Explore the vast range of titles available.

Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca²⁺ channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.

Morphogens are signaling molecules that convey positional information and dictate cell fates during development. Although ectopic expression in model organisms suggests that morphogen gradients form through diffusion, little is known about how morphogen gradients are created and interpreted during mammalian embryogenesis due to the combined difficulties of measuring endogenous morphogen levels and observing development in utero. Here we take advantage of a human gastruloid model to visualize endogenous Nodal protein in living cells, during specification of germ layers. We show that Nodal is extremely short range so that Nodal protein is limited to the immediate neighborhood of source cells. Nodal activity spreads through a relay mechanism in which Nodal production induces neighboring cells to transcribe Nodal. We further show that the Nodal inhibitor Lefty, while biochemically capable of long-range diffusion, also acts locally to control the timing of Nodal spread and therefore of mesoderm differentiation during patterning. Our study establishes a paradigm for tissue patterning by an activator-inhibitor pair. Studying morphogen gradient formation and reception in mammalian development is challenging. Here, the authors show with human gastruloids that Nodal activity in live cells spreads via a relay mechanism with timing that is locally controlled by Lefty, which dictates mesoderm differentiation timing.

Biological systems involving short-range activators and long-range inhibitors can generate complex patterns. Reaction-diffusion models postulate that differences in signaling range are caused by differential diffusivity of inhibitor and activator. Other models suggest that differential clearance underlies different signaling ranges. To test these models, we measured the biophysical properties of the Nodal/Lefty activator/inhibitor system during zebrafish embryogenesis. Analysis of Nodal and Lefty gradients revealed that Nodals have a shorter range than Lefty proteins. Pulse-labeling analysis indicated that Nodals and Leftys have similar clearance kinetics, whereas fluorescence recovery assays revealed that Leftys have a higher effective diffusion coefficient than Nodals. These results indicate that differential diffusivity is the major determinant of the differences in Nodal/Lefty range and provide biophysical support for reaction-diffusion models of activator/inhibitor-mediated patterning.

The establishment of left–right (LR) asymmetry is fundamental to animal development, but the identification of a unifying mechanism establishing laterality across different phyla has remained elusive. A cilia-driven, directional fluid flow is important for symmetry breaking in numerous vertebrates, including zebrafish. Alternatively, LR asymmetry can be established independently of cilia, notably through the intrinsic chirality of the acto-myosin cytoskeleton. Here, we show that Myosin1D (Myo1D), a previously identified regulator of Drosophila LR asymmetry, is essential for the formation and function of the zebrafish LR organizer (LRO), Kupffer’s vesicle (KV). Myo1D controls the orientation of LRO cilia and interacts functionally with the planar cell polarity (PCP) pathway component VanGogh-like2 (Vangl2), to shape a productive LRO flow. Our findings identify Myo1D as an evolutionarily conserved regulator of animal LR asymmetry, and show that functional interactions between Myo1D and PCP are central to the establishment of animal LR asymmetry. Left-right (LR) axis specification is essential for embryonic patterning but a unifying mechanism across organisms has not been identified. Here, the authors show that Myosin1D, known to regulate Drosophila LR asymmetry, controls zebrafish LR Organizer function, and is therefore a conserved regulator of animal laterality.

Enhancers play a critical role in regulating precise gene expression patterns essential for development and cellular identity; however, how gene-enhancer specificity is encoded within the genome is not clearly defined. To investigate how this specificity arises within topologically associated domains (TAD), we performed allele-specific genome editing of sequences surrounding the Lefty1 and Lefty2 paralogs in mouse embryonic stem cells. The Lefty genes arose from a tandem duplication event and these genes interact with each other in chromosome conformation capture assays which place these genes within the same TAD. Despite their physical proximity, we demonstrate that these genes are primarily regulated by separate enhancer elements. Through CRISPR-Cas9 mediated deletions to remove the intervening chromatin between the Lefty genes, we reveal a distance-dependent dosage effect of the Lefty2 enhancer on Lefty1 expression. These findings indicate a role for chromatin distance in insulating gene expression domains in the Lefty locus in the absence of architectural insulation.

Individuals can vary substantially in size, but the proportions of their body plans are often maintained. We generated smaller zebrafish by removing 30% of their cells at the blastula stages and found that these embryos developed into normally patterned individuals. Strikingly, the proportions of all germ layers adjusted to the new embryo size within 2 hours after cell removal. As Nodal–Lefty signalling controls germ-layer patterning, we performed a computational screen for scale-invariant models of this activator–inhibitor system. This analysis predicted that the concentration of the highly diffusive inhibitor Lefty increases in smaller embryos, leading to a decreased Nodal activity range and contracted germ-layer dimensions. In vivo studies confirmed that Lefty concentration increased in smaller embryos, and embryos with reduced Lefty levels or with diffusion-hindered Lefty failed to scale their tissue proportions. These results reveal that size-dependent inhibition of Nodal signalling allows scale-invariant patterning. Almuedo-Castillo et al. show that extirpated embryos are reduced in size but exhibit normal proportions. Following a computational screen, the authors identify an increased concentration of the Nodal inhibitor Lefty to be responsible for the size scaling.

The hindered diffusion model postulates that the movement of a signaling molecule through an embryo is affected by tissue geometry and binding-mediated hindrance, but these effects have not been directly demonstrated in vivo. Here, we visualize extracellular movement and binding of individual molecules of the activator-inhibitor signaling pair Nodal and Lefty in live developing zebrafish embryos using reflected light-sheet microscopy. We observe that diffusion coefficients of molecules are high in extracellular cavities, whereas mobility is reduced and bound fractions are high within cell-cell interfaces. Counterintuitively, molecules nevertheless accumulate in cavities, which we attribute to the geometry of the extracellular space by agent-based simulations. We further find that Nodal has a larger bound fraction than Lefty and shows a binding time of tens of seconds. Together, our measurements and simulations provide direct support for the hindered diffusion model and yield insights into the nanometer-to-micrometer-scale mechanisms that lead to macroscopic signal dispersal. Multiple models have been proposed for how diffusion is regulated to shape morphogen gradients. Here they use single molecule tracking of an activator-inhibitor signaling pair in a developing tissue to show how effective diffusivity is modulated in the extracellular space.

Adipose-derived mesenchymal stem cells (ADSCs) have exhibited promising therapeutic potential in Alzheimer’s disease (AD), although the underlying mechanisms remain poorly understood. Previously established Alzheimer’s disease neuron models derived from Ts21-induced pluripotent stem cells (Ts21-iPSCs) have been shown to exhibit progressive amyloid beta accumulation during neuronal differentiation. In this study, we employed a Transwell co-culture system to investigate the interaction between neurons derived from Ts21-iPSCs and ADSCs. Our findings revealed that co-culture with ADSCs significantly enhanced the survival rate of AD neurons. Proteomics analysis identified significant upregulation of left–right determination factor 2 (LEFTY2) protein in the co-culture medium. Supplementation with 2 nM LEFTY2 markedly improved the survival and growth of AD neurons. Furthermore, LEFTY2 effectively downregulates the expression of apolipoprotein E4 and amyloid beta 1–42, along with attenuating phosphorylated tau231 levels in AD neurons. These results suggest the potential of LEFTY2 as a promising therapeutic candidate for Alzheimer’s disease.

Mammalian primordial germ cells (PGCs) migrate asynchronously through the embryonic hindgut and dorsal mesentery to reach the gonads. We previously found that interaction with different somatic niches regulates mouse PGC proliferation along the migration route. To characterize transcriptional heterogeneity of migrating PGCs and their niches, we performed single-cell RNA sequencing of 13,262 mouse PGCs and 7868 surrounding somatic cells during migration (E9.5, E10.5, E11.5) and in anterior vs posterior locations to enrich for leading and lagging migrants. Analysis of PGCs by position revealed dynamic gene expression changes between faster or earlier migrants in the anterior and slower or later migrants in the posterior at E9.5; these differences include migration-associated actin polymerization machinery and epigenetic reprogramming-associated genes. We furthermore identified changes in signaling with various somatic niches, notably strengthened interactions with hindgut epithelium via non-canonical WNT (ncWNT) in posterior PGCs compared to anterior. Reanalysis of a previously published dataset suggests that ncWNT signaling from the hindgut epithelium to early migratory PGCs is conserved in humans. Trajectory inference methods identified putative differentiation trajectories linking cell states across timepoints and from posterior to anterior in our mouse dataset. At E9.5, we mainly observed differences in cell adhesion and actin cytoskeletal dynamics between E9.5 posterior and anterior migrants. At E10.5, we observed divergent gene expression patterns between putative differentiation trajectories from posterior to anterior, including Nodal signaling response genes Lefty1, Lefty2, and Pycr2 and reprogramming factors Dnmt1, Prc1, and Tet1 . At E10.5, we experimentally validated anterior migrant-specific Lefty1/2 upregulation via whole-mount immunofluorescence staining for LEFTY1/2 and phosphorylated SMAD2/3, suggesting that elevated autocrine Nodal signaling in migrating PGCs occurs as they near the gonadal ridges. Together, this positional and temporal atlas of mouse PGCs supports the idea that niche interactions along the migratory route elicit changes in proliferation, actin dynamics, pluripotency, and epigenetic reprogramming.

Developmental signaling pathways often activate their own inhibitors. Such inhibitory feedback has been suggested to restrict the spatial and temporal extent of signaling or mitigate signaling fluctuations, but these models are difficult to rigorously test. Here, we determine whether the ability of the mesendoderm inducer Nodal to activate its inhibitor Lefty is required for development. We find that zebrafish lefty mutants exhibit excess Nodal signaling and increased specification of mesendoderm, resulting in embryonic lethality. Strikingly, development can be fully restored without feedback: Lethal patterning defects in lefty mutants can be rescued by ectopic expression of lefty far from its normal expression domain or by spatially and temporally uniform exposure to a Nodal inhibitor drug. While drug-treated mutants are less tolerant of mild perturbations to Nodal signaling levels than wild type embryos, they can develop into healthy adults. These results indicate that patterning without inhibitory feedback is functional but fragile. During animal development, a single fertilized cell gives rise to different tissues and organs. This ‘patterning’ process depends on signaling molecules that instruct cells in different positions in the embryo to acquire different identities. To avoid mistakes during patterning, each cell must receive the correct amount of signal at the appropriate time. In a process called ‘inhibitory feedback’, a signaling molecule instructs cells to produce molecules that block its own signaling. Although inhibitory feedback is widely used during patterning in organisms ranging from sea urchins to mammals, its exact purpose is often not clear. In part this is because feedback is challenging to experimentally manipulate. Removing the inhibitor disrupts feedback, but also increases signaling. Since the effects of broken feedback and increased signaling are intertwined, any resulting developmental defects do not provide information about what feedback specifically does. In order to examine the role of feedback, it is therefore necessary to disconnect the production of the inhibitor from the signaling process. In developing embryos, a well-known signaling molecule called Nodal instructs cells to become specific types – for example, a heart or gut cell. Nodal also promotes the production of its inhibitor, Lefty. To understand how this feedback system works, Rogers, Lord et al. first removed Lefty from zebrafish embryos. These embryos had excessive levels of Nodal signaling, did not develop correctly, and could not survive. Bathing the embryos in a drug that inhibits Nodal reduced excess signaling and allowed them to develop successfully. In these drug-treated embryos, inhibitor production is disconnected from the signaling process, allowing the role of feedback to be examined. Drug-treated embryos were less able to tolerate fluctuations in Nodal signaling than normal zebrafish embryos, which could compensate for such disturbances by adjusting Lefty levels. Overall, it appears that inhibitory feedback in this patterning system is important to compensate for alterations in Nodal signaling, but is not essential for development. Understanding the role of inhibitory feedback will be useful for efforts to grow tissues and organs in the laboratory for clinical use. The results presented by Rogers, Lord et al. also suggest the possibility that drug treatments could be developed to help correct birth defects in the womb.