Whole mount nuclear fluorescent imaging: Convenient documentation of embryo morphology

Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. genesis 2012. © 2012 Wiley Periodicals, Inc.     

Extracellular matrix organized in embryonic cavities during induction of the embryonic axis in chick embryo

Extracellular matrix (ECM) is detected as short, disorganized fibrils in the forming embryonic extracellular spaces shortly prior to the first morphogenetic cellular movements and interactions in the early chick embryo. As development progresses, the ECM is organized into an intricate network spanning the embryonic cavities. This dynamic entity undergoes relatively rapid changes in its organization pattern during the developmental period from morula to the induction of the neural plate. The ECM seems to preserve the exquisite architecture of the embryo and could guide migrating cells into defined pathways in the early embryo.     

Wnt/β‐catenin signaling during early vertebrate neural development

The vertebrate central nervous system (CNS) is comprised of vast number of distinct cell types arranged in a highly organized manner. This high degree of complexity is achieved by cellular communication, including direct cell‐cell contact, cell‐matrix interactions, and cell‐growth factor signaling. Among the several developmental signals controlling the development of the CNS, Wnt proteins have emerged as particularly critical and, hence, have captivated the attention of many researchers. With Wnts' evolutionarily conserved function as primordial symmetry breaking signals, these proteins and their downstream effects are responsible for simultaneously establishing cellular diversity and tissue organization. With their expansive repertoire of secreted agonists and antagonists, cell surface receptors, signaling cascades and downstream biological effects, Wnts are ideally suited to control the complex processes underlying vertebrate neural development. In this review, we will describe the mechanisms by which Wnts exert their potent effects on cells and tissues and highlight the many roles of Wnt signaling during neural development, starting from the initial induction of the neural plate, the subsequent patterning along the embryonic axes, to the intricately organized structure of the CNS. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1239–1259, 2017     

Hemicentins assemble on diverse epithelia in the mouse.

Hemicentins are recently described extracellular matrix (ECM) proteins with a single ortholog in C. elegans that assembles into discrete tracks constricting broad regions of epithelial cell contact into adhesive and flexible line-shaped junctions. There are two highly conserved hemicentin genes in most vertebrate species; however, nothing is known about the function or distribution of vertebrate hemicentins. To determine the distribution of vertebrate hemicentins, we used a polyclonal antibody to stain mouse tissue and showed that hemicentins are found in the pericellular ECM of epithelial cells in a number of tissues including embryonic trophectoderm and adult skin and tongue, in addition to the ECM of some, but not all, blood vessels. Hemicentins also assemble on multiple epithelia in the eye, including cornea, lens, and retina. The pericellular localization of vertebrate hemicentins on epithelia and other cell surfaces suggests that vertebrate hemicentins, like their nematode counterpart, are secreted ECM proteins likely to have a role in the architecture of adhesive and flexible cell junctions, particularly in tissues subject to significant amounts of mechanical stress.     

Neural crest cell migration in relation to extracellular matrix organization in the embryonic axolotl trunk

Temporal and regional aspects of early neural crest cell migration in relation to extracellular matrix (ECM) organization and distribution in the embryonic axolotl trunk were studied by light microscopy, TEM, and SEM. The dominating structure of the interstitial ECM is a complex network of fibrils, which are indicated by ruthenium red staining to consist of collagen in association with ruthenium red-positive components, probably including glycosaminoglycans. The ECM fibrils, which are largely used as substratum for locomotion by the crest cells, have a temporally and regionally specific organization and distribution. Increase in ECM fibrils on the neural tube, ahead of the crest cell front, is correlated with initiation of crest cell emigration, and it is suggested that the fibrils may stimulate this process by providing a suitable substratum for cell locomotion. An increase in ECM fibrils in extracellular spaces surrounding the crest cell population is correlated with an expansion of these spaces and with progressing crest cell migration into them. It is proposed that the spatial organization of the ECM fibrils influences crest cell shape and orientation during early migration.     

Structural and mechanical properties of the proliferative zone of the developing murine growth plate cartilage assessed by atomic force microscopy

The growth plate (GP) is a dynamic tissue driving bone elongation through chondrocyte proliferation, hypertrophy and matrix production. The extracellular matrix (ECM) is the major determinant of GP biomechanical properties and assumed to play a pivotal role for chondrocyte geometry and arrangement, thereby guiding proper growth plate morphogenesis and bone elongation. To elucidate the relationship between morphology and biomechanics during cartilage morphogenesis, we have investigated age-dependent structural and elastic properties of the proliferative zone of the murine GP by atomic force microscopy (AFM) from the embryonic stage to adulthood. We observed a progressive cell flattening and arrangement into columns from embryonic day 13.5 until postnatal week 2, correlating with an increasing collagen density and ECM stiffness, followed by a nearly constant cell shape, collagen density and ECM stiffness from week 2 to 4 months. At all ages, we found marked differences in the density and organization of the collagen network between the intracolumnar matrix, and the intercolumnar matrix, associated with a roughly two-fold higher stiffness of the intracolumnar matrix compared to the intercolumnar matrix. This difference in local ECM stiffness may force the cells to arrange in a columnar structure upon cell division and drive bone elongation during embryonic and juvenile development.     

Mice lacking the extracellular matrix adaptor protein matrilin-2 develop without obvious abnormalities.

Matrilins are putative adaptor proteins of the extracellular matrix (ECM) which can form both collagen-dependent and collagen-independent filamentous networks. While all known matrilins (matrilin-1, -2, -3, and -4) are expressed in cartilage, only matrilin-2 and matrilin-4 are abundant in non-skeletal tissues. To clarify the biological role of matrilin-2, we have developed a matrilin-2-deficient mouse strain. Matrilin-2 null mice show no gross abnormalities during embryonic or adult development, are fertile, and have a normal lifespan. Histological and ultrastructural analyses indicate apparently normal structure of all organs and tissues where matrilin-2 is expressed. Although matrilin-2 co-localizes with matrilin-4 in many tissues, Northern hybridization, semiquantitative RT-PCR, immunohistochemistry and biochemical analysis reveal no significant alteration in the steady-state level of matrilin-4 expression in homozygous mutant mice. Immunostaining of wild-type and mutant skin samples indicate no detectable differences in the expression and deposition of matrilin-2 binding partners including collagen I, laminin-nidogen complexes, fibrillin-2 and fibronectin. In addition, electron microscopy reveals an intact basement membrane at the epidermal-dermal junction and normal organization of the dermal collagen fibrils in mutant skin. These data suggest that either matrilin-2 and matrilin-2-mediated matrix-matrix interactions are dispensable for proper ECM assembly and function, or that they are efficiently compensated by other matrix components including wild-type levels of matrilin-4.     

Extracellular matrix assembly in diatoms (Bacillariophyceae). iv. ultrastructure of Achnanthes longipes and Cymbella cistula as revealed by high-pressure freezing/freeze substituton and cryo-field emission scanning electron microscopy

Extracellular matrix (ECM) polymers secreted by the diatoms Achnanthes longipes Ag. and Cymbella cistula (Ehr.) Kirchn. completely encase the cell and are responsible for adhesion and other interactions with the external environment. To preserve details of the highly hydrophilic ECM in the native state and to preserve, with a high degree of fidelity, the intracellular structures involved in synthesis of extracellular polymers, we applied a suite of cryotechniques. The methods included high‐resolution visualization of surfaces using cryo‐field emission SEM (cryo‐FESEM) and preservation for TEM observation of thin sections by high‐pressure freezing (HPF) and freeze substitution (FS). The extracellular structures of diatoms plunge‐frozen in liquid ethane, etched at low temperature, and observed on a cryostage in the FESEM showed overall dimensions and shapes closely comparable to those observed with light microscopy. Cryo‐FESEM demonstrated the pervasive nature of the extracellular polymers and their importance in cell–substratum and cell–cell associations and revealed details of cell attachment processes not visible using other SEM techniques or light microscopy. The layer of ECM coating the frustule and entirely encapsulating cells of A. longipes and C. cistula was shown to have a significant role in initial cell adhesion and subsequent interaction with the environment. Trails of raphe‐associated ECM, generated during cell motility, were shown at high resolution and consist of anastomoses of coiled and linear strands. Cryo‐FESEM revealed a sheet‐like mucilage covering stalks. HPF/FS of A. longipes resulted in excellent preservation of intra‐ and extracellular structures comparable to previous reports for animals and higher plants and revealed several organelles not described previously. Three distinct vesicle types were identified, including a class closely associated with Golgi bodies and postulated to participate in formation of the extracellular adhesive structures. HPF/FS showed a number of continuous diatotepic layers positioned between the plasma membrane and the silicon frustule and revealed that extracellular adhesive extrusion through frustule pores during stalk production was closely related to the diatotepum. The stalks of A. longipes consist of highly organized, multilayered, fine fibrillar materials with an electron‐opaque layer organized as a sheath at the stalk periphery.     

Raman spectroscopy for the non‐contact and non‐destructive monitoring of collagen damage within tissues

The non‐destructive and label‐free monitoring of extracellular matrix (ECM) remodeling and degradation processes is a great challenge. Raman spectroscopy is a non‐contact method that offers the possibility to analyze ECM in situ without the need for tissue processing. Here, we employed Raman spectroscopy for the detection of heart valve ECM, focusing on collagen fibers. We screened the leaflets of porcine aortic valves either directly after dissection or after treatment with collagenase. By comparing the fingerprint region of the Raman spectra of control and treated tissues (400–1800 cm^–1), we detected no significant differences based on Raman shifts; however, we found that increasing collagen degradation translated into decreasing Raman signal intensities. After these proof‐of‐principal experiments, we compared Raman spectra of native and cryopreserved valve tissues and revealed that the signal intensities of the frozen samples were significantly lower compared to those of native tissues, similar to the data seen in the enzymatically‐degraded tissues. In conclusion, our data demonstrate that Raman microscopy is a promising, non‐destructive and non‐contact tool to probe ECM state in situ. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Diversity of extracellular matrix morphology in vertebrate skeletal muscle

Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.     

Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.     

Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies

Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM, which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:212–219, 2015     

Extracellular matrix fluctuations during early embryogenesis

Extracellular matrix (ECM) movements and rearrangements were studied in avian embryos during early stages of development. We show that the ECM moves as a composite material, whereby distinct molecular components as well as spatially separated layers exhibit similar displacements. Using scanning wide field and confocal microscopy we show that the velocity field of ECM displacement is smooth in space and that ECM movements are correlated even at locations separated by several hundred micrometers. Velocity vectors, however, strongly fluctuate in time. The autocorrelation time of the velocity fluctuations is less than a minute. Suppression of the fluctuations yields a persistent movement pattern that is shared among embryos at equivalent stages of development. The high resolution of the velocity fields allows a detailed spatio-temporal characterization of important morphogenetic processes, especially tissue dynamics surrounding the embryonic organizer (Hensen's node).     

HSP90 and eNOS partially co-localize and change cellular localization in relation to different ECM components in 2D and 3D cultures of adult rat cardiomyocytes

Background information. Cultivation techniques promoting three‐dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel‐based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co‐cultures to evaluate, on both two‐ and three‐dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat‐shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase). Results. Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co‐culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel‐cultured cardiomyocytes, areas of high co‐localization between HSP90 and eNOS and co‐localization coefficients, which indicated the highest correlation with respect to the other substrates. Conclusions. Our three‐dimensional adult cardiomyocyte cultures are suitable for both analysing cell—ECM interactions at electron and confocal microscopy levels and monitoring micro‐environment impact on cardiomyocyte phenotype.     

Stimulation of initial neural crest cell migration in the axolotl embryo by tissue grafts and extracellular matrix transplanted on microcarriers

The present experiments were designed to test whether the onset of neural crest cell migration in the embryonic axolotl trunk is stimulated by surrounding tissues and their associated extracellular matrix (ECM). Tissue grafts, or embryonic ECM adsorbed in vivo onto inert "microcarriers" prepared from Nuclepore filters, were placed close to the premigratory neural crest cells, and the embryos were then incubated to a specific stage. The experiments were evaluated with light microscopy, SEM, and TEM. It was found that grafts from the dorsal epidermis were especially effective in locally stimulating initial neural crest cell migration in the region under the graft. The microcarrier experiments showed that the subepidermal ECM alone could initiate neural crest cell migration, implying that the ECM of the epidermal grafts was the stimulating factor. These results indicate that the premigratory neural crest cells along the trunk have migratory capability but that they need to be triggered from the environment, probably from the surrounding ECM, to start migration. It is proposed that ECM, as substrate for cell locomotion, initiates and regulates the onset of neural crest cell migration.     

On the origins of the extracellular matrix in vertebrates.

Extracellular matrix (ECM) is a key metazoan characteristic. In addition to providing structure and orientation to tissues, it is involved in many cellular processes such as adhesion, migration, proliferation and differentiation. Here we provide a comprehensive analysis of ECM molecules focussing on when vertebrate specific matrices evolved. We identify 60 ECM genes and 20 associated processing enzymes in the genome of the urochordate Ciona intestinalis. A comparison with vertebrate and protostome genomes has permitted the identification of both a core set of metazoan matrix genes and vertebrate-specific innovations in the ECM. We have identified a few potential cases of de novo vertebrate ECM gene innovation, but the majority of ECM genes have resulted from duplication of pre-existing genes present in the ancestral vertebrate. In conclusion, the modern complexity we see in vertebrate ECM has come about largely by duplication and modification of pre-existing matrix molecules. Extracellular matrix genes and their processing enzymes appear to be over-represented in the vertebrate genome suggesting that these genes played an active role enabling and underpinning the evolution of vertebrates.     

Three‐dimensional reconstruction of black tiger prawn (Penaeus monodon) spermatozoa using serial block‐face scanning electron microscopy

Serial Block‐Face Scanning Electron Microscopy (SBF‐SEM) was used in this study to examine the ultrastructural morphology of Penaeus monodon spermatozoa. SBF‐SEM provided a large dataset of sequential electron‐microscopic‐level images that facilitated comprehensive ultrastructural observations and three‐dimensional reconstructions of the sperm cell. Reconstruction divulged a nuclear region of the spermatophoral spermatozoon filled with decondensed chromatin but with two apparent levels of packaging density. In addition, the nuclear region contained, not only numerous filamentous chromatin elements with dense microregions, but also large centrally gathered granular masses. Analysis of the sperm cytoplasm revealed the presence of degenerated mitochondria and membrane‐less dense granules. A large electron‐lucent vesicle and “arch‐like” structures were apparent in the subacrosomal area, and an acrosomal core was found in the acrosomal vesicle. The spermatozoal spike arose from the inner membrane of the acrosomal vesicle, which was slightly bulbous in the middle region of the acrosomal vesicle, but then extended distally into a broad dense plate and to a sharp point proximally. This study has demonstrated that SBF‐SEM is a powerful technique for the 3D ultrastructural reconstruction of prawn spermatozoa, that will no doubt be informative for further studies of sperm assessment, reproductive pathology and the spermiocladistics of penaeid prawns, and other decapod crustaceans. J. Morphol. 277:565–574, 2016. © 2016 Wiley Periodicals, Inc.     

Matrix association of latent TGF‐beta binding protein‐2 (LTBP‐2) is dependent on fibrillin‐1

The components of the extracellular matrix (ECM) and their differential expression patterns play important roles in tissue formation. The deposition of latent TGF‐β binding proteins (LTBPs) to the ECM exhibit distinct distribution profiles. We have analyzed here the temporal and spatial ECM association of latent TGF‐β binding protein LTBP‐2 in cultured human embryonic lung fibroblasts. We found that LTBP‐2 was not assembled to the ECM until by confluency of cultures following the deposition of fibronectin (FN) and fibrillin‐1. In 5‐day‐old cultures LTBP‐2 was rapidly secreted from cells and it subsequently associated with the ECM as shown by metabolic labeling and immunoprecipitation. LTBP‐2 colocalized transiently with fibronectin and failed to assemble to the ECM of FN deficient mouse fibroblasts. Analysis of different cultured human cell lines revealed partial colocalization of LTBP‐2 and fibrillin‐1 in the ECM of fibroblasts, MG‐63 osteosarcoma cells and human vascular endothelial cells. Silencing of fibrillin‐1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP‐2. Current results suggest that LTBP‐2 is not an element of the provisional ECM of fibroblasts but is more likely a component of more mature ECM and indicate that matrix association of LTBP‐2 depends on a pre‐formed fibrillin‐1 network. J. Cell. Physiol. 221: 586–593, 2009. © 2009 Wiley‐Liss, Inc.     

Developmental SEM observations on an extracellular matrix in embryogenic calli ofDrosera rotundifolia andZea mays

Summary Primary embryogenic callus ofDrosera rotundifolia and long-term cultured embryogenic callus ofZea mays possess a conspicuous extracellular matrix (ECM) around and between embryogenic cells. The structural arrangement of ECM depends on the developmental stage of the embryogenic cells. Single embryoid cells were covered with, and connected by net-like material. However, surface cells of young globular embryoids were covered with a coherent layer of ECM which forms bridges with net-like material between the cells which was gradually reduced to coarse strands. When protodermis was formed on the surface of globular embryoids, the ECM disappeared completely. The ECM network was never observed on the surface of heart- and torpedo-shaped embryoids. Safranine (especially 0.1%) stabilized the structure of ECM. Digestion with pronase E and proteinase K indicated that the ECM contains proteinaceous components. Similar developmental patterns of ECM were observed in dicotyledonous and monocotyledonous examples. The ECM represents a stable morphological structure even during long-term embryogenic culture in maize.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - Dicamba 3,6-dichloro-o-anisic acid - ECM extracellular matrix - KIN kinetin - SEM scanning electron microscopy - TEM transmission electron microscopy     

Development by three‐dimensional approaches and four‐dimensional imaging: To the knowledge frontier and beyond

Many advances have been taken on elucidating embryonic development and tissue homeostasis and repair by the use of experimental strategies that preserve the three‐dimensional (3D) organization and allow quantitative analysis of images over time (four‐dimensional). Ranging from the understanding about the relationship between blastomeres and the events that take place during gastrulation by the use of time‐lapse imaging through 3D cultures that mimic organogenesis, the advances in this area are of critical value. The studies on embryonic development without disrupting the original architecture and the development of 3D organoid cultures pave a new avenue for unprecedented experimental advances that will positively impact the emergence of new treatments applying regenerative principles for both tissue repair and organ transplant. Birth Defects Research (Part C) 105:1–8, 2015. © 2015 Wiley Periodicals, Inc.