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The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

Kyoto University

Osaka Medical and Pharmaceutical University

Neurons develop dendritic arbors in cell type-specific patterns. Using growing Purkinje cells in culture as a model, we performed a long-term time-lapse observation of dendrite branch dynamics to understand the rules that govern the characteristic space-filling dendrites. We found that dendrite architecture was sculpted by a combination of reproducible dynamic processes, including constant tip elongation, stochastic terminal branching, and retraction triggered by contacts between growing dendrites. Inhibition of protein kinase C/protein kinase D signaling prevented branch retraction and significantly altered the characteristic morphology of long proximal segments. A computer simulation of dendrite branch dynamics using simple parameters from experimental measurements reproduced the time-dependent changes in the dendrite configuration in live Purkinje cells. Furthermore, perturbation analysis to parameters in silico validated the important contribution of dendritic retraction in the formation of the characteristic morphology. We present an approach using live imaging and computer simulations to clarify the fundamental mechanisms of dendrite patterning in the developing brain.

Principles of branch dynamics governing shape characteristics of cerebellar Purkinje cell dendrites.

Most organs scale proportionally with body size through regulation of individual cell size and/or cell number. Here we addressed how postmitotic and morphologically complex cells such as neurons scale with the body size by using the dendritic arbor of one Drosophila sensory neuron as an assay system. In small adults eclosed under a limited-nutrition condition, the wild-type neuron preserved the branching complexity of the arbor, but scaled down the entire arbor, making a "miniature". In contrast, mutant neurons for the Insulin/IGF signaling (IIS) or TORC1 pathway exhibited "undergrowth", which was characterized by decreases in both the branching complexity and the arbor size, despite a normal diet. These contrasting phenotypes hinted that a novel regulatory mechanism contributes to the dendritic scaling in wild-type neurons. Indeed, we isolated a mutation in the gene CHORD/morgana that uncoupled the neuron size and the body size: CHORD mutant neurons generated miniature dendritic arbors regardless of the body size. CHORD encodes an evolutionarily conserved co-chaperone of HSP90. Our results support the notion that dendritic growth and branching are controlled by partly separate mechanisms. The IIS/TORC1 pathways control both growth and branching to avert underdevelopment, whereas CHORD together with TORC2 realizes proportional scaling of the entire arbor.

An evolutionarily conserved protein CHORD regulates scaling of dendritic arbors with body size.

Hippocampal pyramidal cells and dentate granule cells develop morphologically distinct dendritic arbors, yet also share some common features. Both cell types form a long apical dendrite which extends from the apex of the cell soma, while short basal dendrites are developed only in pyramidal cells. Using quantitative morphometric analyses of mouse hippocampal cultures, we evaluated the differences in dendritic arborization patterns between pyramidal and granule cells. Furthermore, we observed and described the final apical dendrite determination during dendritic polarization by time-lapse imaging. Pyramidal and granule cells in culture exhibited similar dendritic patterns with a single principal dendrite and several minor dendrites so that the cell types were not readily distinguished by appearance. While basal dendrites in granule cells are normally degraded by adulthood in vivo, cultured granule cells retained their minor dendrites. Asymmetric growth of a single principal dendrite harboring the Golgi was observed in both cell types soon after the onset of dendritic growth. Time-lapse imaging revealed that up until the second week in culture, final principal dendrite designation was not stabilized, but was frequently replaced by other minor dendrites. Before dendritic polarity was stabilized, the Golgi moved dynamically within the soma and was repeatedly repositioned at newly emerging principal dendrites. Our results suggest that polarized growth of the apical dendrite is regulated by cell intrinsic programs, while regression of basal dendrites requires cue(s) from the extracellular environment in the dentate gyrus. The apical dendrite designation is determined from among multiple growing dendrites of young developing neurons.

Differentiation of apical and basal dendrites in pyramidal cells and granule cells in dissociated hippocampal cultures.

The distribution of mitochondria within mature, differentiated neurons is clearly adapted to their regional physiological needs and can be perturbed under various pathological conditions, but the function of mitochondria in developing neurons has been less well studied. We have studied mitochondrial distribution within developing mouse cerebellar Purkinje cells and have found that active delivery of mitochondria into their dendrites is a prerequisite for proper dendritic outgrowth. Even when mitochondria in the Purkinje cell bodies are functioning normally, interrupting the transport of mitochondria into their dendrites severely disturbs dendritic growth. Additionally, we find that the growth of atrophic dendrites lacking mitochondria can be rescued by activating ATP-phosphocreatine exchange mediated by creatine kinase (CK). Conversely, inhibiting cytosolic CKs decreases dendritic ATP levels and also disrupts dendrite development. Mechanistically, this energy depletion appears to perturb normal actin dynamics and enhance the aggregation of cofilin within growing dendrites, reminiscent of what occurs in neurons overexpressing the dephosphorylated form of cofilin. These results suggest that local ATP synthesis by dendritic mitochondria and ATP-phosphocreatine exchange act synergistically to sustain the cytoskeletal dynamics necessary for dendritic development.

Synergistic action of dendritic mitochondria and creatine kinase maintains ATP homeostasis and actin dynamics in growing neuronal dendrites.

Mitochondria dynamically change their shape by repeated fission and fusion in response to physiological and pathological conditions. Recent studies have uncovered significant roles of mitochondrial fission and fusion in neuronal functions, such as neurotransmission and spine formation. However, the contribution of mitochondrial fission to the development of dendrites remains controversial. We analyzed the function of the mitochondrial fission GTPase Drp1 in dendritic arborization in cerebellar Purkinje cells. Overexpression of a dominant-negative mutant of Drp1 in postmitotic Purkinje cells enlarged and clustered mitochondria, which failed to exit from the soma into the dendrites. The emerging dendrites lacking mitochondrial transport remained short and unstable in culture and in vivo. The dominant-negative Drp1 affected neither the basal respiratory function of mitochondria nor the survival of Purkinje cells. Enhanced ATP supply by creatine treatment, but not reduced ROS production by antioxidant treatment, restored the hypomorphic dendrites caused by inhibition of Drp1 function. Collectively, our results suggest that Drp1 is required for dendritic distribution of mitochondria and thereby regulates energy supply in growing dendritic branches in developing Purkinje cells.

Mitochondrial fission protein Drp1 regulates mitochondrial transport and dendritic arborization in cerebellar Purkinje cells.

Dendritic filopodia of developing neurons function as environmental sensors, regulating the spatial organization of dendrites and proper targeting to presynaptic partners. Dendritic filopodia morphology is determined by the balance of F-actin assembled via two major nucleating pathways, the ARP2/3 complex and formins. The inverse-BAR protein MTSS1 is highly expressed in Purkinje cells (PCs) and has been shown to upregulate ARP2/3 activity. PCs in MTSS1 conditional knockout mice showed dendrite hypoplasia due to excessive contact-induced retraction during development. This phenotype was concomitant with elongated dendritic filopodia and was phenocopied by overactivation of the actin nucleator formin DAAM1 localized in the tips of PC dendritic protrusions. Cell biology assays including single-molecule speckle microscopy demonstrated that MTSS1's C terminus binds to DAAM1 and paused DAAM1-mediated F-actin polymerization. Thus, MTSS1 plays a dual role as a formin inhibitor and ARP2/3 activator in dendritic filopodia, determining final neuronal morphology.

MTSS1 Regulation of Actin-Nucleating Formin DAAM1 in Dendritic Filopodia Determines Final Dendritic Configuration of Purkinje Cells.

A series of porphyrin-fullerene linked molecules has been synthesized to evaluate the effects of substituents and molecular structures on their charge-separation yield and the lifetime of a final charge-separated state in various hydrophilic environments. The selected high-performance molecule effectively achieved depolarization in a plasma cell membrane by visible light as well as two-photon excitation using a near-infrared light laser. Moreover, it was revealed that the depolarization can trigger neuronal firing in rat hippocampal neurons, demonstrating the potential and versatility for controlling cell functions using light.

Optical control of neuronal firing  photoinduced electron transfer in donor-acceptor conjugates.

Cerebellar Purkinje cells arborize unique dendrites that exhibit a planar, fan shape. The dendritic branches fill the space of their receptive field with little overlap. This dendritic arrangement is well-suited to form numerous synapses with the afferent parallel fibers of the cerebellar granule cells in a non-redundant manner. Purkinje cell dendritic arbor morphology is achieved by a combination of dynamic local branch growth behaviors, including elongation, branching, and retraction. Impacting these behaviors, the self-avoidance of each branch terminal is essential to form the non-overlapping dendritic configuration. This review outlines recent advances in our understanding of the cellular and molecular mechanisms of dendrite formation during cerebellar Purkinje cell development.

Dendritic Self-Avoidance and Morphological Development of Cerebellar Purkinje Cells.

The mechanism underlying the geometrical patterning of axon and dendrite wiring remains elusive, despite its crucial importance in the formation of functional neural circuits. The cerebellar Purkinje cell (PC) arborizes a typical planar dendrite, which forms an orthogonal network with granule cell (GC) axons. By using electrospun nanofiber substrates, we reproduce the perpendicular contacts between PC dendrites and GC axons in culture. In the model system, PC dendrites show a preference to grow perpendicularly to aligned GC axons, which presumably contribute to the planar dendrite arborization  We show that βIII spectrin, a causal protein for spinocerebellar ataxia type 5, is required for the biased growth of dendrites. βIII spectrin deficiency causes actin mislocalization and excessive microtubule invasion in dendritic protrusions, resulting in abnormally oriented branch formation. Furthermore, disease-associated mutations affect the ability of βIII spectrin to control dendrite orientation. These data indicate that βIII spectrin organizes the mouse dendritic cytoskeleton and thereby regulates the oriented growth of dendrites with respect to the afferent axons.

βIII spectrin controls the planarity of Purkinje cell dendrites by modulating perpendicular axon-dendrite interactions.

Intestinal epithelial cells (IECs) are crucial for the digestive process and nutrient absorption. The intestinal epithelium is composed of the different cell types of the small intestine (mainly, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, and tuft cells). The small intestine is characterized by the presence of crypt-villus units that are in a state of homeostatic cell turnover. Organoid technology enables an efficient expansion of intestinal epithelial tissue in vitro. Thus, organoids hold great promise for use in medical research and in the development of new treatments. At present, the cholinergic system involved in IECs and intestinal stem cells (ISCs) are attracting a great deal of attention. Thus, understanding the biological processes triggered by epithelial cholinergic activation by acetylcholine (ACh), which is produced and released from neuronal and/or non-neuronal tissue, is of key importance. Cholinergic signaling via ACh receptors plays a pivotal role in IEC growth and differentiation. Here, we discuss current views on neuronal innervation and non-neuronal control of the small intestinal crypts and their impact on ISC proliferation, differentiation, and maintenance. Since technology using intestinal organoid culture systems is advancing, we also outline an organoid-based organ replacement approach for intestinal diseases.

Kazuto Fujishima

Institute for Integrated Cell-Material Sciences (KUIAS-iCeMS)

Kyoto University

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

Kazuto Fujishima

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute for Integrated Cell-Material Sciences (KUIAS-iCeMS)

Kyoto University

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

Institute for Integrated Cell-Material Sciences (KUIAS-iCeMS)