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Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, we investigate how pattern refinement is regulated. We found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate competence after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. We propose that mechano-chemical feedback leads to competition for the developmental signal which plays a critical role in pattern refinement.

The Bone Morphogenetic Protein (BMP) pathway is involved in numerous developmental processes, including cell growth, apoptosis, and differentiation. In mouse embryogenesis, BMP signaling is a well-known morphogen for both mesoderm induction and germ cell development. Recent evidence points to a potential role in development of the extraembryonic compartment, including trophectoderm-derived tissues. In this study, we investigated the effect of BMP signaling in both mouse and human trophoblast stem cells (TSC) in vitro, evaluating the expression and activation of the BMP signaling response machinery, and the effect of BMP signaling manipulation during TSC maintenance and differentiation. Both mouse trophoblast stem cells (mTSC) and human trophoblast stem cells (hTSC) expressed various BMP ligands and the receptors BMPR1A and BMPR2, necessary for BMP response, and displayed maximal active BMP signaling when undifferentiated. We also observed a conserved modulatory role of BMP signaling during trophoblast differentiation, whereby maintenance of active BMP signaling blunted differentiation of TSC in both species. Conversely, the effect of BMP signaling on the undifferentiated state of TSC appeared to be species-specific, with SMAD-independent signaling important in maintenance of mTSC, and a more subtle role for both SMAD-dependent and -independent BMP signaling in hTSC. Altogether, these data establish an autocrine role for the BMP pathway in the trophoblast compartment. As specification and correct differentiation of the extraembryonic compartment are fundamental for implantation and early placental development, insights on the role of the BMP signaling in early development might prove useful in the setting of in vitro fertilization as well as targeting trophoblast-associated placental dysfunction.

Neurulation-stage alcohol exposure (NAE; embryonic day [E] 8–10) is associated with midline craniofacial and CNS defects that likely arise from disruption of morphogen pathways, such as Sonic hedgehog (Shh). Notably, midline anomalies are also a hallmark of genetic ciliopathies such as Joubert syndrome. We tested whether NAE alters Shh pathway signaling and the number and function of primary cilia, organelles critical for Shh pathway transduction. Female C57BL/6 J mice were administered two doses of alcohol (2.9 g/kg/dose) or vehicle on E9. Embryos were collected 6, 12, or 24 h later, and changes to Shh, cell cycle genes, and primary cilia were measured in the rostroventral neural tube (RVNT). Within the first 24 h post-NAE, reductions in Shh pathway and cell cycle gene expression and the ratio of Gli3 forms in the full-length activator state were observed. RVNT volume and cell layer width were reduced at 12 h. In addition, altered expression of multiple cilia-related genes was observed at 6 h post-NAE. As a further test of cilia gene-ethanol interaction, mice heterozygous for Kif3a exhibited perturbed behavior during adolescence following NAE compared to vehicle-treated mice, and Kif3a heterozygosity exacerbated the hyperactive effects of NAE on exploratory activity. These data demonstrate that NAE downregulates the Shh pathway in a region of the neural tube that gives rise to alcohol-sensitive brain structures and identifies disruption of primary cilia function, or a “transient ciliopathy”, as a possible cellular mechanism of prenatal alcohol pathogenesis.

PURPOSE OF REVIEW Primary cilia, the antenna-like organelles on most mammalian cells, host key components of multiple morphogen signal transduction pathways. Mutations in genes responsible for primary cilia assembly and function generally result in pathological conditions known as ciliopathies, which underlie several diseases, including various forms of fibrosis. Primary cilia modulate cellular responses to extracellular cues, including TGF-β and morphogens, such as Hedgehog. Aberrant morphogen signaling is recognized as essential for the transition of mesenchymal progenitor cells to myofibroblasts, the key step in fibrosis. This article aims to provide a critical overview of recent developments and insights in primary cilia biology relevant to fibrosis. RECENT FINDINGS Several studies have highlighted the association of altered primary cilia with various forms of fibrosis. In a rather complex manner, the presence of primary cilia seems to be required for initiation of myofibroblast transition, whereas its loss promotes myofibroblast transition at a later stage. Recent evidence also suggested that noncanonical functions of ciliary transport proteins may influence, such cellular transitions independently of primary cilia. The possibility of opposing signaling regulations being topologically separated between primary cilia and plasma membrane could also be critical for fibrosis. SUMMARY Recent progress in the field suggests that primary cilia are critical mediators of the pathogenesis of fibrosis. Understanding the potential role of primary cilia in fibrosis and the underlying mechanisms may pave the way for entirely new approaches for fibrosis prevention and treatment of SSc.

Morphogens of the Hh-family trigger gene expression changes of receiving cells in a concentration-dependent manner. The outputs of the pathway include regulation of cell identity, proliferation, death or metabolism, depending on the tissue or organ. This variety of responses relies on a conserved signaling pathway. Its internal logic includes a negative feedback loop involving the Hh receptor Ptc. In this paper, we use experiments and computational models to study and compare the different spatial signaling profiles downstream of Hh in several developing Drosophila organs. We show that the spatial distribution of Ptc and the activator form of the Gli transcription factor, CiA, in wing, antenna and ocellus show similar features, but markedly different from that in the compound eye (CE). We show that these two profile types represent two time points along the signaling dynamics, and that the interplay between the spatial displacement of the Hh source in the CE and the negative feedback loop maintains the receiving cells effectively in an earlier stage of signaling. These results indicate that the dynamics of the Hh source strongly influences the signaling profile Hh elicits in receiving cells, and show how the interaction between spatial and temporal dynamics of signaling and differentiation processes can contribute to the informational versatility of the conserved Hh signaling pathway.

Retinoic acid (RA) is a fundamental regulator of cell cycle and cell differentiation. Using a leukemic patient-derived in vitro model of a non-APL AML, we previously found that RA evokes activation of a macromolecular signaling complex, a signalosome, built of numerous MAPK-pathway-related signaling molecules; and this signaling enabled Retinoic-Acid-Response-Elements (RAREs) to regulate gene expression that results in cell differentiation/cell cycle arrest. Toward mechanistic insight into the nature of this novel signaling, we now find that the NUMB cell fate determinant protein is an apparent scaffold for the signalosome. Numb exists in the cell bound to an ensemble of signalosome molecules, including Raf, Lyn, Slp-76, and Vav. Addition of RA induces the expression of Fgr. Fgr binds NUMB, which is associated with (p-tyr)phosphorylation of NUMB and enhanced NUMB-binding and (p-tyr)phosphorylation of select signalosome components, thereby betraying signalosome activation. Signalosome activation is associated with cell differentiation along the myeloid lineage and G1/0 cell cycle arrest. If RA-induced Fgr expression is ablated by a CRISPR-KO; then the RA-induced (p-tyr) phosphorylation of NUMB and enhanced NUMB-binding and (p-tyr)phosphorylation of select signalosome components are lost. The cells now fail to undergo RA-induced differentiation or G1/0 arrest. In sum we find that NUMB acts as a scaffold for a signaling machine that functions to propel RA-induced differentiation and G1/0 arrest, and that Fgr binding to NUMB turns the function on. The Numb fate determinant protein thus appears to regulate the retinoic acid embryonic morphogen using the Fgr Src-Family-Kinase. These mechanistic insights suggest therapeutic targets for a hitherto incurable AML.

Sonic hedgehog acts as a key mitogen to drive cell proliferation and as a morphogen to direct cell differentiation during embryonic development and adult tissue maintenance by controlling the activities of its transcriptional effectors Glis. We previously reported that RNF220 controls the nuclear translocation and subcellular localization of Glis by promoting their K63-linked polyubiquitination, through which it fine tunes Shh/Gli gradients during ventral spinal cord patterning. RNF220 also epigenetically regulates Shh signaling by targeting epifactor EED in cerebellar development. Here, we continued to study the molecular events underlying RNF220-mediated Shh regulation in the cytoplasm. The results showed that HDAC6 is required for RNF220-induced Gli accumulation in the cytoskeletal fraction and inclusion in aggresomes. In the cytoplasm, Glis polyubiquitinated by RNF220 are prone to interact with p62 and destined for autophagy-mediated degradation. Additionally, we showed that RNF220 inhibits the processing of Gli2 and Gli3 both in vitro and in vivo. Collectively, our studies shed new light on Shh signaling regulation.

Background: Tendons heal by fibrotic repair, increasing the likelihood of reinjury. Animal tendon injury and overuse models have identified transforming growth factor beta (TGF-β) and bone morphogenetic proteins (BMPs) as growth factors actively involved in the development of fibrosis, by mediating extracellular matrix synthesis and cell differentiation. Purpose: To understand how TGF-β and BMPs contribute to fibrotic processes using tendon-derived cells isolated from healthy and diseased human tendons. Study Design: Controlled laboratory study. Methods: Tendon-derived cells were isolated from patients with a chronic rotator cuff tendon tear (large to massive, diseased) and healthy hamstring tendons of patients undergoing anterior cruciate ligament repair. Isolated cells were incubated with TGF-β1 (10 ng/mL) or BMP-2 (100 ng/mL) for 3 days. Gene expression was measured by real-time quantitative polymerase chain reaction. Cell signaling pathway activation was determined by Western blotting. Results: TGF-β1 treatment induced ACAN mRNA expression in both cell types but less in the diseased compared with healthy cells ( P < .05). BMP-2 treatment induced BGN mRNA expression in healthy but not diseased cells ( P < .01). In the diseased cells, TGF-β1 treatment induced increased ACTA2 mRNA expression ( P < .01) and increased small mothers against decapentaplegic (SMAD) signaling ( P < .05) compared with those of healthy cells. Moreover, BMP-2 treatment induced ACTA2 mRNA expression in the diseased cells only ( P < .05). Conclusion: Diseased tendon–derived cells show reduced expression of the proteoglycans aggrecan and biglycan in response to TGF-β1 and BMP-2 treatments. These same treatments induced enhanced fibrotic differentiation and canonical SMAD cell signaling in diseased compared with healthy cells. Clinical Relevance: Findings from this study suggest that diseased tendon–derived cells respond differently than healthy cells in the presence of TGF-β1 and BMP-2. The altered responses of diseased cells may influence fibrotic repair processes during tendon healing.

One of the major events in animal morphogenesis is the emergence of a polar body axis. Here, we combine classic grafting techniques with live imaging to study the emergence of body axis polarity during whole body regeneration in Hydra. Composite tissues are made by fusing two rings, excised from separate animals, in different configurations that vary in the relative polarity and original position of the rings along the body axis of the parent animals. We find that under frustrating initial configurations, body axis polarity that is otherwise stably inherited from the parent animal, can become labile and even be reversed. The site of head regeneration exhibits a bias for the edges of the fused doublets, even when this involves polarity reversal in the tissue, emphasizing the importance of structural factors in head formation. The doublets’ edges invariably contain defects in the organization of the supra-cellular actin fibers, which form as the tissue ring seals on each side. We suggest that the presence of a defect can act as an “attractor” for head formation at the edge, even though a defect is neither required nor sufficient for head formation. The observation of head formation at an originally distal edge of the tissue upon polarity reversal, is not compatible with models of Hydra regeneration based solely on preexisting morphogen gradients. Rather, our results suggest that body axis determination is a dynamic process that involves mechanical feedback and signaling processes that are sensitive to the original polarity and position of the excised tissues. Significance statement The formation of a polar body axis is one of the most basic steps in defining the body plan of a developing animal. Here we study the emergence of polarity in Hydra regenerating from composite tissue segments under constraints. We show that these frustrating conditions expose non-trivial dynamics, reflecting the integration of the memory of the tissue’s original polarity and position in the parent animal with dynamic biochemical and mechanical processes. In particular, we demonstrate that the organization of the cytoskeleton participates together with biochemical signaling processes in feedback loops that eventually result in the formation and stabilization of the animal’s body axis. We conclude that preexisting biochemical gradients in the tissue cannot by themselves explain axis determination.

Cells within developing tissues rely on morphogens to assess positional information. Passive diffusion is the most parsimonious transport model for long-range morphogen gradient formation but does not, on its own, readily explain scaling, robustness and planar transport. Here, we argue that diffusion is sufficient to ensure robust morphogen gradient formation in a variety of tissues if the interactions between morphogens and their extracellular binders are considered. A current challenge is to assess how the affinity for extracellular binders, as well as other biophysical and cell biological parameters, determines gradient dynamics and shape in a diffusion-based transport system. Technological advances in genome editing, tissue engineering, live imaging and in vivo biophysics are now facilitating measurement of these parameters, paving the way for mathematical modelling and a quantitative understanding of morphogen gradient formation and modulation.

Recent advances in human pluripotent stem cells (hPSCs)-derived in vitro models open a new avenue for studying early stage human development. While current approaches leverage the self-organizing capability of hPSCs, it remains unclear whether extrinsic morphogen gradients are sufficient to pattern neuroectoderm tissues in vitro. While microfluidics or hydrogel-based approaches to generate chemical gradients are well-established, these systems either require continuous pumping or encapsulating cells in gels, making it difficult for adaptation in standard biology laboratories and downstream analysis. In this work, we report a new device design that leverages localized passive diffusion, or LPaD for short, to generate a stable chemical gradient in an open environment. As LPaD is operated simply by media changing, common issues for microfluidic systems such as leakage, bubble formation, and contamination can be avoided. The device contains a slit carved in a film filled with solid gelatin and connected to a static aqueous morphogen reservoir. Concentration gradients generated by the device were visualized via DAPI fluorescent intensity and were found to be stable for up to 168 h. Using this device, we successfully induced cellular response of Madin-Darby canine kidney (MDCK) cells to the concentration gradient of a small-molecule drug, cytochalasin D. Furthermore, we efficiently patterned the dorsal-ventral axis of hPSC-derived forebrain neuroepithelial cells with the sonic hedgehog (Shh) signal gradient generated by the LPaD devices. Together, LPaD devices are powerful tools to control the local chemical microenvironment for engineering organotypic structures in vitro.

Background Organogenesis is regulated by morphogen signaling and transcription networks. These networks differ between organs, and identifying the organ-specific network is important to clarify the molecular mechanisms of development and regeneration of organs. Several studies have been conducted to identify salivary gland-specific networks using a mouse submandibular gland model. The submandibular glands (SMGs) of mice manifest as a thickening of the oral epithelium at embryonic day 11.5 and invaginate into the underlying mesenchyme. The network between Fgf10 and Sox9 is involved in SMG development in mice. Highlight Sox9, a member of the Sox family, is expressed in the SMG in mice from the embryonic stage to the adult stage, although the distribution changes during development. A null mutation of mouse Sox9 is lethal during the neonatal period due to respiratory failure, whereas deletion of Sox9 in the oral epithelium using the Cre/lox P system, can lead to smaller initial buds of SMGs in conditional knockout (cKO) mice than in normal mice. In addition, we showed that adenoviral transduction of Sox9 and Foxc1 genes into mouse embryonic stem cell–derived oral ectoderm could induce salivary gland rudiment in an organoid culture system. ChIP-sequencing revealed that Sox9 possibly regulates several tube- and branching-formation–related genes. Conclusion Sox9 may serve as an essential transcription factor for salivary gland development. The Sox9-mediated pathway can be a promising candidate for regenerating damaged salivary glands.

Over the last decade, scientists have begun to model CNS development, function, and disease in vitro using human pluripotent stem cell (hPSC)-derived organoids. Using traditional protocols, these 3D tissues are generated by combining the innate emergent properties of differentiating hPSC aggregates with a bioreactor environment that induces interstitial transport of oxygen and nutrients and an optional supportive hydrogel extracellular matrix (ECM). During extended culture, the hPSC-derived neural organoids (hNOs) obtain millimeter scale sizes with internal microscale cytoarchitectures, cellular phenotypes, and neuronal circuit behaviors mimetic of those observed in the developing brain, eye, or spinal cord. Early studies evaluated the cytoarchitectural and phenotypical character of these organoids and provided unprecedented insight into the morphogenetic processes that govern CNS development. Comparisons to human fetal tissues revealed their significant similarities and differences. While hNOs have current disease modeling applications and significant future promise, their value as anatomical and physiological models is limited because they fail to form reproducibly and recapitulate more mature in vivo features. These include biomimetic macroscale tissue morphology, positioning of morphogen signaling centers to orchestrate appropriate spatial organization and intra- and inter-connectivity of discrete tissue regions, maturation of physiologically relevant neural circuits, and formation of vascular networks that can support sustained in vitro tissue growth. To address these inadequacies scientists have begun to integrate organoid culture with bioengineering techniques and methodologies including genome editing, biomaterials, and microfabricated and microfluidic platforms that enable spatiotemporal control of cellular differentiation or the biochemical and biophysical cues that orchestrate organoid morphogenesis. This review will examine recent advances in hNO technologies and culture strategies that promote reproducible in vitro morphogenesis and greater biomimicry in structure and function.

Although gene expression is tightly regulated during embryonic development, the impact of translational control has received less experimental attention. Here, we find that eukaryotic translation initiation factor-3 (eIF3) is required for Shh-mediated tissue patterning. Analysis of loss-of-function eIF3 subunit c (Eif3c) mice reveal a unique sensitivity to the Shh receptor Patched 1 (Ptch1) dosage. Genome-wide in vivo enhanced cross-linking immunoprecipitation sequence (eCLIP-seq) shows unexpected specificity for eIF3 binding to a pyrimidine rich motif present in subsets of 5’-UTRs and a corresponding change in the translation of these transcripts by ribosome profiling in Eif3c loss-of-function embryos. We further find that while Eif3c loss-of-function embryos do not show a global decrease in protein synthesis, translation of Ptch1 through this pyrimidine rich motif is specifically sensitive to eIF3 amount. Altogether, this work uncovers hidden specificity of housekeeping translation initiation machinery for the translation of key developmental signaling transcripts.

Developmental regulation of the vertebrate visual system has been a focus of investigation for generations as understanding this critical time period has direct implications on our understanding of congenital blinding disease. The majority of studies to date have focused on transcriptional regulation mediated by morphogen gradients and signaling pathways. However, recent studies of post translational regulation during ocular development have shed light on the role of the ubiquitin proteasome system (UPS). This rather ubiquitous yet highly diverse system is well known for regulating protein function and localization as well as stability via targeting for degradation by the 26S proteasome. Work from many model organisms has recently identified UPS activity during various milestones of ocular development including retinal morphogenesis, retinal ganglion cell function as well as photoreceptor homeostasis. In particular work from flies and zebrafish has highlighted the role of the E3 ligase enzyme family, Seven in Absentia Homologue (Siah) during these events. In this review, we summarize the current understanding of UPS activity during Drosophila and vertebrate ocular development, with a major focus on recent findings correlating Siah E3 ligase activity with two major developmental stages of vertebrate ocular development, retinal morphogenesis and photoreceptor specification and survival.

Primary cilia are immotile appendages that have evolved to receive and interpret a variety of different extracellular cues. Cilia play crucial roles in intercellular communication during development and defects in cilia affect multiple tissues accounting for a heterogeneous group of human diseases called ciliopathies. The Hedgehog (Hh) signaling pathway is one of these cues and displays a unique and symbiotic relationship with cilia. Not only does Hh signaling require cilia for its function but the majority of the Hh signaling machinery is physically located within the cilium-centrosome complex. More specifically, cilia are required for both repressing and activating Hh signaling by modifying bifunctional Gli transcription factors into repressors or activators. Defects in balancing, interpreting or establishing these repressor/activator gradients in Hh signaling either require cilia or phenocopy disruption of cilia. Here, we will summarize the current knowledge on how spatiotemporal control of the molecular machinery of the cilium allows for a tight control of basal repression and activation states of the Hh pathway. We will then discuss several paradigms on how cilia influence Hh pathway activity in tissue morphogenesis during development. Last, we will touch on how cilia and Hh signaling are being reactivated and repurposed during adult tissue regeneration. More specifically, we will focus on mesenchymal stem cells within the connective tissue and discuss the similarities and differences of how cilia and ciliary Hh signaling control the formation of fibrotic scar and adipose tissue during fatty fibrosis of several tissues.

In this manuscript, we developed a two-fold symmetric linchpin (TSL) that converts readily available phage-displayed peptides libraries made of 20 common amino acids to genetically-encoded libraries of bicyclic peptides displayed on phage. TSL combines an aldehyde-reactive group and two thiol-reactive groups; it bridges two side chains of cysteine [C] with an N-terminal aldehyde group derived from the N-terminal serine [S], yielding a novel bicyclic topology that lacks a free N-terminus. Phage display libraries of SX1CX2X3X4X5X6X7C sequences, where X is any amino acid but Cys, were converted to a library of bicyclic TSL-[S]X1[C]X2X3X4X5X6X7[C] peptides in 45 ± 15% yield. Using this library and protein morphogen NODAL as a target, we discovered bicyclic macrocycles that specifically antagonize NODAL-induced signaling in cancer cells. At a 10 μM concentration, two discovered bicyclic peptides completely suppressed NODAL-induced phosphorylation of SMAD2 in P19 embryonic carcinoma cells. The TSL-[S]Y[C]KRAHKN[C] bicycle inhibited NODAL-induced proliferation of NODAL-TYK-nu ovarian carcinoma cells with apparent IC50 of 1 μM. The same bicycle at 10 μM concentration did not affect the growth of the control TYK-nu cells. TSL-bicycles remained stable over the course of the 72 hour-long assays in a serum-rich cell-culture medium. We further observed general stability in mouse serum and in a mixture of proteases (Pronase™) for 21 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. TSL-constrained peptides to expand the previously reported repertoire of phage-displayed bicyclic architectures formed by cross-linking Cys side chains. We anticipate that it will aid the discovery of proteolytically stable bicyclic inhibitors for a variety of protein targets., A two-fold symmetric linchpin (TSL) converts readily available phage-displayed disulfide peptide libraries to proteolytically stable bicyclic peptides. The bicyclic phage library was screened to discover an antagonist of NODAL morphogen.

Cell specification in the ventral spinal cord is a well-studied model system to understand how tissue pattern develops in response to a morphogen gradient. Ventral cell types including motor neurons (MNs) are induced in the neural tube in response to graded Sonic Hedgehog (Shh) signaling. We performed a forward genetic screen in the mouse that incorporated a GFP-expressing transgene to visualize MNs to identify genes regulating ventral patterning. Here we contrast the neural patterning phenotypes of two mouse lines carrying induced mutations in ciliary trafficking genes. We show that a hypomorphic mutation in the gene Tubby-like protein 3 (Tulp3) resulted in a dorsal expansion of MNs consistent with an up-regulation of Shh signaling. Interestingly, patterning defects in Tulp3 mutants were restricted to posterior regions of the spinal cord as patterning was similar to WT in the anterior spinal cord. In contrast, a mutation in the ciliary trafficking gene cytoplasmic dynein 2 heavy chain 1 (Dync2h1), led to a complete loss of MNs in anterior regions of the spinal cord, indicating a strong down-regulation of Shh signaling. However, this severe phenotype was restricted to the cervical region as MNs developed posteriorly. Mutations in cilia trafficking genes affect Shh-dependent signaling in the neural tube differentially along the anterior-posterior (A-P) axis in a process that is not understood.

The stereotyped dimensions of animal bodies and their component parts result from tight constraints on growth. Yet, the mechanisms that stop growth when organs reach the right size are unknown. Growth of the Drosophila wing—a classic paradigm—is governed by two morphogens, Decapentaplegic (Dpp, a BMP) and Wingless (Wg, a Wnt). Wing growth during larval life ceases when the primordium attains full size, concomitant with the larval-to-pupal molt orchestrated by the steroid hormone ecdysone. Here, we block the molt by genetically dampening ecdysone production, creating an experimental paradigm in which the wing stops growing at the correct size while the larva continues to feed and gain body mass. Under these conditions, we show that wing growth is limited by the ranges of Dpp and Wg, and by ecdysone, which regulates the cellular response to their signaling activities. Further, we present evidence that growth terminates because of the loss of two distinct modes of morphogen action: 1) maintenance of growth within the wing proper and 2) induced growth of surrounding “pre-wing” cells and their recruitment into the wing. Our results provide a precedent for the control of organ size by morphogen range and the hormonal gating of morphogen action.

Convergence of paradigms yields patterns In embryo development, spatial patterns of distinct cell types arise reproducibly. In the zebrafish spinal cord, neural progenitors form stereotypic stripe patterns despite the noisy instructive signals and large-scale cellular rearrangement required during morphogenesis. Tsai et al. show that a cell type–specific adhesion code, regulated by a Shh morphogen gradient composed of three adhesion molecules, provides adhesion specificity for three neural progenitor types and mediates patterning robustness in the zebrafish spinal cord. Although insufficient on their own, the integration of the morphogen gradient and differential adhesion mechanisms enables robust pattern formation during tissue morphogenesis. Science , this issue p. 113

Differentiation of human pluripotent stem cells (hPSCs) into ectoderm provides neurons and glia useful for research, disease modeling, drug discovery, and potential cell therapies. In current protocols, hPSCs are traditionally differentiated into an obligate rostro‐dorsal ectodermal fate expressing PAX6 after 6 to 12 days in vitro when protected from mesendoderm inducers. This rate‐limiting step has performed a long‐standing role in hindering the development of rapid differentiation protocols for ectoderm‐derived cell types, as any protocol requires 6 to 10 days in vitro to simply initiate. Here, we report efficient differentiation of hPSCs into a naive early ectodermal intermediate within 24 hours using combined inhibition of bone morphogenic protein and fibroblast growth factor signaling. The induced population responds immediately to morphogen gradients to upregulate rostro‐caudal neurodevelopmental landmark gene expression in a generally accelerated fashion. This method can serve as a new platform for the development of novel, rapid, and efficient protocols for the manufacture of hPSC‐derived neural lineages., Efficient conversion into primal ectoderm by treatment with BGJ398 and LDN193189 accelerates neural induction in human pluripotent stem cells.

Ventralization, a major patterning process in the developing vertebrate neural tube (central nervous system, CNS), depends on Sonic hedgehog (SHH) as a main signaling morphogen. We studied the CNS of late larval and young adult zebrafish in a transgenic shh-GFP line revealing increased neuroanatomical detail due to the progressed differentiation state compared to earlier stages. Some major findings emerge from the present study. (a) shh -GFP is still expressed along the adult zebrafish CNS neuraxis in most locations seen in larvae. (b) We newly identify a ventroposterior shh pallidal domain representing the basal telencephalic signaling center important for basal ganglia development known in other vertebrates (i.e., the anterior entopeduncular area-basal medial ganglionic eminence of mammals). (c) We further show late-emerging shh-GFP positive radial glia cells in the medial zone of the dorsal telencephalon (i.e., the teleostan pallial amygdala). (d) Immunostains for tyrosine hydroxylase demonstrate that there is selective colocalization in adult dopamine cells with shh-GFP in the posterior tuberculum, including in projection cells to striatum, which represents a striking parallel to amniote mesodiencephalic dopamine cell origin from shh expressing floor plate cells. (e) There is no colocalization of shh and islet1 as shown by respective shh-GFP and islet1-GFP lines. (f) The only radially far migrated shh-GFP cells are located in the preglomerular area. (g) There are no adult cerebellar and tectal shh-GFP cells confirming their exclusive role during early development as previously reported by our laboratory.

Summary Developmental biology studies using model organisms suggest that the emergence of spatial patterning in the ectoderm is mediated by the morphogen gradients. However, it is still unclear whether the morphogen gradient is necessary and dominates the cell spatial patterning, particularly in human genetic background. Here, we demonstrate that human pluripotent stem cells can self-organize to concentric rings of all major cell types in ectoderm when cultured on micropatterned surfaces in a chemically defined condition. We reveal that modulating the dynamics of NODAL, BMP, and WNT signals is sufficient to control the spatial order of different cell types. Our mathematical model suggests that changes in wavelength and phase of signaling patterns formed via reaction-diffusion may be the mechanism by which temporal information is translated into spatial information. Together, our work demonstrates that in vitro human ectoderm microtissues have great potential in understanding the mechanisms of early-stage human development.

Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal–ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal. This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.

In a developing animal, morphogen gradients act to pattern tissues into distinct domains of cell types. However, despite their prevalence in development, morphogen gradient formation is a matter of debate. In our recent publication, we showed that the Dorsal/NF-κB morphogen gradient, which patterns the DV axis of the early Drosophila embryo, is partially established by a mechanism of facilitated diffusion. This mechanism, also known as "shuttling," occurs when a binding partner of the morphogen facilitates the diffusion of the morphogen, allowing it to accumulate at a given site. In this case, the inhibitor Cactus/IκB facilitates the diffusion of Dorsal/NF-κB. In the fly embryo, we used computation and experiment to not only show that shuttling occurs in the embryo, but also that it enables the viability of embryos that inherit only one copy of dorsal maternally. In this commentary, we further discuss our evidence behind the shuttling mechanism, the previous literature data explained by the mechanism, and how it may also be critical for robustness of development. Finally, we briefly provide additional experimental data pointing toward an interaction between Dorsal and BMP signaling that is likely affected by shuttling.

PURPOSE OF REVIEW: Stem cells are exquisitely sensitive to biophysical and biochemical cues within the native microenvironment. This review focuses on emerging strategies to manipulate neural cell behavior using these influences in three-dimensional (3D) culture systems. RECENT FINDINGS: Traditional systems for neural cell differentiation typically produce heterogeneous populations with limited diversity rather than the complex, organized tissue structures observed in vivo. Advancements in developing engineering tools to direct neural cell fates can enable new applications in basic research, disease modeling, and regenerative medicine. SUMMARY: This review article highlights engineering strategies that facilitate controlled presentation of biophysical and biochemical cues to guide differentiation and impart desired phenotypes on neural cell populations. Specific highlighted examples include engineered biomaterials and microfluidic platforms for spatiotemporal control over the presentation of morphogen gradients.

To study the mechanisms underlying developmental pattern formation in a tissue, one needs to analyze the dynamics of the regulators in time and space across the tissue of a specific architecture. This problem is essential for the developmental regulators (morphogens) that distribute over the tissues anisotropically, forming there maxima and gradients and guiding cellular processes in a dose-dependent manner. Here we present the PlantLayout pipeline for MATLAB software, which facilitates the computational studies of tissue patterning. With its help, one can build a structural model of a two-dimensional tissue, embed it into a mathematical model in ODEs, perform numerical simulations, and visualize the obtained results - everything on the same platform. As a result, one can study the concentration dynamics of developmental regulators over the cell layout reconstructed from the real tissue. PlantLayout allows studying the dynamics and the output of gene networks guided by the developmental regulator in specific cells. The gene networks could be different for different cell types. One of the obstacles that PlantLayout removes semi-automatically is the determination of the cell wall orientation which is relevant when cells in the tissue have a polarity. Additionally, PlantLayout allows automatically extracting other quantitative and qualitative features of the cells and the cell walls, which might help in the modeling of a developmental pattern, such as the length and the width of the cell walls, the set of the neighboring cells, cell volume and cell perimeter. We demonstrate PlantLayout performance on the model of phytohormone auxin distribution over the plant root tip.Для изучения механизмов, лежащих в основе формирования паттернов развития в тканях, необходим анализ динамики распределения регуляторов во времени и пространстве в тканях сложной структуры. Наибольшее значение это имеет в случае регуляторов развития (морфогенов), которые распределены в ткани неравномерно, образуя максимумы и градиенты, и регулируют клеточные процессы по-разному в зависимости от дозы. Настоящая работа посвящена описанию программного средства PlantLayout на языке MATLAB, которое облегчает исследование формирования распределений регуляторов в тканях различного строения. С его помощью можно построить двумерную структурную модель ткани, внедрить ее в математическую модель, описанную в терминах обычных дифференциальных уравнений, выполнить численные расчеты модели и визуализировать полученные результаты – всё на одной платформе. В результате можно изучать динамику работы генных сетей и изменения концентрации регуляторов в каждой клетке клеточного ансамбля, воспроизводящего строение реальной ткани. Исследуемые генные сети могут различаться для разных типов клеток. Одной из задач, которую решает PlantLayout в полуавтоматическом режиме, является определение ориентации клеточной стенки, что имеет значение в случае присутствия в ткани поляризованных клеток. Кроме того, PlantLayout позволяет автоматически определять другие качественные и количественные характеристики клеток и клеточных стенок, такие как длина и ширина клеточных стенок, площадь сечения и периметр клеток, которые могут помочь в моделировании паттернов распределения регуляторов развития, а также список соседних клеток для каждой клетки. В данной работе продемонстрирована эффективность разработанного программного средства PlantLayout применительно к моделированию распределения фитогормона ауксина в кончике корня растения.

Significance Understanding how mammalian organisms achieve the full diversity of cell types in the adult organism is a central goal of developmental cell biology. Recent work has shown that some embryonic precursor cells can self-organize into developmental structures but the mechanisms of gene regulation that contribute to this process remain unknown. Here we show embryonic stem cells self-organize into distinct gene expression states that resemble developmental gene programs. We find that microRNAs, small noncoding regulators of gene expression, play a critical role in organizing fluctuations across gene networks to help achieve this organization into distinct expression states., Pluripotent embryonic stem cells (ESCs) contain the potential to form a diverse array of cells with distinct gene expression states, namely the cells of the adult vertebrate. Classically, diversity has been attributed to cells sensing their position with respect to external morphogen gradients. However, an alternative is that diversity arises in part from cooption of fluctuations in the gene regulatory network. Here we find ESCs exhibit intrinsic heterogeneity in the absence of external gradients by forming interconverting cell states. States vary in developmental gene expression programs and display distinct activity of microRNAs (miRNAs). Notably, miRNAs act on neighborhoods of pluripotency genes to increase variation of target genes and cell states. Loss of miRNAs that vary across states reduces target variation and delays state transitions, suggesting variable miRNAs organize and propagate variation to promote state transitions. Together these findings provide insight into how a gene regulatory network can coopt variation intrinsic to cell systems to form robust gene expression states. Interactions between intrinsic heterogeneity and environmental signals may help achieve developmental outcomes.

A critical juncture in early development is the partitioning of cells that will adopt different fates into three germ layers: the ectoderm, the mesoderm, and the endoderm. This step is achieved through the internalization of specified cells from the outermost surface layer, through a process called gastrulation. In Drosophila, gastrulation is achieved through cell shape changes (i.e., apical constriction) that change tissue curvature and lead to the folding of a surface epithelium. Folding of embryonic tissue results in mesoderm and endoderm invagination, not as individual cells, but as collective tissue units. The tractability of Drosophila as a model system is best exemplified by how much we know about Drosophila gastrulation, from the signals that pattern the embryo to the molecular components that generate force, and how these components are organized to promote cell and tissue shape changes. For mesoderm invagination, graded signaling by the morphogen, Spätzle, sets up a gradient in transcriptional activity that leads to the expression of a secreted ligand (Folded gastrulation) and a transmembrane protein (T48). Together with the GPCR Mist, which is expressed in the mesoderm, and the GPCR Smog, which is expressed uniformly, these signals activate heterotrimeric G-protein and small Rho-family G-protein signaling to promote apical contractility and changes in cell and tissue shape. A notable feature of this signaling pathway is its intricate organization in both space and time. At the cellular level, signaling components and the cytoskeleton exhibit striking polarity, not only along the apical–basal cell axis, but also within the apical domain. Furthermore, gene expression controls a highly choreographed chain of events, the dynamics of which are critical for primordium invagination; it does not simply throw the cytoskeletal “on” switch. Finally, studies of Drosophila gastrulation have provided insight into how global tissue mechanics and movements are intertwined as multiple tissues simultaneously change shape. Overall, these studies have contributed to the view that cells respond to forces that propagate over great distances, demonstrating that cellular decisions, and, ultimately, tissue shape changes, proceed by integrating cues across an entire embryo.

Leveraging the developmental potential and self-organizing property of human pluripotent stem (hPS) cells, researchers have developed tractable models of human embryonic development. Owing to their compatibility to live imaging, genome editing, mechanical perturbation and measurement, these models offer promising quantitative experimental platforms to advance human embryology and regenerative medicine. Herein, we provide a review of recent progress in using hPS cells to generate models of early human neural development or neurulation, including neural induction and regional patterning of the neural tube. These models, even in their nascent developmental stages, have already revealed intricate cell-cell signaling and mechanoregulation mechanisms likely involved in tissue patterning during early neural development. We also discuss future opportunities in modeling early neural development by incorporating bioengineering tools to control precisely neural tissue morphology and architecture, morphogen dynamics, intracellular signaling events, and cell-cell interactions to further the development of this emerging field and expand its applications.

In the spider, determination of the dorsal-ventral body (DV) axis depends on the interplay of the dorsal morphogen encoding genedecapentaplegic(Dpp) and its antagonist,short gastrulation(sog), a gene that is involved in the correct establishment of ventral tissues. Recent work demonstrated that the forkhead domain encoding geneFoxBis involved in dorsal-ventral axis formation in spider limbs. Here, Dpp likely acts as a dorsal morphogen, and FoxB is likely in control of ventral tissues as RNAi-mediated knockdown ofFoxBcauses dorsalization of the limbs. In this study, we present phenotypes ofFoxBknockdown that demonstrate a function in the establishment of the DV body axis. Knockdown of FoxB function leads to embryos with partially duplicated median germ bands (Duplicitas media) that are possibly the result of ectopic activation of Dpp signalling. Another class of phenotypes is characterized by unnaturally slim (dorsal-ventrally compressed) germ bands in which ventral tissue is either not formed, or is specified incorrectly, likely a result of Dpp over-activity. These results suggest that FoxB functions as an antagonist of Dpp signalling during body axis patterning, similarly as it is the case in limb development. FoxB thus represents a general player in the establishment of dorsal-ventral structures during spider ontogeny.

Macroalgal microbiomes have core functions related to biofilm formation, growth, and morphogenesis of seaweeds. In particular, the growth and development of the sea lettuce Ulva spp. (Chlorophyta) depend on bacteria releasing morphogenetic compounds. Under axenic conditions, the macroalga Ulva mutabilis develops a callus-like phenotype with cell wall protrusions. However, co-culturing with Roseovarius sp. (MS2) and Maribacter sp. (MS6), which produce various stimulatory chemical mediators, completely recovers morphogenesis. This ecological reconstruction forms a tripartite community which can be further studied for its role in cross-kingdom interactions. Hence, our study sought to identify algal growth- and morphogenesis-promoting factors (AGMPFs) capable of phenocopying the activity of Maribacter spp. We performed bioassay-guided solid-phase extraction in water samples collected from U. mutabilis aquaculture systems. We uncovered novel ecophysiological functions of thallusin, a sesquiterpenoid morphogen, identified for the first time in algal aquaculture. Thallusin, released by Maribacter sp., induced rhizoid and cell wall formation at a concentration of 11 pmol l−1. We demonstrated that gametes acquired the iron complex of thallusin, thereby linking morphogenetic processes with intracellular iron homeostasis. Understanding macroalgae–bacteria interactions permits further elucidation of the evolution of multicellularity and cellular differentiation, and development of new applications in microbiome-mediated aquaculture systems., Thallusin was isolated from sea lettuce aquacultures and assigned novel morphogenesis-inducing properties. Specifically, thallusin promoted rhizoidal zone and cell wall formation; thus, it plays crucial roles in cross-kingdom interactions.

Local endometrial stem cells play an important role in regulating endometrial thickness, which is an essential factor for successful embryo implantation and pregnancy outcomes. Importantly, defects in endometrial stem cell function can be responsible for thin endometrium and subsequent recurrent pregnancy losses. Therefore, many researchers have directed their efforts toward finding a novel stimulatory factor that can enhance the regenerative capacity of endometrial stem cells. Sonic hedgehog (SHH) is a morphogen that plays a key role in regulating pattern formation throughout embryonic limb development. In addition to this canonical function, we identified for the first time that SHH is actively secreted as a stem cell-activating factor in response to tissue injury and subsequently stimulates tissue regeneration by promoting various beneficial functions of endometrial stem cells. Our results also showed that SHH exerts stimulatory effects on endometrial stem cells via the FAK/ERK1/2 and/or phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. More importantly, we also observed that endometrial stem cells stimulated with SHH showed markedly enhanced differentiation and migratory capacities and subsequent in vivo therapeutic effects in an endometrial ablation animal model.

Significance A central issue in robust patterning mediated by morphogens is how to buffer variability in morphogen distribution between embryos. We previously described a class of mechanisms that buffers variability through “distal pinning,” a global feedback mechanism that modulates the spread of the gradient and terminates only when morphogen values at a distal position reach some given, fixed level. We have previously shown in Drosophila embryonic dorso-ventral patterning that the secreted WntD protein modulates Toll pathway activation. We have altered the wntD enhancer and demonstrate a global contraction of the Toll gradient. The demonstration of a direct link between the enhancer properties of the modulator gene and the morphogen activation profile establishes a molecular mechanism for buffering variability in morphogen distribution.

The Hedgehog (Hh) morphogen regulates growth and patterning. Since Hh signaling is also implicated in carcinogenesis, it is conceivable that de novo Hh-secreting organizers, if formed in association with oncogenic hit could be tumor-cooperative. Here we validate this hypothesis using the Drosophila model of cooperative epithelial carcinogenesis. We generate somatic clones with simultaneous loss of tumor suppressor, Lgl, and gain of the posterior compartment selector, Engrailed (En), known to induce synthesis of Hh. We show that lgl UAS-en clones in the anterior wing compartment trigger Hh signaling cascade via cross-talk with their Ci-expressing wild type cell neighbors. Hh-Dpp signaling from clone boundaries of such ectopically formed de novo organizers in turn drive lgl carcinogenesis. By contrast, Ci-expressing lgl clones transform by autocrine and/or juxtracine activation of Hh signaling in only the posterior compartment. We further show that sequestration of the Hh ligand or loss of Dpp receptor, Tkv, in these Hh-sending or –receiving lgl clones arrested their carcinogenesis. Our results therefore reveal a hitherto unrecognized mechanism of tumor cooperation by developmental organizers, which are induced fortuitously by oncogenic hits.

The notions of positional information and positional value describe the role of cell position in cell development and pattern formation. Despite their frequent usage in literature, their definitions are blurry, and are interpreted differently by different researchers. Through reflection on previous definitions and usage, and analysis of related experiments, we propose three clear and verifiable criteria for positional information/value. Then we reviewed literature on molecular mechanisms of cell development and pattern formation, to search for a possible molecular basis of positional information/value, including those used in theoretical models. We conclude that although morphogen gradients and cell-to-cell contacts are involved in the pattern formation process, complete molecular explanations of positional information/value are still far from reality.

During the development of all metazoans, the Hedgehog (Hh) signaling pathway provides instructional cues influencing a variety of cellular processes. The pathway ligand, Hh, is dually lipidated by cholesterol and palmitate, which effectively anchors the molecule to the lipid bilayer of the signal producing cell. To complicate the Hh pathway induction process, the Hh ligand is often produced at a significant distance from the cells it influences. Only one known conserved molecule, Dispatched (Disp), can alleviate the membrane tethering imparted by Hh lipidation. Underscoring the importance of Disp protein during development, knockout animals succumb to lethality at E9.5, an exact phenocopy of the knockout of the essential signal transducer of the pathway: Smoothened. Furthermore, mutations within Disp have been found in patients with Holoprosencephaly, the most common cause of human forebrain malformations, which is frequently caused by inhibition of Hh signaling during development. Very little is known regarding the functional or regulatory mechanisms enabling Disp to transport and release Hh ligand. This dissertation aimed to narrow this gap and began with investigation into the role of Disp in a largely ignored mechanism of Hh ligand transport known as cytoneme-mediated morphogen transport. This method of morphogen transport utilizes fragile, thin cytoplasmic extensions which deliver cargo directly from the source of production to ligand responding cells. Through the use of a modified electron microscopy fixative, which we named MEM-fix, to maintain cytonemes for traditional cell biological analysis, I established an in vitro cytoneme system capable of modeling in vivo cytoneme biology. This in vitro cytoneme system uses Schneider 2 cells, an embryonically derived Drosphila cell line, which demonstrate competency to produce and utilize cytonemes as a mechanism of Hh transport. Equipped with this tool, I investigated the relationship between cytonemes and Hh pathway components. In doing so, I uncovered a previously unknown Disp requirement in cytoneme-mediated transport of Hh ligand and subsequently a Disp-mediated cytoneme stabilizing effect. Through these studies into Disp-mediated cytoneme transport, I identified a Disp cleavage event of Disp that regulates the ability to release Hh ligand. The second part of this dissertation details a collaborative effort in which we discovered that the Furin family of proprotein convertases facilitate the cleavage of Disp at a conserved site of the first extracellular loop. The Furin family has been implicated in cleaving ligands and receptors of other developmental signaling pathways, but no reports exist linking the Furin family to regulation of the Hh pathway. Therefore, we investigated the functional consequence of this Disp cleavage event, and our results suggest that this cleavage likely influences the proper trafficking of Hh for efficient release both in vitro and in vivo. To our knowledge, this is the first report of a regulatory protein partner controlling the activity of Disp in releasing Hh.

Despite variability in embryo size, the tissue, organ and body plan develop in proportion with embryo size, known as the scaling phenomenon. Scale-invariant patterning of gene expression is a common feature in development and regeneration, and can be generated by mechanisms such as scaling morphogen gradient and dynamic oscillation. However, whether and how static non-scaling morphogens (input) can induce a scaling gene expression (output) across the entire embryo is not clear. Here we show that scaling requirement sets severe constraints on the geometric structure of the input-output relation (the decoder), from which information about the regulation and mutants’ behavior can be deduced without going into any molecular details. We demonstrate that the Drosophila gap gene system achieves scaling in the way that is entirely consistent with our theory. Remarkably, following the geometry dictated by scaling, a parameter-free decoder correctly and quantitatively accounts for the gap gene expression patterns in nearly all morphogen mutants. Furthermore, the regulation logic and the coding/decoding strategy of the gap gene system can also be revealed from the decoder geometry. Our work provides a general theoretical framework on a large class of problems where scaling output is induced by non-scaling input, as well as a unified understanding of scaling, mutants’ behavior and regulation in the Drosophila gap gene and related systems. Significance Statement Within a given species, fluctuation in egg or embryo size is unavoidable. Despite this, the gene expression pattern and hence the embryonic structure often scale in proportion with the body length. This scaling phenomenon is very common in development and regeneration, and has long fascinated scientists. In this paper, the authors address the question of whether and how a scaling gene expression pattern can originate from non-scaling signals (morphogens). They found that scaling has profound implications in the developmental programming -- properties and behaviors of the underlying gene network can be deduced from the scaling requirement. They demonstrated that the scaling in fruit fly embryogenesis indeed works in this way. Thus, although biological regulatory systems are very complex in general, it can be forced to exhibit simple macroscopic behaviors due to selection pressure, as demonstrated in this study.

The development of an organism from an undifferentiated single cell into a spatially complex structure requires spatial patterning of cell fates across tissues. Positional information, proposed by Lewis Wolpert in 1969, has led to the characterization of many components involved in regulating morphogen signaling activity. However, how morphogen gradients are established, maintained, and interpreted by cells still is not fully understood. Quantitative and systems-based approaches are increasingly needed to define general biological design rules that govern positional information systems in developing organisms. This short review highlights a selective set of studies that have investigated the roles of physiological signaling in modulating and mediating morphogen-based pattern formation. Similarities between neural transmission and morphogen-based pattern formation mechanisms suggest underlying shared principles of active cell-based communication. Within larger tissues, neural networks provide directed information, via physiological signaling, that supplements positional information through diffusion. Further, mounting evidence demonstrates that physiological signaling plays a role in ensuring robustness of morphogen-based signaling. We conclude by highlighting several outstanding questions regarding the role of physiological signaling in morphogen-based pattern formation. Elucidating how physiological signaling impacts positional information is critical for understanding the close coupling of developmental and cellular processes in the context of development, disease, and regeneration.

Cell-cell adhesion is essential for tissue growth and multicellular pattern formation and crucial for the cellular dynamics during embryogenesis and cancer progression. Understanding the dynamical gene regulation of cell adhesion molecules (CAMs) responsible for the emerging spatial tissue behaviors is a current challenge because of the complexity of these nonlinear interactions and feedback loops at different levels of abstraction—from genetic regulation to whole-organism shape formation. To extend our understanding of cell and tissue behaviors due to the regulation of adhesion molecules, here we present a novel, to our knowledge, model for the spatial dynamics of cellular patterning, growth, and shape formation due to the differential expression of CAMs and their regulation. Capturing the dynamic interplay between genetic regulation, CAM expression, and differential cell adhesion, the proposed continuous model can explain the complex and emergent spatial behaviors of cell populations that change their adhesion properties dynamically because of inter- and intracellular genetic regulation. This approach can demonstrate the mechanisms responsible for classical cell-sorting behaviors, cell intercalation in proliferating populations, and the involution of germ layer cells induced by a diffusing morphogen during gastrulation. The model makes predictions on the physical parameters controlling the amplitude and wavelength of a cellular intercalation interface, as well as the crucial role of N-cadherin regulation for the involution and migration of cells beyond the gradient of the morphogen Nodal during zebrafish gastrulation. Integrating the emergent spatial tissue behaviors with the regulation of genes responsible for essential cellular properties such as adhesion will pave the way toward understanding the genetic regulation of large-scale complex patterns and shapes formation in developmental, regenerative, and cancer biology.

In 1952, Alan Turing proposed a theory showing how morphogenesis could occur from a simple two morphogen reaction–diffusion system [Turing, A. M. (1952) Phil. Trans. R. Soc. Lond. A 237 , 37–72. (doi:10.1098/rstb.1952.0012)]. While the model is simple, it has found diverse applications in fields such as biology, ecology, behavioural science, mathematics and chemistry. Chemistry in particular has made significant contributions to the study of Turing-type morphogenesis, providing multiple reproducible experimental methods to both predict and study new behaviours and dynamics generated in reaction–diffusion systems. In this review, we highlight the historical role chemistry has played in the study of the Turing mechanism, summarize the numerous insights chemical systems have yielded into both the dynamics and the morphological behaviour of Turing patterns, and suggest future directions for chemical studies into Turing-type morphogenesis. This article is part of the theme issue ‘Recent progress and open frontiers in Turing’s theory of morphogenesis’.

Dorsoventral (DV) embryonic patterning relies on precisely controlled interpretation of morphogen signaling. In all vertebrates, DV axis specification is informed by gradients of bone morphogenetic proteins (BMPs). We developed a 3D single-molecule mRNA quantification method in whole-mount zebrafish to quantify the inputs and outputs in this pathway. In combination with 3D computational modeling of zebrafish embryo development, data from this method revealed that sizzled (Szl), shaped by BMP and Nodal signaling, maintained a consistent inhibition level with chordin (Chd) to maintain the BMP morphogen gradient. Intriguingly, intrinsic BMP morphogen expression is highly noisy at the ventral marginal layer in the early zebrafish gastrula, where the gradient for DV patterning is established, which implies an unexpected role for noise in gradient shaping.

BackgroundA central problem in developmental and synthetic biology is understanding the mechanisms by which cells in a tissue or a Petri dish process external cues and transform such information into a coherent response, e.g., a terminal differentiation state. It was long believed that this type of positional information could be entirely attributed to a gradient of concentration of a specific signaling molecule (i.e., a morphogen). However, advances in experimental methodologies and computer modeling have demonstrated the crucial role of the dynamics of a cell’s gene regulatory network (GRN) in decoding the information carried by the morphogen, which is eventually translated into a spatial pattern. This morphogen interpretation mechanism has gained much attention in systems biology as a tractable system to investigate the emergent properties of complex genotype-phenotype maps.MethodsIn this study, we apply a Markov chain Monte Carlo (MCMC)-like algorithm to probe the design space of three-node GRNs with the ability to generate a band-like expression pattern (target phenotype) in the middle of an arrangement of 30 cells, which resemble a simple (1-D) morphogenetic field in a developing embryo. Unlike most modeling studies published so far, here we explore the space of GRN topologies with nodes having the potential to perceive the same input signal differently. This allows for a lot more flexibility during the search space process, and thus enables us to identify a larger set of potentially interesting and realizable morphogen interpretation mechanisms.ResultsOut of 2061 GRNs selected using the search space algorithm, we found 714 classes of network topologies that could correctly interpret the morphogen. Notably, the main network motif that generated the target phenotype in response to the input signal was the type 3 Incoherent Feed-Forward Loop (I3-FFL), which agrees with previous theoretical expectations and experimental observations. Particularly, compared to a previously reported pattern forming GRN topologies, we have uncovered a great variety of novel network designs, some of which might be worth inquiring through synthetic biology methodologies to test for the ability of network design with minimal regulatory complexity to interpret a developmental cue robustly.Author summarySystems biology is a fast growing field largely powered by advances in high-performance computing and sophisticated mathematical modeling of biological systems. Based on these advances, we are now in a position to mechanistically understand and accurately predict the behavior of complex biological processes, including cell differentiation and spatial pattern formation during embryogenesis. In this article, we use an in silico approach to probe the design space of multi-input, three-node Gene Regulatory Networks (GRNs) capable of generating a striped gene expression pattern in the context of a simplified 1-D morphogenetic field.

In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name “neighbourhood watch” model) illuminates the classic “French Flag Problem” and how positional information is interpreted by a sheet of cells in a large developing system.Summary statementIn a large developing system, the chick embryo before gastrulation, cells interpret gradients of positional signals relative to their neighbours to position the primitive streak, establishing bilateral symmetry.

The processes of primitive streak formation and fate specification in the mammalian epiblast rely on complex interactions between morphogens and tissue organization. Little is known about how these instructive cues functionally interact to regulate gastrulation. We interrogated the interplay between tissue organization and morphogens by using human induced pluripotent stem cells (hiPSCs) down-regulated for the morphogen regulator GLYPICAN-4, in which defects in tight junctions result in areas of disrupted epithelial integrity. Remarkably, this phenotype does not affect hiPSC stemness, but impacts on cell fate acquisition processes. Strikingly, cells within disrupted areas become competent to perceive BMP4 and ACTIVIN A/NODAL gastrulation signals and thus, differentiate into mesendoderm. Yet, disruption of epithelial integrity prolongs temporal activation of BMP4 and ACTIVIN A/NODAL downstream effectors and correlates with enhanced hiPSC endoderm/mesoderm differentiation potential. Altogether, our results disclose epithelial cell integrity as a key determinant of TGF-β activity and highlight a new mechanism guiding morphogen sensing and spatial cell fate change within an epithelium.

SummaryBacterial cell wall, a universal pathogen-associated molecular pattern (PAMP), crosses the placenta into the fetal brain. We determined that PAMPs interact with TLR2/6 on murine fetal neural progenitor cells (NPCs) to induce overexpansion of all neocortical layers leading to a larger, folded cortex and abnormal postnatal behavior. The NPC overexpansion originated at E10 and targeted ventricular radial glia (vRG), the primary NPC, by shortening cell cycle and increasing self-renewal. The mechanism involved two novel signaling pathways in NPCs mediated by recognition of bacterial PAMPs by TLR2/6 including: a) loss of primary cilia, activation of hedgehog signaling, and increased FOXG1 and b) increased PI3K/AKT activity. These findings reveal PAMP/TLR2/6 acts as a morphogen in fetal neurodevelopment. In addition, the loss ofTlr2orTlr6without pathogenic challenge, increased the number of neurons, establishing the requirement for an endogenous TLR2 signal for normal neurodevelopment in the embryo.

Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. Here we show that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal quantitative regulatory dynamics from a complex genetic network in vivo. We engineer light-controlled variants of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. Our results recapitulate known relationships, including rapid Bicoid-dependent expression of giant and hunchback and delayed repression of Krüppel. In contrast, we find that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a previously unreported role for Bicoid in suppressing knirps expression. Acute modulation of transcription factor concentration while simultaneously recording output gene activity represents a powerful approach for studying how gene circuit elements are coupled to cell identification and complex body pattern formation in vivo.

The intestinal epithelium is a tissue with high cell turnover, supported by adult intestinal stem cells. Intestinal homeostasis is underpinned by crypt basal columnar stem cells, marked by expression of the LGR5 gene. However, recent research has demonstrated considerable stem cell plasticity following injury, with dedifferentiation of a range of other intestinal cell populations, induced by a permissive microenvironment in the regenerating mucosa. The regulation of this profound adaptive cell reprogramming response is the subject of current research. There is a demonstrable contribution from disruption of key homeostatic signaling pathways such as wingless-related integration site and bone morphogenetic protein, and an emerging signaling hub role for the mechanoreceptor transducers Yes-associated protein 1/transcriptional coactivator with PDZ-binding motif, negatively regulated by the Hippo pathway. However, a number of outstanding questions remain, including a need to understand how tissues sense damage, and how pathways intersect to mediate dynamic changes in the stem cell population. Better understanding of these pathways, associated functional redundancies, and how they may be both enhanced for recovery of inflammatory diseases, and co-opted in neoplasia development, may have significant clinical implications, and could lead to development of more targeted molecular therapies which target individual stem or stem-like cell populations.

Glypicans influence signaling pathways by regulating morphogen trafficking and reception. However, the underlying mechanisms in vertebrates are poorly understood. In zebrafish, Glypican 4 (Gpc4) is required for convergence and extension (C&E) of both the mesoderm and endoderm. Here, we show that transgenic expression of GFP-Gpc4 in the endoderm of gpc4 mutants rescued C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2 and depended on signaling filopodia rather than on cleavage of the Gpc4 GPI anchor. Gpc4 bound both Wnt5b and Wnt11f2 and regulated formation of the filopodia that transport Wnt5b and Wnt11f2 to neighboring cells. Moreover, this rescue was suppressed by blocking signaling filopodia that extend from endodermal cells. Thus, GFP-Gpc4–labeled protrusions that emanated from endodermal cells transported Wnt5b and Wnt11f2 to other germ layers, rescuing the C&E defects caused by a gpc4 deficiency. Our study reveals a new mechanism that could explain in vivo morphogen distribution involving Gpc4.

The 12-pass transmembrane protein Dispatched (DISP) is essential for Sonic Hedgehog (SHH) release from ligand-producing cells and is indispensable for establishment of the SHH morphogen gradient during tissue patterning. Regulatory events controlling DISP release of SHH are not yet fully characterized. We recently demonstrated that DISP is cleaved by FURIN proprotein convertase at a conserved site in its first extracellular loop. Mutation of the cleavage site attenuates DISP-mediated SHH release, which indicates that Furin cleavage is a positive step toward DISP protein maturation. In this chapter, we present a ligand release/retention protocol that allows for the analysis of DISP cleavage, DISP-mediated release of SHH ligand from producing cells, and secretion-dependent signal induction in target cells.