Exploring the principles of embryonic mammary gland branching morphogenesis.

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.

Opto-Thermal-Tension Mediated Precision Large-Scale Particle Manipulation and Flexible Patterning.

Large-scale particle manipulation with single-particle precision and further flexible patterning into functional structures is of huge potentials in many fields including bio-optoelectronic sensing, colloidal lithography, and wearable devices. However, it is very challenging for the precision manipulation and flexible patterning of particles on complicated curved and functional substrates. In this work, opto-thermal-tension (OTT) mediated precision large-scale particle manipulation and flexible patterning based on soap film are reported. Flexible manipulation and subsequent patterning of particles with single-particle resolution is realized by optothermal regulated surface tension on soap films. Reconfigurable patterning of particle structures with different shapes as well as large-scale ordered structures (up to 2000 particles) with particle sizes spanning two orders of magnitude (0.5-20 µm) is realized using this OTT mediation method. Importantly, due to the high flexibility of soap films, the patterned large-scale particle structures can be non-destructively transferred to curved and rough substrates, including rough iron pipe surface, leaf and skin surface. This OTT mediated method provides a new method for precision large-scale particle manipulation and flexible patterning with high versatility on complicated functional substrates, with great potentials for optoelectronic and biophotonic sensing and wearable device design on different curved and rough functional substrates.

Perturbation of periodic spot-generation balance leads to diversified pigmentation patterning of harlequin Phalaenopsis orchids: in silico prediction.

BACKGROUND: A retrotransposon HORT1 in the promoter of the anthocyanin activator gene PeMYB11, microRNA858 (miR858) that targets PeMYB11, and a repressor PeMYBx have been implicated in pigmentation patterning diversity of harlequin Phalaenopsis orchids. However, the interrelationship among them remains to be elucidated. RESULTS: To understand how these factors interact to generate anthocyanin spots in Phalaenopsis, we successfully developed a mathematical model based on the known reaction-diffusion system to simulate their interplay and refined the conceptual biological model. Intriguingly, the expression of both PeMYBx and PeMYB11 were in phase for purple spot formation, even though they showed adverse effects on anthocyanin accumulations. An increase in the self-activation rate of PeMYB11 resulted in the increased size of purple spots, but no effects on spot fusion. Decreased degradation rate of miR858 in the purple regions, led to disruption of the formation of spotted pigmentation patterning and a full-red pigmentation pattern. Significantly, the reduced miR858 level promotes the fusion of large dark purple dots induced by the solo-LTR of HORT1, eventually generating the purple patches. In addition, the spatially heterogeneous insertion of HORT1 caused by the remnant solo-LTR of HORT1 derived from random homologous unequal recombination of HORT1 in individual cells of floral organs could explain the diverse pigmentation patterning of harlequin Phalaenopsis. CONCLUSIONS: This devised model explains how HORT1 and miR858 regulate the formation of the pigmentation patterning and holds great promise for developing efficient and innovative approaches to breeding harlequin Phalaenopsis orchids.

Greater aperture counteracts effects of reduced stomatal density on WUE: a case study on sugarcane and meta-analysis.

Stomata regulate CO2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (gsw). However, the quantitative relationship between reduced SD, gsw, and the mechanisms underlying it is poorly understood. We addressed this knowledge gap using low-SD sugarcane (Saccharum spp. hybrid) as a case study alongside a meta-analysis of data from 10 species. Transgenic expression of EPIDERMAL PATTERNING FACTOR 2 from Sorghum bicolor (SbEFP2) in sugarcane reduced SD by 26-38% but did not affect gsw compared to wildtype. Further, no changes occurred in stomatal complex size or proxies for photosynthetic capacity. Measurements of gas exchange at low CO2 concentrations that promote complete stomatal opening to normalize aperture size between genotypes were combined with modeling of maximum gsw from anatomical data. These data suggest that increased stomatal aperture is the only possible explanation for maintaining gsw when SD is reduced. Meta-analysis across C3 dicots, C3 monocots, and C4 monocots revealed engineered reductions in SD are strongly correlated with lower gsw (r2=0.60-0.98), but this response is damped relative to the change in anatomy.

Coumarin-Based Photodegradable Hydrogels Enable Two-Photon Subtractive Biofabrication at 300†mm s-1.

Spatiotemporally controlled two-photon photodegradation of hydrogels has gained increasing attention for high-precision subtractive tissue engineering. However, conventional photolabile hydrogels often have poor efficiency upon two-photon excitation in the near-infrared (NIR) region and thus require high laser dosage that may compromise cell activity. As a result, high-speed two-photon hydrogel erosion in the presence of cells remains challenging. Here we introduce the design and synthesis of efficient coumarin-based photodegradable hydrogels to overcome these limitations. A set of photolabile coumarin-functionalized polyethylene glycol linkers are synthesized through a Passerini multicomponent reaction. After mixing these linkers with thiolated hyaluronic acid, semi-synthetic photodegradable hydrogels are formed in situ via Michael addition crosslinking. The efficiency of photodegradation in these hydrogels is significantly higher than that in nitrobenzyl counterparts upon two-photon irradiation at 780 nm. A complex microfluidic network mimicking the bone microarchitecture is successfully fabricated in preformed coumarin hydrogels at high speeds of up to 300 mm s-1 and low laser dosage down to 10 mW. Further, we demonstrate fast two-photon printing of hollow microchannels inside a hydrogel to spatiotemporally direct cell migration in 3D. Collectively, these hydrogels may open new avenues for fast laser-guided tissue fabrication at high spatial resolution.

Recapitulation and investigation of human brain development with neural organoids.

Organoids are 3D cultured tissues derived from stem cells that resemble the structure of living organs. Based on the accumulated knowledge of neural development, neural organoids that recapitulate neural tissue have been created by inducing self-organized neural differentiation of stem cells. Neural organoid techniques have been applied to human pluripotent stem cells to differentiate 3D human neural tissues in culture. Various methods have been developed to generate neural tissues of different regions. Currently, neural organoid technology has several significant limitations, which are being overcome in an attempt to create neural organoids that more faithfully recapitulate the living brain. The rapidly advancing neural organoid technology enables the use of living human neural tissue as research material and contributes to our understanding of the development, structure and function of the human nervous system, and is expected to be used to overcome neurological diseases and for regenerative medicine.

Deep learning for quantifying spatial patterning and formation process of early differentiated human-induced pluripotent stem cells with micropattern images.

Micropatterning is reliable method for quantifying pluripotency of human-induced pluripotent stem cells (hiPSCs) that differentiate to form a spatial pattern of sorted, ordered and nonoverlapped three germ layers on the micropattern. In this study, we propose a deep learning method to quantify spatial patterning of the germ layers in the early differentiation stage of hiPSCs using micropattern images. We propose decoding and encoding U-net structures learning labelled Hoechst (DNA-stained) hiPSC regions with corresponding Hoechst and bright-field micropattern images to segment hiPSCs on Hoechst or bright-field images. We also propose a U-net structure to extract extraembryonic regions on a micropattern, and an algorithm to compares intensities of the fluorescence images staining respective germ-layer cells and extract their regions. The proposed method thus can quantify the pluripotency of a hiPSC line with spatial patterning including cell numbers, areas and distributions of germ-layer and extraembryonic cells on a micropattern, and reveal the formation process of hiPSCs and germ layers in the early differentiation stage by segmenting live-cell bright-field images. In our assay, the cell-number accuracy achieved 86% and 85%, and the cell region accuracy 89% and 81% for segmenting Hoechst and bright-field micropattern images, respectively. Applications to micropattern images of multiple hiPSC lines, micropattern sizes, groups of markers, living and fixed cells show the proposed method can be expected to be a useful protocol and tool to quantify pluripotency of a new hiPSC line before providing it to the scientific community.

3D printing assisted surface patterning process on acrylated hydrogels for contact guidance of fibroblasts.

Generating stable and customizable topography on hydrogel surfaces with contact guidance potential is critical as it can direct/influence cell growth. This necessitates the development of new techniques for surface patterning of the hydrogels. We report on the design of a square grid template for surface patterning hydrogels. The template was 3-D printed and has the diameter of a well in a 24-well plate. Hyaluronic acid methacrylate (HA) hydrogel precursor solutions were cast on the 3D printed template's surface, which generated 3D square shape topographies on the HA hydrogel surface upon demolding. The 3D Laser Microscopy has shown the formation of a periodic array of 3D topographies on hydrogel surfaces. 3D Laser and Electron Microscopy Imaging have revealed that this new method has increased the surface area and exposed the underlying pore structure of the HA hydrogels. To demonstrate the method's versatility, we have successfully applied this technique to generate 3D topography on two more acrylate hydrogel formulations, gelatin Methacrylate and polyethylene glycol dimethacrylate. Human neonatal dermal fibroblast cells were used as a model cell line to evaluate the cell guidance potential of patterned HA hydrogel. Confocal fluorescence microscopy imaging has revealed that the 3D surface topographies on HA hydrogels can guide and align the actin filaments of the fibroblasts presumably due to the contact guidance mechanism. The newly developed methodology of 3D topography generation in acrylate hydrogels may influence the cell responses on hydrogel surfaces which can impact biomedical applications such as tissue engineering, wound healing, and disease modeling.

Amphicarpic development in Cardamine chenopodiifolia.

Amphicarpy is an unusual trait where two fruit types develop on the same plant: one above and the other belowground. This trait is not found in conventional model species. Therefore, its development and molecular genetics remain under-studied. Here, we establish the allooctoploid Cardamine chenopodiifolia as an emerging experimental system to study amphicarpy. We characterized C. chenopodiifolia development, focusing on differences in morphology and cell wall histochemistry between above- and belowground fruit. We generated a reference transcriptome with PacBio full-length transcript sequencing and analysed differential gene expression between above- and belowground fruit valves. Cardamine chenopodiifolia has two contrasting modes of seed dispersal. The main shoot fails to bolt and initiates floral primordia that grow underground where they self-pollinate and set seed. By contrast, axillary shoots bolt and develop exploding seed pods aboveground. Morphological differences between aerial explosive fruit and subterranean nonexplosive fruit were reflected in a large number of differentially regulated genes involved in photosynthesis, secondary cell wall formation and defence responses. Tools established in C. chenopodiifolia, such as a reference transcriptome, draft genome assembly and stable plant transformation, pave the way to study amphicarpy and trait evolution via allopolyploidy.

Comparing the Representation of a Simple Visual Stimulus across the Cerebellar Network.

The cerebellum is a conserved structure of the vertebrate brain involved in the timing and calibration of movements. Its function is supported by the convergence of fibers from granule cells (GCs) and inferior olive neurons (IONs) onto Purkinje cells (PCs). Theories of cerebellar function postulate that IONs convey error signals to PCs that, paired with the contextual information provided by GCs, can instruct motor learning. Here, we use the larval zebrafish to investigate (1) how sensory representations of the same stimulus vary across GCs and IONs and (2) how PC activity reflects these two different input streams. We use population calcium imaging to measure ION and GC responses to flashes of diverse luminance and duration. First, we observe that GCs show tonic and graded responses, as opposed to IONs, whose activity peaks mostly at luminance transitions, consistently with the notion that GCs and IONs encode context and error information, respectively. Second, we show that GC activity is patterned over time: some neurons exhibit sustained responses for the entire duration of the stimulus, while in others activity ramps up with slow time constants. This activity could provide a substrate for time representation in the cerebellum. Together, our observations give support to the notion of an error signal coming from IONs and provide the first experimental evidence for a temporal patterning of GC activity over many seconds.

Laser-Patterning of Micromagnets for Immuno-Magnetophoretic Exosome Capture.

Efficient isolation and patterning of biomolecules is a vital step within sample preparation for biomolecular analysis, with numerous diagnostic and therapeutic applications. For exosomes, nanoscale lipid-bound biomolecules, efficient isolation is challenging due to their minute size and resultant behavior within biofluids. This study presents a method for the rapid isolation and patterning of magnetically tagged exosomes via rationally designed micromagnets. Micromagnet fabrication utilizes a novel, scalable, and high-throughput laser-based fabrication approach that enables patterning at microscale lateral resolution (<50 µm) without lithographic processing and is agnostic to micromagnet geometry. Laser-based processing allows for flexible and tunable device configurations, and herein magnetophoretic capture within both an open-air microwell and an enclosed microfluidic system is demonstrated. Patterned micromagnets enhance localized gradient fields throughout the fluid medium, resulting in rapid and high efficiency magnetophoretic separation, with capture efficiencies nearing 70% after just 1s within open-air microwells, and throughputs upward of 3 mL h-1 within enclosed microfluidic systems. Using this microchip architecture, immunomagnetic exosome isolation and patterning directly from undiluted plasma samples is further achieved. Lastly, a FEA-based modeling workflow is introduced to characterize and optimize micromagnet unit cells, simulating magnetophoretic capture zones for a given micromagnet geometry.

Highly parallel production of designer organoids by mosaic patterning of progenitors.

Organoids derived from human stem cells are a promising approach for disease modeling, regenerative medicine, and fundamental research. However, organoid variability and limited control over morphological outcomes remain as challenges. One open question is the extent to which engineering control over culture conditions can guide organoids to specific compositions. Here, we extend a DNA "velcro" cell patterning approach, precisely controlling the number and ratio of human induced pluripotent stem cell-derived progenitors contributing to nephron progenitor (NP) organoids and mosaic NP/ureteric bud (UB) tip cell organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints. We then show emergent trends in organoid tissue proportions that depend on initial progenitor cell composition. These include higher nephron and stromal cell representation in mosaic NP/UB organoids vs. NP-only organoids and a "goldilocks" initial cell ratio in mosaic organoids that optimizes the formation of proximal tubule structures.

Development of Light-Scribing Process Using L-Ascorbic Acid for Graphene Micro-Supercapacitor.

The rapid development of smart technologies is accelerating the growing demand for microscale energy storage devices. This work reports a facile and practical approach to fabricating interdigitated graphene micro-patterns through the LSC process accompanied by the l-ascorbic acid (L-AA) and preheating treatment. Our work offered a higher degree of GO reduction than the conventional microfabrication. It significantly shortened the overall processing time to obtain the micro-patterns with improved electrical and electrochemical performances. The interdigitated MSC composed of 16 electrodes exhibited a high capacitance of 14.1 F/cm3, energy density of 1.78 mWh/cm3, and power density of 69.9 mW/cm3. Furthermore, the fabricated MSC device demonstrated excellent cycling stability of 88.2% after 10,000 GCD cycles and a high rate capability of 81.1% at a current density of 1.00 A/cm3. The fabrication process provides an effective means for producing high-performance MSCs for miniaturized electronic devices.

Putting heads together: Developmental genetics of the Asteraceae capitulum.

Inflorescence architecture is highly variable across plant lineages yet is critical for facilitating reproductive success. The capitulum-type inflorescence of the Asteraceae is marked as a key morphological innovation that preceded the family's diversification and expansion. Despite its evolutionary significance, our understanding of capitulum development and evolution is limited. This review highlights our current perspective on capitulum evolution through the lens of both its molecular and developmental underpinnings. We attempt to summarize our understanding of the capitulum by focusing on two key characteristics: patterning (arrangement of florets on a capitulum) and floret identity specification. Note that these two features are interconnected such that the identity of florets depends on their position along the inflorescence axis. Phytohormones such as auxin seemingly determine both pattern progression and floret identity specification through unknown mechanisms. Floret morphology in a head is controlled by differential expression of floral symmetry genes regulating floret identity specification. We briefly summarize the applicability of the ABCE quartet model of flower development in regulating the floret organ identity of a capitulum in Asteraceae. Overall, there have been promising advancements in our understanding of capitula; however, comprehensive functional genetic analyses are necessary to fully dissect the molecular pathways and mechanisms involved in capitulum development.

Precision Chemistry of Metallofullerenes and Graphene: Recent Advances.

Precision chemistry of synthetic carbon allotropes including fullerene and graphene, characterized by a well-controlled and spatially resolved addends bonding, has received widespread attention owing to its capability to tailor their physicochemical properties for high-end applications. In the context of fullerene, particularly endohedral metallofullerenes (EMFs), precision chemistry emphasizes the regioselective binding of a specific number of moieties to the fullerene cage. In the case of graphene, precision chemistry focuses on achieving precise patterning and tailored modifications. Inspired by their intriguing advantages, the precision chemistry of these two members has witnessed rapid advancements. While existing reviews have outlined advancements in the precision chemistry of EMFs and graphene, this review uniquely concentrates on the most recent progress. Finally, the prospects in this field, with a special focus on the potential for creating functional materials through strategically patterned binding of fullerene and graphene networks are envisioned.

Sall4 regulates downstream patterning genes during limb regeneration.

Many salamanders can completely regenerate a fully functional limb. Limb regeneration is a carefully coordinated process involving several defined stages. One key event during the regeneration process is the patterning of the blastema to inform cells of what they must differentiate into. Although it is known that many genes involved in the initial development of the limb are re-used during regeneration, the exact molecular circuitry involved in this process is not fully understood. Several large-scale transcriptional profiling studies of axolotl limb regeneration have identified many transcription factors that are up-regulated after limb amputation. Sall4 is a transcription factor that has been identified to play essential roles in maintaining cells in an undifferentiated state during development and also plays a unique role in limb development. Inactivation of Sall4 during limb bud development results in defects in anterior-posterior patterning of the limb. Sall4 has been found to be up-regulated during limb regeneration in both Xenopus and salamanders, but to date it function has been untested. We confirmed that Sall4 is up-regulated during limb regeneration in the axolotl using qRT-PCR and identified that it is present in the skin cells and also in cells within the blastema. Using CRISPR technology we microinjected gRNAs specific for Sall4 complexed with cas9 protein into the blastema to specifically knockout Sall4 in blastema cells only. This resulted in limb regenerate defects, including missing digits, fusion of digit elements, and defects in the radius and ulna. This suggests that during regeneration Sall4 may play a similar role in regulating the specification of anterior-proximal skeletal elements.

Cell wall-mediated maternal control of apical-basal patterning of the kelp Undaria pinnatifida.

The role of maternal tissue in embryogenesis remains enigmatic in many complex organisms. Here, we investigate the contribution of maternal tissue to apical-basal patterning in the kelp embryo. Focussing on Undaria pinnatifida, we studied the effects of detachment from the maternal tissue using microsurgery, staining of cell wall modifications, morphometric measurements, flow cytometry, genotyping and a modified kelp fertilisation protocol synchronising kelp embryogenesis. Detached embryos are rounder and often show aberrant morphologies. When a part of the oogonial cell wall remains attached to the zygote, the apical-basal patterning is rescued. Furthermore, the absence of contact with maternal tissue increases parthenogenesis, highlighting the critical role of maternal signals in the initial stages of development. These results show a key role for the connection to the maternal oogonial cell wall in apical-basal patterning in kelps. This observation is reminiscent of another brown alga, Fucus, where the cell wall directs the cell fate. Our findings suggest a conserved mechanism across phylogenetically distant oogamous lineages, where localised secretion of sulphated F2 fucans mediates the establishment of the apical-basal polarity. In this model, the maternal oogonial cell wall mediates basal cell fate determination by providing an extrinsic patterning cue to the future kelp embryo.

Growth patterns of caudal fin rays are informed by both external signals from the regenerating organ and remembered identity autonomous to the local tissue.

Regenerating tissues must remember or interpret their spatial position, using this information to restore original size and patterning. The external skeleton of the zebrafish caudal fin is composed of 18 rays; after any portion of the fin is amputated, position-dependent regenerative growth restores each ray to its original length. We tested for transcriptional differences during regeneration of proximal versus distal tissues and identified 489 genes that differed in proximodistal expression. Thyroid hormone directs multiple aspects of ray patterning along the proximodistal axis, and we identified 364 transcripts showing a proximodistal expression pattern that was dependent on thyroid hormone context. To test what aspects of ray positional identity are directed by extrinsic environental cues versus remembered identity autonomous to the tissue, we transplanted distal portions of rays to proximal environments and evaluated regeneration within the new location. Native regenerating proximal tissue showed robust expression of scpp7, a transcript with thyroid-regulated proximal enrichment; in contrast, regenerating rays originating from transplanted distal tissue showed reduced (distal-like) expression during outgrowth. These distal-to-proximal transplants regenerated far beyond the length of the graft itself, indicating that cues from the proximal environment promoted additional growth. Nonetheless, these transplants initiated regeneration at a much slower rate compared to controls, suggesting memory of distal identity was retained by the transplanted tissue. This early growth retardation caused rays that originated from transplants to grow noticeably shorter than neighboring native rays. While several aspects of fin ray morphology (bifurcation, segment length) were found to be determined by the environment, we found that both regeneration speed and ray length are remembered autonomously by tissues, and that persist through multiple rounds of amputation and regeneration.

Multiplexed in situ hybridization reveals distinct lineage identities for major and minor vein initiation during maize leaf development.

Leaves of flowering plants are characterized by diverse venation patterns. Patterning begins with the selection of vein-forming procambial initial cells from within the ground meristem of a developing leaf, a process which is considered to be auxin-dependent, and continues until veins are anatomically differentiated with functional xylem and phloem. At present, the mechanisms responsible for leaf venation patterning are primarily characterized in the model eudicot Arabidopsis thaliana which displays a reticulate venation network. However, evidence suggests that vein development may proceed via a different mechanism in monocot leaves where venation patterning is parallel. Here, we employed Molecular Cartography, a multiplexed in situ hybridization technique, to analyze the spatiotemporal localization of a subset of auxin-related genes and candidate regulators of vein patterning in maize leaves. We show how different combinations of auxin influx and efflux transporters are recruited during leaf and vein specification and how major and minor vein ranks develop with distinct identities. The localization of the procambial marker PIN1a and the spatial arrangement of procambial initial cells that give rise to major and minor vein ranks further suggests that vein spacing is prepatterned across the medio-lateral leaf axis prior to accumulation of the PIN1a auxin transporter. In contrast, patterning in the adaxial-abaxial axis occurs progressively, with markers of xylem and phloem gradually becoming polarized as differentiation proceeds. Collectively, our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves.

Long-range polymer ordering by directional coating to remarkably enhance the charge carrier mobility in PCDTPT-based organic field-effect transistors.

With the wide potential of organic field-effect transistors in all the modern electronic circuitries, researchers are grappling with the challenge of poor charge transport and hence lower mobility in organic polymers. Low-charge carrier mobility is mainly due to disorder in the molecular packing of organic semiconductors along with other factors, such as impurities, defects and interactions between molecules. The current research work has been conducted to align the molecular chains of poly[4-(4,4-dihexadecyl-4H-cyclopenta[1,2-|||b:5,4-|b']|dithiophen-2-yl)-alt-[1,2,5]thiadiazolo-[3,4-c]pyridine] (PCDTPT) using directional coating techniques such as dip coating and brush coating on nano-grooved substrates. Long-range order of polymer chains was clearly observed along the direction of brush coating and nanogrooves in optical and atomic force microscope (AFM) images while transmission spectra confirmed decreased pi-pi stacking for the polymer films deposited by this technique. By comparing the mobility performance of brush-coated devices with other techniques, we found a remarkable mobility enhancement of 90 times that of conventional spin-coated device and 24 times enhancement compared with the dip-coated device for the case when the alignment of polymer chains was parallel to the channel. All the fabrication and characterizations were performed in the ambient environment. This study demonstrates a potential approach to align the polymers on long and short ranges hence providing a route for high-performing devices in ambient conditions.