically induced by callus-inducing medium （CIM） in multiple organs. Ectopic expression of each of the four LBD genes in Arabidopsis is sufficient to trigger spontaneous callus formation without exogenous phytohormones, whereas suppression of LBD function inhibits the callus formation induced by CIM. Moreover, the callus triggered by LBD resembles that induced by CIM by characteristics of ectopically activated root meristem genes and efficient regeneration capacity. These findings define LBD transcription factors as key regulators in the callus induction process, thereby establishing a molecular link between auxin signaling and the plant regeneration program.

The organization of muscle is the product of functional adaptation over several length scales spanning from the sarcomere to the muscle bundle. One possible strategy for solving this multiscale coupling problem is to physically constrain the muscle cells in microenvironments that potentiate the organization of their intracellular space. We hypothesized that boundary conditions in the extracellular space potentiate the organization of cytoskeletal scaffolds for directed sarcomeregenesis. We developed a quantitative model of how the cytoskeleton of neonatal rat ventricular myocytes organizes with respect to geometric cues in the extracellular matrix. Numerical results and in vitro assays to control myocyte shape indicated that distinct cytoskeletal architectures arise from two temporally-ordered, organizational processes: the interaction between actin fibers, premyofibrils and focal adhesions, as well as cooperative alignment and parallel bundling of nascent myofibrils. Our results suggest that a hierarchy of mechanisms regulate the self-organization of the contractile cytoskeleton and that a positive feedback loop is responsible for initiating the break in symmetry, potentiated by extracellular boundary conditions, is required to polarize the contractile cytoskeleton.

Collective cell migrations are essential in several physiological processes and are driven by both chemical and mechanical cues. The roles of substrate stiffness and confinement on collective migrations have been investigated in recent years, however few studies have addressed how geometric shapes influence collective cell migrations. Here, we address the hypothesis that the relative position of a cell within the confinement influences its motility. Monolayers of two types of epithelial cells--MCF7, a breast epithelial cancer cell line, and MDCK, a control epithelial cell line--were confined within circular, square, and cross-shaped stencils and their migration velocities were quantified upon release of the constraint using particle image velocimetry. The choice of stencil geometry allowed us to investigate individual cell motility within convex, straight and concave boundaries. Cells located in sharp, convex boundaries migrated at slower rates than those in concave or straight edges in both cell types. The overall cluster migration occurred in three phases: an initial linear increase with time, followed by a plateau region and a subsequent decrease in cluster speeds. An acto-myosin contractile ring, present in the MDCK but absent in MCF7 monolayer, was a prominent feature in the emergence of leader cells from the MDCK clusters which occurred every ~125 μm from the vertex of the cross. Further, coordinated cell movements displayed vorticity patterns in MDCK which were absent in MCF7 clusters. We also used cytoskeletal inhibitors to show the importance of acto-myosin bounding cables in collective migrations through translation of local movements to create long range coordinated movements and the creation of leader cells within ensembles. To our knowledge, this is the first demonstration of how bounding shapes influence long-term migratory behaviours of epithelial cell monolayers. These results are important for tissue engineering and may also enhance our understanding of cell movements during developmental patterning and cancer metastasis.

The separation of cells with distinct fates and functions is important for tissue and organ formation during animal development. Regions of different fates within tissues are often separated from another along straight boundaries. These compartment boundaries play a crucial role in tissue patterning and growth by stably positioning organizers. In Drosophila, the wing imaginal disc is subdivided into a dorsal and a ventral compartment. Cells of the dorsal, but not ventral, compartment express the selector gene apterous. Apterous expression sets in motion a gene regulatory cascade that leads to the activation of Notch signaling in a few cell rows on either side of the dorsoventral compartment boundary. Both Notch and apterous mutant clones disturb the separation of dorsal and ventral cells. Maintenance of the straight shape of the dorsoventral boundary involves a local increase in mechanical tension at cell bonds along the boundary. The mechanisms by which cell bond tension is locally increased however remain unknown. Here we use a combination of laser ablation of cell bonds, quantitative image analysis, and genetic mutants to show that Notch and Apterous are required to increase cell bond tension along the dorsoventral compartment boundary. Moreover, clonal expression of the Apterous target gene capricious results in cell separation and increased cell bond tension at the clone borders. Finally, using a vertex model to simulate tissue growth, we find that an increase in cell bond tension at the borders of cell clones, but not throughout the cell clone, can lead to cell separation. We conclude that Apterous and Notch maintain the characteristic straight shape of the dorsoventral compartment boundary by locally increasing cell bond tension.