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Pelizaeus-Merzbacher disease (PMD, currently known as hypomyelinating leukodystrophy type 1 [HLD1]) is a hereditary hypomyelinating and/or demyelinating disease associated with the proteolipid protein 1 (plp1) gene in the central nervous system (CNS). One of the major causes of this condition is incomplete or defective oligodendroglial cell myelin sheath formation triggered by endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR). The HLD1-associated Ala-243-to-Val mutation (p.Ala243Val) of PLP1 is widely recognized to trigger defective oligodendroglial cell morphological differentiation, primarily due to ER stress. We have previously reported that knockdown of Rab7B (also known as Rab42), a small GTP/GDP-binding protein involved in intracellular vesicle trafficking around the lysosome, can recover chemical ER stress-induced incomplete cell shapes in the FBD-102b cell line, a model of oligodendroglial cell morphological differentiation. Here, we present findings indicating that incomplete cell shapes induced by PLP1 p.Ala243Val can be restored by knockdown of Rab7B using the clustered regularly interspaced short palindromic repeats (CRISPR) and CasRx (also known as Cas13d) system. Also, the knockdown promoted the trafficking of PLP1 p.Ala243Val to lysosome-associated membrane protein 1 (LAMP1)-positive organelles. These results highlight the unique role of Rab7B knockdown in modulating oligodendroglial cell morphological changes and potentially facilitating the transport of mutated PLP1 to LAMP1-positive organelles, suggesting its potential as a therapeutic target for alleviating HLD1 phenotypes, at least in part, at the molecular and cellular levels.

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Skull morphogenesis is a complex, dynamic process involving two different germ layers and progressing to the coordinated, directional growth of individual bones. The mechanisms underlying directional growth toward the apex are not completely understood. Here, a microfluidic chip-based approach is utilized to test whether calvarial osteoblast progenitors undergo haptotaxis on a gradient of Fibronectin1 (FN1) via lamellipodia. Mimicking the embryonic cranial mesenchyme's FN1 pattern, FN1 gradients is established in the chip using computer modeling and fluorescent labeling. Primary mouse calvarial osteoblast progenitors are plated in the chip along an array of segmented gradients of adsorbed FN1. The study performs single-cell tracking and measures protrusive activity. Haptotaxis is observed at an intermediate FN1 concentration, with an average directional migration index (yFMI) of 0.07, showing a significant increase compared to the control average yFMI of -0.01. A significant increase in protrusive activity is observed during haptotaxis. Haptotaxis is an Arp2/3-dependent, lamellipodia-mediated process. Calvarial osteoblast progenitors treated with the Arp2/3 (Actin Related Protein 2/3 complex) inhibitor CK666 show significantly diminished haptotaxis, with an average yFMI of 0.01. Together, these results demonstrate haptotaxis on an FN1 gradient as a new mechanism in the apical expansion of calvarial osteoblast progenitors during development and shed light on the etiology of calvarial defects.

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Bacterial shape and division rely on the dynamics of cell wall assembly, which involves regulated synthesis and cleavage of the peptidoglycan. In ovococci, these processes are coordinated within an annular mid-cell region with nanometric dimensions. More precisely, the cross-wall synthesized by the divisome is split to generate a lateral wall, whose expansion is insured by the insertion of the so-called peripheral peptidoglycan by the elongasome. Septum cleavage and peripheral peptidoglycan synthesis are, thus, crucial remodeling events for ovococcal cell division and elongation. The structural DivIVA protein has long been known as a major regulator of these processes, but its mode of action remains unknown. Here, we integrate click chemistry-based peptidoglycan labeling, direct stochastic optical reconstruction microscopy, and in silico modeling, as well as epifluorescence and stimulated emission depletion microscopy to investigate the role of DivIVA in Streptococcus pneumoniae cell morphogenesis. Our work reveals two distinct phases of peptidoglycan remodeling during the cell cycle that are differentially controlled by DivIVA. In particular, we show that DivIVA ensures homogeneous septum cleavage and peripheral peptidoglycan synthesis around the division site and their maintenance throughout the cell cycle. Our data additionally suggest that DivIVA impacts the contribution of the elongasome and class A penicillin-binding proteins to cell elongation. We also report the position of DivIVA on either side of the septum, consistent with its known affinity for negatively curved membranes. Finally, we take the opportunity provided by these new observations to propose hypotheses for the mechanism of action of this key morphogenetic protein.IMPORTANCEThis study sheds light on fundamental processes governing bacterial growth and division, using integrated click chemistry, advanced microscopy, and computational modeling approaches. It addresses cell wall synthesis mechanisms in the opportunistic human pathogen Streptococcus pneumoniae, responsible for a range of illnesses (otitis, pneumonia, meningitis, septicemia) and for one million deaths every year worldwide. This bacterium belongs to the morphological group of ovococci, which includes many streptococcal and enterococcal pathogens. In this study, we have dissected the function of DivIVA, which is a structural protein involved in cell division, morphogenesis, and chromosome partitioning in Gram-positive bacteria. This work unveils the role of DivIVA in the orchestration of cell division and elongation along the pneumococcal cell cycle. It not only enhances our understanding of how ovoid bacteria proliferate but also offers the opportunity to consider how DivIVA might serve as a scaffold and sensor for particular membrane regions, thereby participating in various cell cycle processes.

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Candida albicans is an opportunistic fungal pathogen that can cause systemic infections in immunocompromised individuals. Morphological transition and biofilm formation are major virulence factors of C. albicans. Moreover, biofilm enhances resistance to antifungal agents. Therefore, it is urgent to identify new and effective compounds to target the biofilm of C. albicans. In the present study, the antifungal activities of equol against C. albicans were investigated. In vitro, the microdilution analysis and spot assay result showed that equol exhibited potent inhibitory activities against C. albicans. Further investigations confirmed that the antifungal effects of equol involved interference with the transition from yeast to hypha and biofilm formation of C. albicans. In addition, transcriptome sequencing and reverse transcription-quantitative PCR (qRT-PCR) analysis showed that equol significantly downregulated the expression of several genes in the Ras1-cAMP-PKA pathway related to hyphae and biofilm formation and significantly upregulated the expression of the negative transcriptional repressors RFG1 and TUP1. Moreover, equol effectively reduced the production of cAMP, a key messenger in the Ras1-cAMP-PKA pathway, while supplementation with cAMP partly rescued the equol-induced defects in hyphal development. Furthermore, in a mouse model of systemic candidiasis (SC), equol treatment significantly decreased the fungal burden (liver, kidneys, and lung) in mice and local tissue damage, while enhancing the production of interleukin-10 (IL-10). Together, these findings confirm that equol is a potentially effective agent for treatment of SC.

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Cdon and boc are members of the cell adhesion molecule subfamily III Ig/fibronectin. Although they have been reported to be involved in muscle and neural development at late developmental stage, their early roles in embryonic development remain unknown. Here, we discovered that in zebrafish, cdon, but not boc, is expressed in dorsal forerunner cells (DFCs) and the epithelium of Kupffer's vesicle (KV), suggesting a potential role for cdon in organ left-right (LR) patterning. Further data showed that liver and heart LR patterning were disrupted in cdon morphants and cdon mutants. Mechanistically, we found that loss of cdon function led to defect in DFCs clustering, reduced KV lumen, and defective cilia, resulting in randomized Nodal/spaw signaling and subsequent organ LR patterning defects. Additionally, predominant distribution of a cdon morpholino (MO) in DFCs caused defects in DFC clustering, KV morphogenesis, cilia number/length, Nodal/spaw signaling, and organ LR asymmetry, similar to those observed in cdon morphants and cdon -/- embryos, indicating a cell-autonomous role for cdon in regulating KV formation during LR patterning. In conclusion, our data demonstrate that during gastrulation and early somitogenesis, cdon is essential for proper DFC clustering, KV formation, and normal cilia, thereby playing a critical role in establishing organ LR asymmetry.

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Ciliates in the subclass Hypotrichia have long been difficult to classify as they are one of the most polymorphic and highly differentiated groups, leading to their systematics remaining unresolved. Phylogenetic relationships within the hypotrich family Strongylidiidae have been ambiguous due to discordance between the morphological and genetic data. In this study, a new strongylidiid genus Heterouroleptus is established, mainly based on the novel mode of origin of the ventral cirral rows: left ventral cirral row (LVR) originates from frontal-ventral-transverse cirral anlagen (FVTA) III (anterior portion), IV (middle portion), and V (rear portion); right ventral cirral row comes from the entire FVTA VI. A new species, Heterouroleptus weishanensis gen. nov., sp. nov., is investigated along with the morphometric and molecular data from a population of Strongylidium wuhanense. Eight new sequences and nuclear gene markers (single-gene and multi-gene) are provided to analyze the phylogenetic relationships of strongylidiids, with the COI gene utilized to uncover further genetic information at species level and below. The results reveal that: (1) Strongylidiidae is monophyletic and has a close relationship with Dorsomarginalia; (2) Heterouroleptus gen. nov. forms a clade that is sister to all the other strongylidiids; (3) Hemiamphisiella Foissner, 1988 and Pseudouroleptus Hemberger, 1985 should not be synonyms, and both genera should be subdivided due to their variable morphological characteristics; (4) LVR originating from three anlagen is a plesiomorphy of Strongylidiidae. The discovery of the origin of the LVR not only contributes to the establishment of the genus Heterouroleptus, but also helps to improve the diagnosis of the family Strongylidiidae. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00243-z.

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Embryonic muscle activity is involved in various aspects of bone morphogenesis and growth. Normal mechanical stimuli of muscle contraction are important in most cases, and when the muscles are immobilized, the developing bones are abnormally shaped. In chick embryos, a characteristic curved deformity is reproducibly induced in the developing tibiotarsus using the bone-weakening agent, beta-aminopropionitrile (bAPN). In this study, we applied decamethonium bromide (DMB), a well-established neuromuscular blocking agent, to embryos treated with bAPN, to test the hypothesis that the deformity is triggered and formed depending on the balance between the decrease in stiffness of the bAPN-affected tibiotarsus and the normal physiological increase in embryonic skeletal muscle activity. The occurrence of curved morphology induced by bAPN administered at 4 or 8 days of incubation (embryonic day [ED]) was temporally consistent with the posterior displacement of the leg muscles, which occurred just before ED8. The displaced muscles were assumed to exert a contraction force comparable to that of untreated normal muscles. When treated with DMB at ED8, the muscles atrophied and exhibited degenerative changes, and the degree of curved morphology was alleviated and reduced to 50% or more in the morphometric evaluation at ED10. These findings indicated that the coordinated development of skeletal element stiffness and muscle activity must be temporally regulated, particularly during the early stages of skeletogenesis.

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The aroma is critical in the reproductive biology of truffles and in their commercial quality. However, previous research has almost exclusively focused on characterizing ripe ascocarps. We characterized the volatilome of the highly-prized black truffle (Tuber melanosporum) ascocarps from July, in an early development stage, to March, in the late harvesting season, and investigated the relationships among aroma, ascocarp growth and morphogenetic development. The aroma profile was analyzed using a head space gas chromatography technique coupled with mass spectrometer. Seventy-one volatile compounds were identified and three development stages were clearly distinguished according to the volatile profile. In unripe ascocarps of July-September, the profile was dominated by methanethiol (19 %), 4-penten-2-ol (11 %) and acetone (11 %), the monthly mean weight of ascocarps ranged 2-20 g, and morphogenetic stages 4-6a were prevalent. In unripe ascocarps of October-December, the most abundant volatiles were 4-penten-2-ol (21 %), methanethiol (20 %) and ethanol (13 %), the monthly mean ascocarp weight ranged 28-43 g, and morphogenetic stages 6a, 6b-c were prevalent. In ripe ascocarps (December-March), the most abundant volatiles were 4-penten-2-ol (17 %), dimethyl sulfide (16 %) and ethanol (10 %), ascocarp weight did not increase significantly, and 6b-c was practically the sole morphogenetic stage. Thirty volatiles were associated to one of these three development stages. Amongst those with higher occurrence, 4-penten-2-ol, dimethyl sulfide, ethyl acetate, 2-pentanol and 2-butanone were associated to ripe truffles, whereas methanethiol, isobutyl isobutyrate, butanedione and 3-methylanisole were associated to unripe truffles.

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As a kind of tonic Chinese medicine with dual use in medicine and food, there is a large market demanding for Codonopsis pilosula. Taking one-year-old C. pilosula seedlings as materials, we conducted a field experiment to examine the effect of compound fertilizer (750 kg·hm-2), organic fertilizer (15 t·hm-2) and Streptomyces pactum Act12 agent (9 t·hm-2 Act12+10 t·hm-2 organic fertilizer) treatments on root morphology, secondary metabolite content and expression level of lobetyolin metabolic pathway gene of C. pilosula, to clarify the effects of three fertilizers on the root morphology and medicinal quality. Compared to the control (10 t·hm-2 organic fertilizer, conventional fertilization), three fertilization treatments could promote root growth and formation. All fertilization treatments promoted the accumulation of C. pilosula polysaccharides and secondary metabolites. Act12 agent significantly increased the content of lobetyolin, atractylenolideIII, and 5-hydroxymethylfurfural. The qRT-PCR analysis indicated that three fertilization treatments increased the expression level of lobetyolin metabolic pathway genes, with Act12 agent treatment showing the most significant effect. Pearson correlation analysis demonstrated that the expression level of CpHCT and CpFAD genes was significantly positively correlated with atractylenolide III content. In conclusion, three fertilization treatments could effectively improve the yield and quality of C. pilosula. Among the three treatments, Act12 agent performed better than that of compound fertilizer and organic fertilizer, which was an effective measure to increase the yield and quality of C. pilosula.

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Salivary gland branching morphogenesis is regulated by the functional integration of neuronal signaling, but the underlying mechanisms are not fully understood in aging accelerated klotho-deficient (Kl-/-) mice. Here, we investigated whether the neuropeptides substance P (SP) and neuropeptide Y (NPY) affect the branching morphogenesis of embryonic salivary glands in aging Kl-/- mice. In the salivary glands of embryonic Kl-/- mice, morphological analysis and immunostaining revealed that epithelial bud formation, neuronal cell proliferation/differentiation, and the expression of the salivary gland functional marker ZO-1 were decreased in embryonic ductal cells. Incubation with SP/NPY at E12-E13d promoted branching morphogenesis, parasympathetic innervation, and epithelial proliferation in salivary glands of embryonic Kl-/- mice. The ERK inhibitor U0126 specifically inhibited neuronal substance-induced epithelial bud formation in the embryonic salivary gland. RNA-seq profiling analysis revealed that the expression of fibroblast growth factors/fibroblast growth factors (FGFs/FGFRs) and their receptors was significantly regulated by SP/NPY treatment in the embryonic salivary gland (E15). The FGFR inhibitor BGJ389 inhibited new branching formation induced by SP and NPY treatment and ERK1/2 expression. These results showed that aging may affect virtually the development of salivary gland by neuronal dysfunction. The neuropeptides SP/NPY induced embryonic salivary gland development through FGF/FGFR/ERK1/2-mediated signaling.

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Hox transcription factors play crucial roles in organizing developmental patterning across metazoa, but how these factors trigger regional morphogenesis has largely remained a mystery. In the developing gut, Hox genes help demarcate identities of intestinal subregions early in embryogenesis, which ultimately leads to their specialization in both form and function. Although the midgut forms villi, the hindgut develops sulci that resolve into heterogeneous outgrowths. Combining mechanical measurements of the embryonic chick intestine and mathematical modeling, we demonstrate that the posterior Hox gene HOXD13 regulates biophysical phenomena that shape the hindgut lumen. We further show that HOXD13 acts through the transforming growth factor ß (TGF-ß) pathway to thicken, stiffen, and promote isotropic growth of the subepithelial mesenchyme-together, these features lead to hindgut-specific surface buckling. TGF-ß, in turn, promotes collagen deposition to affect mesenchymal geometry and growth. We thus identify a cascade of events downstream of positional identity that direct posterior intestinal morphogenesis.

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Study of morphogenesis and its regulation requires analytical tools that enable simultaneous assessment of processes operating at cellular level, such as synthesis of transcription factors (TF), with their effects at the tissue scale. Most current studies conduct histological, cellular and immunochemical (IHC) analyses in separate steps, introducing inevitable biases in finding and alignment of areas of interest at vastly distinct scales of organization, as well as image distortion associated with image repositioning or file modifications. These problems are particularly severe for longitudinal analyses of growing structures that change size and shape. Here we introduce a python-based application for automated and complete whole-slide measurement of expression of multiple TFs and associated cellular morphology. The plugin collects data at customizable scale from the cell-level to the entire structure, records each data point with positional information, accounts for ontogenetic transformation of structures and variation in slide positioning with scalable grid, and includes a customizable file manager that outputs collected data in association with full details of image classification (e.g., ontogenetic stage, population, IHC assay). We demonstrate the utility and accuracy of this application by automated measurement of morphology and associated expression of eight TFs for more than six million cells recorded with full positional information in beak tissues across 12 developmental stages and 25 study populations of a wild passerine bird. Our script is freely available as an open-source Fiji plugin and can be applied to IHC slides from any imaging platforms and transcriptional factors.

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Organoids are quickly becoming an accepted model for understanding human biology and disease. Pluripotent stem cells (PSC) provide a starting point for many organs and enable modeling of the embryonic development and maturation of such organs. The foundation of PSC-derived organoids can be found in elegant developmental studies demonstrating the remarkable ability of immature cells to undergo histogenesis even when taken out of the embryo context. PSC-organoids are an evolution of earlier methods such as embryoid bodies, taken to a new level with finer control and in some cases going beyond tissue histogenesis to organ-like morphogenesis. But many of the discoveries that led to organoids were not necessarily planned, but rather the result of inquisitive minds with freedom to explore. Protecting such curiosity-led research through flexible funding will be important going forward if we are to see further ground-breaking discoveries.

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Astrocytes have gained increasing recognition as key elements of a broad array of nervous system functions. These include essential roles in synapse formation and elimination, synaptic modulation, maintenance of the blood-brain barrier, energetic support, and neural repair after injury or disease of the nervous system. Nevertheless, our understanding of mechanisms underlying astrocyte development and maturation remains far behind that of neurons and oligodendrocytes. Early efforts to understand astrocyte development focused primarily on their specification from embryonic progenitors and the molecular mechanisms driving the switch from neuron to glial production. Considerably, less is known about postnatal stages of astrocyte development, the period during which they are predominantly generated and mature. Notably, this period is coincident with synapse formation and the emergence of nascent neural circuits. Thus, a greater understanding of astrocyte development is likely to shed new light on the formation and maturation of synapses and circuits. Here, we highlight key foundational principles of embryonic and postnatal astrocyte development, focusing largely on what is known from rodent studies.

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BACKGROUND: Uveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye and vasculature enters. The Hedgehog (Hh) signaling pathway regulates optic fissure development: loss-of-function mutations in the Hh receptor ptch2 produce overactive Hh signaling and can result in coloboma. We previously proposed a model where overactive Hh signaling disrupts optic fissure formation by upregulating transcriptional targets acting both cell- and non-cell-autonomously. Here, we examine the Netrin family of secreted ligands as candidate Hh target genes. RESULTS: We find multiple Netrin ligands upregulated in the zebrafish ptch2 mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find that netrin1a or netrin1b overexpression is sufficient to cause coloboma and disrupt wild-type optic fissure formation. We used loss-of-function alleles, CRISPR/Cas9 mutagenesis, and morpholino knockdown to test if loss of Netrin can rescue coloboma in the ptch2 mutant: loss of netrin genes does not rescue the ptch2 mutant phenotype. CONCLUSION: These results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in the ptch2 mutant.

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Formation of fluid-filled lumina by epithelial tissues is essential for organ development. How cells control the hydraulic and cortical forces to control lumen morphology is not well understood. Here, we quantified the mechanical role of tight junctions in lumen formation using MDCK-II cysts. We found that the paracellular ion barrier formed by claudin receptors is not required for the hydraulic inflation of a lumen. However, the depletion of the zonula occludens scaffold resulted in lumen collapse and folding of apical membranes. Combining quantitative measurements of hydrostatic lumen pressure and junctional tension with modeling enabled us to explain lumen morphologies from the pressure-tension force balance. Tight junctions promote lumen inflation by decreasing cortical tension via the inhibition of myosin. In addition, our results suggest that excess apical area contributes to lumen opening. Overall, we provide a mechanical understanding of how epithelial cells use tight junctions to modulate tissue and lumen shape.

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Branching morphogenesis is a characteristic feature of many essential organs, such as the lung and kidney, and most glands, and is the net result of two tissue behaviors: branch point initiation and elongation. Each branched organ has a distinct architecture customized to its physiological function, but how patterning occurs in these ramified tubular structures is a fundamental problem of development. Here, we use quantitative 3D morphometrics, time-lapse imaging, manipulation of ex vivo cultured mouse embryonic organs and mice deficient in the planar cell polarity component Vangl2 to address this question in the developing mammary gland. Our results show that the embryonic epithelial trees are highly complex in topology owing to the flexible use of two distinct modes of branch point initiation: lateral branching and tip bifurcation. This non-stereotypy was contrasted by the remarkably constant average branch frequency, indicating a ductal growth invariant, yet stochastic, propensity to branch. The probability of branching was malleable and could be tuned by manipulating the Fgf10 and Tgfß1 pathways. Finally, our in vivo data and ex vivo time-lapse imaging suggest the involvement of tissue rearrangements in mammary branch elongation.

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In vertebrate retina, individual neurons of the same type are distributed regularly across the tissue in a pattern known as a mosaic. Establishment of mosaics during development requires cell-cell repulsion among homotypic neurons, but the mechanisms underlying this repulsion remain unknown. Here, we show that two mouse retinal cell types, OFF and ON starburst amacrine cells, establish mosaic spacing by using their dendritic arbors to repel neighboring homotypic somata. Using transgenic tools and single-cell labeling, we identify a developmental period when starburst somata are contacted by neighboring starburst dendrites; these serve to exclude somata from settling within the neighbor's dendritic territory. Dendrite-soma exclusion is mediated by MEGF10, a cell-surface molecule required for starburst mosaic patterning. Our results implicate dendrite-soma exclusion as a key mechanism underlying starburst mosaic spacing and raise the possibility that this could be a general mechanism for mosaic patterning across many cell types and species.

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Stem cells often rely on signals from a niche, which in many tissues adopts a precise morphology. What remains elusive is how niches are formed and how morphology impacts function. To address this, we leverage the Drosophila gonadal niche, which affords genetic tractability and live-imaging. We have previously shown mechanisms dictating niche cell migration to their appropriate position within the gonad and the resultant consequences on niche function. Here, we show that once positioned, niche cells robustly polarize filamentous actin (F-actin) and non-muscle myosin II (MyoII) toward neighboring germ cells. Actomyosin tension along the niche periphery generates a highly reproducible smoothened contour. Without contractility, niches are misshapen and exhibit defects in their ability to regulate germline stem cell behavior. We additionally show that germ cells aid in polarizing MyoII within niche cells and that extrinsic input is required for niche morphogenesis and function. Our work reveals a feedback mechanism where stem cells shape the niche that guides their behavior.

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Cancer stem cells (CSC) are thought to be responsible for cancer phenotypes and cellular heterogeneity. Here we demonstrate that the human colon cancer cell line DLD1 contains two types of CSC-like cells that undergo distinct morphogenesis in the reconstituted basement membrane gel Matrigel. In our method with cancer cell spheroids, the parent cell line (DLD1-P) developed grape-like budding structures, whereas the other (DLD1-Wm) and its single-cell clones dynamically developed worm-like ones. Gene expression analysis suggested that the former mimicked intestinal crypt-villus morphogenesis, while the latter mimicked embryonic hindgut development. The organoids of DLD1-Wm cells rapidly extended in two opposite directions by expressing dipolar proteolytic activity. The invasive morphogenesis required the expression of MMP-2 and CD133 genes and ROCK activity. These cells also exhibited gastrula-like morphogenesis even in two-dimensional cultures without Matrigel. Moreover, the two DLD1 cell lines showed clear differences in cellular growth, tumor growth and susceptibility to paclitaxel. This study also provides a simple organoid culture method for human cancer cell lines. HT-29 and other cancer cell lines underwent characteristic morphogenesis in direct contact with normal fibroblasts. Such organoid cultures would be useful for investigating the nature of CSCs and for screening anti-cancer drugs. Our results lead to the hypothesis that CSC-like cells with both invasive activity and a fetal phenotype, i. e. oncofetal CSCs, are generated in some types of colon cancers.