Morphogenic Cues Research Articles

Ankrd26 is a retinoic acid-responsive plasma membrane-binding and -shaping protein critical for proper cell differentiation

Morphogens are important triggers for differentiation processes. Yet, downstream effectors that organize cell shape changes in response to morphogenic cues, such as retinoic acid, largely remain elusive. Additionally, derailed plasma membrane-derived signaling often is associated with cancer. We identify Ankrd26 as a critical player in cellular differentiation and as plasma membrane-localized protein able to self-associate and form clusters at the plasma membrane in response to retinoic acid. We show that Ankrd26 uses an N-terminal amphipathic structure for membrane binding and bending. Importantly, in an acute myeloid leukemia-associated Ankrd26 mutant, this critical structure was absent, and Ankrd26's membrane association and shaping abilities were impaired. In line with this, the mutation rendered Ankrd26 inactive in both gain-of-function and loss-of-function/rescue studies addressing retinoic acid/brain-derived neurotrophic factor (BDNF)-induced neuroblastoma differentiation. Our results highlight the importance and molecular details of Ankrd26-mediated organizational platforms for cellular differentiation at the plasma membrane and how impairment of these platforms leads to cancer-associated pathomechanisms involving these Ankrd26 properties.

A chemo-mechanical model of endoderm movements driving elongation of the amniote hindgut.

Although mechanical and biochemical descriptions of development are each essential, integration of upstream morphogenic cues with downstream tissue mechanics remains understudied during vertebrate morphogenesis. Here, we developed a two-dimensional chemo-mechanical model to investigate how mechanical properties of the endoderm and transport properties of fibroblast growth factor (FGF) regulate avian hindgut morphogenesis in a coordinated manner. Posterior endoderm cells convert a gradient of FGF ligands into a contractile force gradient, leading to a force imbalance that drives collective cell movements that elongate the forming hindgut tube. We formulated a 2D reaction-diffusion-advection model describing the formation of an FGF protein gradient as a result of posterior displacement of cells transcribing unstable Fgf8 mRNA during axis elongation, coupled with translation, diffusion and degradation of FGF protein. The endoderm was modeled as an active viscous fluid that generates contractile stresses in proportion to FGF concentration. With parameter values constrained by experimental data, the model replicates key aspects of hindgut morphogenesis, suggests that graded isotropic contraction is sufficient to generate large anisotropic cell movements, and provides new insight into how chemo-mechanical coupling across the mesoderm and endoderm coordinates hindgut elongation with axis elongation.

Generating high-fidelity cochlear organoids from human pluripotent stem cells.

Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues invitro; however, derivation of cochlear cell types has proven non-trivial. Here, using 3D cultures of human pluripotent stem cells, we sought to replicate key differentiation cues of cochlear specification. We found that timed modulations of Sonic Hedgehog and WNT signaling promote ventral gene expression in otic progenitors. Ventralized otic progenitors subsequently give rise to elaborately patterned epithelia containing hair cells with morphology, marker expression, and functional properties consistent with both outer and inner hair cells in the cochlea. These results suggest that early morphogenic cues are sufficient to drive cochlear induction and establish an unprecedented system to model the human auditory organ.

Ontogenetic rules for the molecular diversification of hypothalamic neurons.

The hypothalamus is an evolutionarily conserved endocrine interface that, among other roles, links central homeostatic control to adaptive bodily responses by releasing hormones and neuropeptides from its many neuronal subtypes. In its preoptic, anterior, tuberal and mammillary subdivisions, a kaleidoscope of magnocellular and parvocellular neuroendocrine command neurons, local-circuit neurons, and neurons that project to extrahypothalamic areas are intermingled in partially overlapping patches of nuclei. Molecular fingerprinting has produced data of unprecedented mass and depth to distinguish and even to predict the synaptic and endocrine competences, connectivity and stimulus selectivity of many neuronal modalities. These new insights support eminent studies from the past century but challenge others on the molecular rules that shape the developmental segregation of hypothalamic neuronal subtypes and their use of morphogenic cues for terminal differentiation. Here, we integrate single-cell RNA sequencing studies with those of mouse genetics and endocrinology to describe key stages of hypothalamus development, including local neurogenesis, the direct terminal differentiation of glutamatergic neurons, transition cascades for GABAergic and GABAergic cell-derived dopamine cells, waves of local neuronal migration, and sequential enrichment in neuropeptides and hormones. We particularly emphasize how transcription factors determine neuronal identity and, consequently, circuit architecture, and whether their deviations triggered by environmental factors and hormones provoke neuroendocrine illnesses.

Deep neural net tracking of human pluripotent stem cells reveals intrinsic behaviors directing morphogenesis.

SummaryLineage tracing is a powerful tool in developmental biology to interrogate the evolution of tissue formation, but the dense, three-dimensional nature of tissue limits the assembly of individual cell trajectories into complete reconstructions of development. Human induced pluripotent stem cells (hiPSCs) can recapitulate aspects of developmental processes, providing an in vitro platform to assess the dynamic collective behaviors directing tissue morphogenesis. Here, we trained an ensemble of neural networks to track individual hiPSCs in time-lapse microscopy, generating longitudinal measures of cell and cellular neighborhood properties on timescales from minutes to days. Our analysis reveals that, while individual cell parameters are not strongly affected by pluripotency maintenance conditions or morphogenic cues, regional changes in cell behavior predict cell fate and colony organization. By generating complete multicellular reconstructions of hiPSC behavior, our tracking pipeline enables fine-grained understanding of morphogenesis by elucidating the role of regional behavior in early tissue formation.

Mono- and multispecies biofilms from a crustose coralline alga induce settlement in the scleractinian coral Leptastrea purpurea

Microorganisms have been reported to induce settlement in various marine invertebrate larvae but their specificity of inductive capacities for the settlement of coral larvae remains poorly understood. In this study, we isolated 56 microbial strains from the crustose coralline alga (CCA) Hydrolithon reinboldii using five different media either with or without additional antibiotics and/or spiked CCA extract. We tested the isolates for their potential to induce settlement behavior in larvae of the brooding scleractinian coral Leptastrea purpurea. From these 56 CCA-associated microbial strains, we identified six bacterial classes and 18 families. The culturable bacterial community associated with H. reinboldii was dominated by Gammaproteobacteria, Actinobacteria, and Alphaproteobacteria while the Illumina MiSeq analysis showed that the culture-independent bacterial community was dominated by Gammaproteobacteria, Alphaproteobacteria, and Flavobacteria. Furthermore, we found no correlation between inductive settlement capacities and phylogenetic relationships. Instead, settlement behavior of L. purpurea larvae was induced by specific isolated species. Strains #1792 (Pseudovibrio denitrificans), #1678 (Acinetobacter pittii), #1633 (Pseudoalteromonas phenolica), #1772 (Marine bacterium LMG1), #1721 (Microbulbifer variabilis), and #1783 (Pseudoalteromonas rubra) induced settlement behavior in coral larvae at mostly high and significant levels (≥ 40%) but the remaining isolates strongly varied in their activity. Multispecies biofilms consisting of four strains (#1792, #1678, #1633, and #1721) were observed to synergistically increase settlement behavior levels (> 90%); however, the addition of #1772 to the multispecies biofilms negatively affected coral larvae and resulted in a total loss of inducing activity. The findings provide new insights into the role of bacteria in the settlement process of scleractinian corals and may help to identify the true nature of bacteria-derived morphogenic cues.

Directed Differentiation of Human Pluripotent Stem Cells into Inner Ear Organoids.

The sensory epithelia of the inner ear contain mechanosensitive hair cells that detect sound and head acceleration. This protocol details a 3D differentiation method to generate inner ear organoids containing sensory epithelia with hair cells. Human pluripotent stem cells are aggregated in low-binding 96-well plates and treated in chemically defined media with extracellular matrix to promote epithelialization. Small molecules and recombinant proteins are applied in a stepwise manner to recapitulate the morphogenic cues (BMP, TGF-β, FGF, and WNT) present during inner ear development in vivo. These treatments induce the sequential formation of nonneural ectoderm, otic-epibranchial progenitor domain, and otic placodes. The derived otic placodes then undergo self-guided morphogenesis to form otic vesicles, which eventually give rise to sensory epithelia containing hair cells and supporting cells, as well as neurons with synaptic formations to hair cells. This human stem cell-derived inner ear organoid system provides an ideal platform to study human inner ear development and disease in vitro.

The elephant in the lung: Integrating lineage-tracing, molecular markers, and single cell sequencing data to identify distinct fibroblast populations during lung development and regeneration

During lung development, the mesenchyme and epithelium are dependent on each other for instructive morphogenic cues that direct proliferation, cellular differentiation and organogenesis. Specification of epithelial and mesenchymal cell lineages occurs in parallel, forming cellular subtypes that guide the formation of both transitional developmental structures and the permanent architecture of the adult lung. While epithelial cell types and lineages have been relatively well-defined in recent years, the definition of mesenchymal cell types and lineage relationships has been more challenging. Transgenic mouse lines with permanent and inducible lineage tracers have been instrumental in identifying lineage relationships among epithelial progenitor cells and their differentiation into distinct airway and alveolar epithelial cells. Lineage tracing experiments with reporter mice used to identify fibroblast progenitors and their lineage trajectories have been limited by the number of cell specific genes and the developmental timepoint when the lineage trace was activated. In this review, we discuss major developmental mesenchymal lineages, focusing on time of origin, major cell type, and other lineage derivatives, as well as the transgenic tools used to find and define them. We describe lung fibroblasts using function, location, and molecular markers in order to compare and contrast cells with similar functions. The temporal and cell-type specific expression of fourteen “fibroblast lineage” genes were identified in single-cell RNA-sequencing data from LungMAP in the LGEA database. Using these lineage signature genes as guides, we clustered murine lung fibroblast populations from embryonic day 16.5 to postnatal day 28 (E16.5-PN28) and generated heatmaps to illustrate expression of transcription factors, signaling receptors and ligands in a temporal and population specific manner.

Vascular Development.

The vascular system forms as a branching network of endothelial cells that acquire identity as arterial, venous, hemogenic, or lymphatic. Endothelial specification depends on gene targets transcribed by Ets domain-containing factors, including Ets variant gene 2 (Etv2), together with the activity of chromatin-remodeling complexes containing Brahma-related gene-1 (Brg1). Once specified and assembled into vessels, mechanisms regulating lumen diameter and axial growth ensure that the structure of the branching vascular network matches the need for perfusion of target tissues. In addition, blood vessels provide important morphogenic cues that guide or direct the development of organs forming around them. As the embryo grows and lumen diameters increase, smooth muscle cells wrap around the nascent vessel walls to provide mechanical strength and vasomotor control of the circulation. Increasing mechanical stretch and wall strain promote smooth muscle cell differentiation via coupling of actin cytoskeletal remodeling to myocardin and serum response factor-dependent transcription. Remodeling of artery walls by developmental signaling pathways reappears in postnatal blood vessels during physiological and pathological adaptation to vessel wall injury, inflammation, or chronic hypoxia. Recent reports providing insights into major steps in vascular development are reviewed here with a particular emphasis on studies that have been recently published in Arteriosclerosis, Thrombosis, and Vascular Biology.

Context clues: the importance of stem cell-material interactions.

Understanding the processes by which stem cells give rise to de novo tissues is an active focus of stem cell biology and bioengineering disciplines. Instructive morphogenic cues surrounding the stem cell during morphogenesis create what is referred to as the stem cell microenvironment. An emerging paradigm in stem cell bioengineering involves "biologically driven assembly," in which stem cells are encouraged to largely define their own morphogenesis processes. However, even in the case of biologically driven assembly, stem cells do not act alone. The properties of the surrounding microenvironment can be critical regulators of cell fate. Stem cell-material interactions are among the most well-characterized microenvironmental effectors of stem cell fate and establish a signaling "context" that can define the mode of influence for morphogenic cues. Here we describe illustrative examples of cell-material interactions that occur during in vitro stem cell studies, with an emphasis on how cell-material interactions create instructive contexts for stem cell differentiation and morphogenesis.

Establishing epithelial glandular polarity: interlinked roles for ARF6, Rac1, and the matrix microenvironment

Epithelial cysts comprise the structural units of the glandular epithelium. Although glandular inversion in epithelial tumors is thought to be a potential mechanism for the establishment of metastatic disease, little is known about the morphogenic cues and signaling pathways that govern glandular polarity and organization. Using organotypic cultures of Madin-Darby canine kidney cells in reconstituted basement membrane, we show that cellular depletion of the small GTP-binding protein ARF6 promotes the formation of inverted cysts, wherein the apical cell membrane faces the cyst exterior, and the basal domain faces the central lumen, while individual cell polarity is maintained. These cysts are also defective in interactions with laminin at the cyst-matrix interface. This inversion of glandular orientation is accompanied by Rac1 inactivation during early cystogenesis, and temporal activation of Rac1 is sufficient to recover the normal cyst phenotype. In an unnatural collagen I microenvironment, ARF6-depleted, inverted epithelial cysts exhibit some loss of cell polarity, a marked increase in Rho activation and Rac1 inactivation, and striking rearrangement of the surrounding collagen I matrix. These studies demonstrate the importance of ARF6 as a critical determinant of glandular orientation and the matrix environment in dictating structural organization of epithelial cysts.

Modeling disease using three dimensional cell culture multi-lumen and inverted cyst phenotypes

Three-dimensional cell culture provides a unique system to investigate intrinsic mechanisms and micro environmental cues involved in the morphogenesis of epithelial glandular architectures. While this culture system allows insight into normal tissue development, it is also is readily amenable to manipulations that permit cellular modeling of various disease states. Here, we discuss a range of cellular and genetic alterations that result in two distinct cyst phenotypes, the multi-lumen cyst and the inverted cyst, both of which involve defects in cell polarity and lumen formation. Multi-lumen cyst formation results from disturbances in the mechanisms that regulate cell polarity, apical assembly, and the rate of lumen clearance. In the inverted cyst, the apical domain is oriented adjacent to the matrix, markedly affecting the morphogenic cues the matrix provides for cystogenesis. Both of these abnormal glandular phenotypes are highly reminiscent of histological patterns used to classify a number of diseases. A better understanding of the causes of multi-lumen and inverted cysts will provide insights into the origin and progression of epithelial diseases, potentially leading to the development of new therapies.

Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation

Embryonic stem cells (ESCs) are pluripotent cells capable of differentiating into all somatic and germ cell types. The intrinsic ability of pluripotent cells to generate a vast array of different cells makes ESCs a robust resource for a variety of cell transplantation and tissue engineering applications, however, efficient and controlled means of directing ESC differentiation is essential for the development of regenerative therapies. ESCs are commonly differentiated in vitro by spontaneously self-assembling in suspension culture into 3D cell aggregates called embryoid bodies (EBs), which mimic many of the hallmarks of early embryonic development, yet the 3D organization and structure of EBs also presents unique challenges to effectively direct the differentiation of the cells. ESC differentiation is strongly influenced by physical and chemical signals comprising the local extracellular microenvironment, thus current methods to engineer EB differentiation have focused primarily on spatially controlling EB size, adding soluble factors to the media, or culturing EBs on or within natural or synthetic extracellular matrices. Although most such strategies aim to influence differentiation from the exterior of EBs, engineering the microenvironment directly within EBs enables new opportunities to efficiently direct the fate of the cells by locally controlling the presentation of morphogenic cues.

Efficacy of solvent extraction methods for acellularization of embryoid bodies

The ability of embryonic stem cells (ESCs) to differentiate into all somatic cell types makes them an attractive cell source for regenerative medicine and tissue-engineering applications. In addition to their potential to restore cellularity of injured or diseased tissues, molecular factors produced by stem cells may also directly influence tissue morphogenesis, thereby providing therapeutic benefit independent of stem cell differentiation. In order to examine this hypothesis, it is necessary to separate the cells from the molecular factors they are capable of producing. One potential method of separation is to acellularize clusters of differentiating ESCs, referred to as embryoid bodies (EBs), from the extracellular matrix they synthesize. Thus, the objective of this study was to examine the effectiveness of different reagents, including peracetic acid, sodium dodecyl sulfate, Triton X-100 and DNase, to acellularize EBs. The efficiency of acellularization was assessed based on cell viability and retention of overall mass, DNA and protein, as well as histological examination of the resulting acellular matrix. Initial studies suggested that sequential treatments of Triton X-100 and DNase successfully yielded a cohesive acellular product that retained protein content and significantly reduced levels of DNA. Additional optimization studies were performed with combinations of Triton X-100 and DNase to assess the specific effects of reagent concentration, treatment duration and solvent volume/EB ratios. These results establish methods to effectively obtain novel acellular matrices from differentiating ESCs that may contain morphogenic cues and have potential applications in regenerative medicine.

Molecular Insights into Segmentation along the Proximal–Distal Axis of the Nephron

The structure of a mammalian kidney is parsed into large collections of polarized nephrons, and each segment is home to a diverse community of cells that specialize in renal endocrine and excretory functions. Early developmental lengthening and diversification of nephron segments along a proximal--distal axis initiate all subsequent facets of tubular growth and function. Morphogenic cues and biochemical interactions that are critical to this process are starting to emerge. The underlying principles of regional cell signaling and transcriptional control organizing early segmentation are the subject of this review.