Tissue Morphogenesis Through Dynamic Cell and Matrix Interactions.

Multicellular organisms generate tissues of diverse shapes and functions from cells and extracellular matrices. Their adhesion molecules mediate cell-cell and cell-matrix interactions, which not only play crucial roles in maintaining tissue integrity but also serve as key regulators of tissue morphogenesis. Cells constantly probe their environment to make decisions: They integrate chemical and mechanical information from the environment via diffusible ligand- or adhesion-based signaling to decide whether to release specific signaling molecules or enzymes, to divide or differentiate, to move away or stay, or even whether to live or die. These decisions in turn modify their environment, including the chemical nature and mechanical properties of the extracellular matrix. Tissue morphology is the physical manifestation of the remodeling of cells and matrices by their historical biochemical and biophysical landscapes. We review our understanding of matrix and adhesion molecules in tissue morphogenesis, with an emphasis on key physical interactions that drive morphogenesis.

Budding epithelial morphogenesis driven by cell-matrix versus cell-cell adhesion.

Many embryonic organs undergo epithelial morphogenesis to form tree-like hierarchical structures. However, it remains unclear what drives the budding and branching of stratified epithelia, such as in the embryonic salivary gland and pancreas. Here, we performed live-organ imaging of mouse embryonic salivary glands at single-cell resolution to reveal that budding morphogenesis is driven by expansion and folding of a distinct epithelial surface cell sheet characterized by strong cell-matrix adhesions and weak cell-cell adhesions. Profiling of single-cell transcriptomes of this epithelium revealed spatial patterns of transcription underlying these cell adhesion differences. We then synthetically reconstituted budding morphogenesis by experimentally suppressing E-cadherin expression and inducing basement membrane formation in 3D spheroid cultures of engineered cells, which required ß1-integrin-mediated cell-matrix adhesion for successful budding. Thus, stratified epithelial budding, the key first step of branching morphogenesis, is driven by an overall combination of strong cell-matrix adhesion and weak cell-cell adhesion by peripheral epithelial cells.

A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions.

Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here, we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by the density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to the emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition.

The kinase ZYG-1 phosphorylates the cartwheel protein SAS-5 to drive centriole assembly in C. elegans.

Centrioles organize centrosomes, the cell's primary microtubule-organizing centers (MTOCs). Centrioles double in number each cell cycle, and mis-regulation of this process is linked to diseases such as cancer and microcephaly. In C. elegans, centriole assembly is controlled by the Plk4 related-kinase ZYG-1, which recruits the SAS-5-SAS-6 complex. While the kinase activity of ZYG-1 is required for centriole assembly, how it functions has not been established. Here we report that ZYG-1 physically interacts with and phosphorylates SAS-5 on 17 conserved serine and threonine residues in vitro. Mutational scanning reveals that serine 10 and serines 331/338/340 are indispensable for proper centriole assembly. Embryos expressing SAS-5S10A exhibit centriole assembly failure, while those expressing SAS-5S331/338/340A possess extra centrioles. We show that in the absence of serine 10 phosphorylation, the SAS-5-SAS-6 complex is recruited to centrioles, but is not stably incorporated, possibly due to a failure to coordinately recruit the microtubule-binding protein SAS-4. Our work defines the critical role of phosphorylation during centriole assembly and reveals that ZYG-1 might play a role in preventing the formation of excess centrioles.

A high-resolution C. elegans essential gene network based on phenotypic profiling of a complex tissue.

High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach-profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes-pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.

A high-content imaging approach to profile C. elegans embryonic development.

The Caenorhabditis elegans embryo is an important model for analyzing mechanisms of cell fate specification and tissue morphogenesis. Sophisticated lineage-tracing approaches for analyzing embryogenesis have been developed but are labor intensive and do not naturally integrate morphogenetic readouts. To enable the rapid classification of developmental phenotypes, we developed a high-content method that employs two custom strains: a Germ Layer strain that expresses nuclear markers in the ectoderm, mesoderm and endoderm/pharynx; and a Morphogenesis strain that expresses markers labeling epidermal cell junctions and the neuronal cell surface. We describe a procedure that allows simultaneous live imaging of development in 80-100 embryos and provide a custom program that generates cropped, oriented image stacks of individual embryos to facilitate analysis. We demonstrate the utility of our method by perturbing 40 previously characterized developmental genes in variants of the two strains containing RNAi-sensitizing mutations. The resulting datasets yielded distinct, reproducible signature phenotypes for a broad spectrum of genes that are involved in cell fate specification and morphogenesis. In addition, our analysis provides new in vivo evidence for MBK-2 function in mesoderm fate specification and LET-381 function in elongation.

Dorsal approach with Glissonian approach for laparoscopic right anatomic liver resections.

BACKGROUND: Laparoscopic anatomic hepatectomy (LAH) has gradually become a routine surgical procedure. However, how to expose the whole hepatic vein and avoid the hepatic vein laceration is still a challenge because of the caudate lobe, particularly in right hepatectomy. We adopted a dorsal approach combined with Glissionian appraoch to perform laparoscopic right anatomic hepatectomy (LRAH). METHODS: Twenty patients who underwent LRAH from January 2017 to November 2018 were retrospectively analysed. Of these patients, seven patients underwent laparoscopic right hemihepatectomy (LRH group), seven patients who underwent laparoscopic right posterior hepatectomy (LRPH group), and six patients who underwent laparoscopic hepatectomy for segment 7 (LS7 group). The paracaval portion of caudate lobe could be transected firstly through dorsal approach and the corresponding major hepatic vein could be exposed from its root to the peripheral branches safely. Due to exposure along the major hepatic vein trunk, the remaining liver parenchyma could be quickly transected from dorsal to cranial side. RESULTS: The mean age of the patients was 53.8 years and the male: female ratio was 8:12. The median operation time was 306.0 ± 58.2 min and the mean estimated volume of blood loss was 412.5 ± 255.4 mL. The mean duration of postoperative hospital stay was 10.2 days. The mean Pringle maneuver time was 64.8 ± 27.7 min. Five patients received transfusion of 2-4 U of red blood cells. Two patients suffered from transient hepatic dysfunction and one suffered from pleural effusion. None of the patients underwent conversion to an open procedure. The operative duration, volume of the blood loss, Pringle maneuver time, and postoperative hospital stay duration did not differ significantly among the LRH, LRPH, and LS7 groups (P > 0.05). CONCLUSIONS: Dorsal approach combined with Glissonian approach for right lobe is feasible and effective in laparoscopic right anatomic liver resections.

Btbd7 is essential for region-specific epithelial cell dynamics and branching morphogenesis in vivo.

Branching morphogenesis of developing organs requires coordinated but poorly understood changes in epithelial cell-cell adhesion and cell motility. We report that Btbd7 is a crucial regulator of branching morphogenesis in vivo. Btbd7 levels are elevated in peripheral cells of branching epithelial end buds, where it enhances cell motility and cell-cell adhesion dynamics. Genetic ablation of Btbd7 in mice disrupts branching morphogenesis of salivary gland, lung and kidney. Btbd7 knockout results in more tightly packed outer bud cells, which display stronger E-cadherin localization, reduced cell motility and decreased dynamics of transient cell separations associated with cleft formation; inner bud cells remain unaffected. Mechanistic analyses using in vitro MDCK cells to mimic outer bud cell behavior establish that Btbd7 promotes loss of E-cadherin from cell-cell adhesions with enhanced migration and transient cell separation. Btbd7 can enhance E-cadherin ubiquitination, internalization, and degradation in MDCK and peripheral bud cells for regulating cell dynamics. These studies show how a specific regulatory molecule, Btbd7, can function at a local region of developing organs to regulate dynamics of cell adhesion and motility during epithelial branching morphogenesis.

A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans.

Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues - the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons - where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.

Extracellular matrix dynamics in cell migration, invasion and tissue morphogenesis.

This review describes how direct visualization of the dynamic interactions of cells with different extracellular matrix microenvironments can provide novel insights into complex biological processes. Recent studies have moved characterization of cell migration and invasion from classical 2D culture systems into 1D and 3D model systems, revealing multiple differences in mechanisms of cell adhesion, migration and signalling-even though cells in 3D can still display prominent focal adhesions. Myosin II restrains cell migration speed in 2D culture but is often essential for effective 3D migration. 3D cell migration modes can switch between lamellipodial, lobopodial and/or amoeboid depending on the local matrix environment. For example, "nuclear piston" migration can be switched off by local proteolysis, and proteolytic invadopodia can be induced by a high density of fibrillar matrix. Particularly, complex remodelling of both extracellular matrix and tissues occurs during morphogenesis. Extracellular matrix supports self-assembly of embryonic tissues, but it must also be locally actively remodelled. For example, surprisingly focal remodelling of the basement membrane occurs during branching morphogenesis-numerous tiny perforations generated by proteolysis and actomyosin contractility produce a microscopically porous, flexible basement membrane meshwork for tissue expansion. Cells extend highly active blebs or protrusions towards the surrounding mesenchyme through these perforations. Concurrently, the entire basement membrane undergoes translocation in a direction opposite to bud expansion. Underlying this slowly moving 2D basement membrane translocation are highly dynamic individual cell movements. We conclude this review by describing a variety of exciting research opportunities for discovering novel insights into cell-matrix interactions.

Laennec's approach for laparoscopic anatomic hepatectomy based on Laennec's capsule.

BACKGROUND: Although isolating Glissonean pedicles and hepatic veins are critical procedures during anatomical hepatectomy, there is no standardized approach. We propose the novel Laennec's approach for laparoscopic anatomic hepatectomy (LAH) based on Laennec's capsule, which serves as the anatomic landmark for LAH. The aim of this study was to elucidate that the natural gap between Laennec's capsule and the adjacent tissues contributes to standardization of the surgical technique for LAH. METHODS: Eighty-four cases were enrolled in this observable clinical trial. They underwent LAH for liver diseases. Laennec's approach was proposed for LAH based on Laennec's capsule. The liver tissues close to Glissonean pedicle, hepatic veins, naked area, and inferior vena cava were collected for hematoxylin and eosin, resorcinol-fuchsin staining, and immunohistochemistry. RESULTS: The staining revealed capsule packaging of the whole liver independent of the adjacent tissues and intrahepatic vessels. A natural gap was found between Laennec's capsule and the adjacent tissues at different sites. Laennec's capsule served as the landmark for isolating Glissonean pedicle and hepatic veins, mobilizing the liver, and performing Hanging maneuver. Eighty-four cases underwent LAH for liver diseases using this strategy. The operation time was 277.23 min. The mean of hospital days was 9.8. CONCLUSIONS: Laennec's approach based on Laennec's capsule contributes to standardization of the surgical technique for LAH, and brings innovations that facilitates safe and effective liver resection under laparoscopy.

Salivary gland developmental mechanics.

The salivary gland undergoes branching morphogenesis to elaborate into a tree-like structure with numerous saliva-secreting acinar units, all joined by a hierarchical ductal system. The expansive epithelial surface generated by branching morphogenesis serves as the structural basis for the efficient production and delivery of saliva. Here, we elucidate the process of salivary gland morphogenesis, emphasizing the role of mechanics. Structurally, the developing salivary gland is characterized by a stratified epithelium tightly encased by the basement membrane, which is in turn surrounded by a mesenchyme consisting of a dense network of interstitial matrix and mesenchymal cells. Diverse cell types and extracellular matrices bestow this developing organ with organized, yet spatially varied mechanical properties. For instance, the surface epithelial sheet of the bud is highly fluidic due to its high cell motility and weak cell-cell adhesion, rendering it highly pliable. In contrast, the inner core of the bud is more rigid, characterized by reduced cell motility and strong cell-cell adhesion, which likely provide structural support for the tissue. The interactions between the surface epithelial sheet and the inner core give rise to budding morphogenesis. Furthermore, the basement membrane and the mesenchyme offer mechanical constraints that could play a pivotal role in determining the higher-order architecture of a fully mature salivary gland.

Differential effects of AKT1 and AKT2 on sleep-wake activity under basal conditions and in response to LPS challenge in mice.

Infectious challenge can trigger alterations in sleep-wake behavior. Accumulating evidence has shown that the serine/threonine kinases Akt1 and Akt2 are important targets in both physiological and infectious signaling processes. However, the involvement of Akt1 and Akt2 in sleep-wake activity under basal conditions and in response to inflammatory stimulation has not been established. In the present study, we assessed the precise role of Akt1 and Akt2 in sleep-wake behavior using electroencephalography (EEG)/electromyography (EMG) data from Akt1- and Akt2-deficient mice and wild-type (WT) mice. The results showed that both Akt1 and Akt2 deficiency affect sleep-wake activity, as indicated by reduced nonrapid eye movement (NREM) sleep and increased wakefulness in mutant mice compared to WT mice. Sleep amount and intensity (delta, theta and alpha activity) at night were also drastically attenuated in Akt1- and Akt2-deficient mice. Moreover, since Akt1 and Akt2 are involved in immune responses, we assessed their roles in the sleep response to the inflammatory stimulus lipopolysaccharide (LPS) throughout the following 24 h. We observed that the decrease in wakefulness and increase in NREM sleep induced by LPS were restored in Akt1 knockout mice but not in Akt2 knockout mice. Correspondingly, the decrease in the number of positive orexin-A neurons induced by LPS was abrogated in Akt1 knockout mice but not in Akt2 knockout mice. Our results revealed that both Akt1 and Akt2 deficiency affect the sleep response under basal conditions, but only Akt1 deficiency protects against the aberrant changes in sleep behavior induced by peripheral immune challenge. Supplementary Information: The online version contains supplementary material available at 10.1007/s41105-024-00519-y.

Microtubule-dependent apical polarization of basement membrane matrix mRNAs in mouse epithelial cells.

The basement membrane (BM) demarcating epithelial tissues undergoes rapid expansion to accommodate tissue growth and morphogenesis during embryonic development. To facilitate the secretion of bulky BM proteins, their mRNAs are polarized basally in the follicle epithelial cells of the Drosophila egg chamber to position their sites of production close to their deposition. In contrast, we observed the apical rather than basal polarization of all major BM mRNAs in the outer epithelial cells adjacent to the BM of mouse embryonic salivary glands using single-molecule RNA fluorescence in situ hybridization (smFISH). Moreover, electron microscopy and immunofluorescence revealed apical polarization of both the endoplasmic reticulum (ER) and Golgi apparatus, indicating that the site of BM component production was opposite to the site of deposition. At the apical side, BM mRNAs colocalized with ER, suggesting they may be co-translationally tethered. After microtubule inhibition, the BM mRNAs and ER became uniformly distributed rather than apically polarized, but they remained unchanged after inhibiting myosin II, ROCK, or F-actin, or after enzymatic disruption of the BM. Because Rab6 is generally required for Golgi-to-plasma membrane trafficking of BM components, we used lentivirus to express an mScarlet-tagged Rab6a in salivary gland epithelial cultures to visualize vesicle trafficking dynamics. We observed extensive bidirectional vesicle movements between Golgi at the apical side and the basal plasma membrane adjacent to the BM. Moreover, we showed that these vesicle movements depend on the microtubule motor kinesin-1 because very few vesicles remained motile after treatment with kinesore to compete for cargo-binding sites on kinesin-1. Overall, our work highlights the diverse strategies that different organisms use to secrete bulky matrix proteins: while Drosophila follicle epithelial cells strategically place their sites of BM protein production close to their deposition, mouse embryonic epithelial cells place their sites of production at the opposite end. Instead of spatial proximity, they use the microtubule cytoskeleton to mediate this organization as well as for the apical-to-basal transport of BM proteins.

Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore.

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

CRISPR/Cas9-Mediated Gene Knockout in Cells and Tissues Using Lentivirus.

CRISPR-Cas9 has become a powerful and popular gene editing tool. However, successful application of this tool in the lab can still be quite daunting to many newcomers to molecular biology, mostly because it is a relatively lengthy process involving multiple steps with variations of each step. Here, we provide a reliable, stepwise, and newcomer-friendly protocol to knock out a target gene in wild-type human fibroblasts. This protocol involves sgRNA design using CRISPOR, construction of an "all-in-one" vector expressing both sgRNA and Cas9 using Golden Gate cloning, streamlined production of high-titer lentiviruses in 1 week after molecular cloning, and transduction of cells to generate a knockout cell pool. We further introduce a protocol for lentiviral transduction of ex vivo mouse embryonic salivary epithelial explants. In summary, our protocol is useful for new researchers to apply CRISPR-Cas9 to generate stable gene knockout cells and tissue explants using lentivirus. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning sgRNA in plasmid vector containing Cas9 encoding sequence using golden gate cloning Basic Protocol 3: Lentivirus packaging Basic Protocol 4: Lentivirus transduction of cells Basic Protocol 5: Lentivirus transduction of salivary gland epithelial buds.

Distally-extending muscle fibers across involved joints: study of long muscles and tendons of wrist and ankle in late-term fetuses and adult cadavers.

It is unclear whether forearm and crural muscle fibers extend distally across the wrist and ankle joints, respectively. We hypothesized, in late-term fetuses, an over-production of muscle bellies extending over the joint. Muscle fibers in histological sections from unilateral wrists and ankles of 16 late-term fetuses (30-40 weeks) were examined and compared with 15 adult cadavers. Muscle fibers of the flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS) in fetuses, especially muscle bellies to the third and fourth fingers, were found to extend far distally beyond the radiocarpal joint. The extensor digitorum and extensor pollicis longus on the extensor side of the wrist were found to carry distally-extending muscle fibers, but these fibers did not extend beyond the distal end of the radius. In the ankle, most muscle bundles in the flexor hallucis longus (FHL), fibularis brevis (FB) and extensor digitorum longus extended distally beyond the talocrural joint, with most FB muscle fibers reaching the level of the talocalcaneal joint. In adult cadavers, muscle fibers of the FDP and FHL did not reach the levels of the radiocarpal and talocrural joints, respectively, whereas the FB muscle belly always reached the talocalcaneal joint. Similarly, some of the FDS reached the level of the radiocarpal joint. Generally, infants' movements at the wrist and ankle could result in friction injury to over-extended muscle. However, the calcaneal and FDP tendons might protect the FB and FDS tendons, respectively, from friction stress.

Mta3-NuRD complex is a master regulator for initiation of primitive hematopoiesis in vertebrate embryos.

Metastasis-associated antigens 1/2/3 (Mta1/2/3) are components of nucleosome remodeling and deacetylase (NuRD) complexes and have been found to play roles in embryonic development and homeostasis. However, their functions in primitive hematopoiesis are unknown. In this study, we demonstrate that knockdown of mta3 by antisense morpholinos abolishes primitive hematopoietic lineages and causes abnormal angiogenesis in zebrafish embryos. However, the expression of the pronephric duct and paraxial mesoderm markers is unaltered and the specification of angioblasts is unaffected in mta3 morphants. The results suggest that mta3 is specifically required for primitive hematopoiesis. Furthermore, inhibition of deacetylase activity with the inhibitors valproic acid (VPA) or trichostatin A (TSA) in zebrafish embryos completely blocks primitive hematopoiesis, resulting in hematopoietic defects almost identical to those seen in mta3 morphants. Importantly, overexpression of scl or scl and lmo2, 2 master genes for primitive hematopoiesis, is able to overturn effects of mta3 knockdown or VPA/TSA treatment; and overexpression of mta3, and human MBD3 or HDAC1, 2 other components of NuRD complex, enhances the expression of scl and lmo2 in the posterior lateral plate mesoderm during early primitive hematopoiesis. We conclude that Mta3-NuRD complex is essential for the initiation of primitive hematopoiesis. Thus, our findings provide new insight into the regulatory hierarchy of primitive hematopoiesis in vertebrates.