Organ Assembly Research Articles

Centering Margins in Organ Assembly

Centering Margins in Organ Assembly

Glycan characteristics of human heart constituent cells maintaining organ function: relatively stable glycan profiles in cellular senescence

Cell surface glycoproteins, which are good indicators of cellular types and biological function; are suited for cell evaluation. Tissue remodeling using various cells is a key feature of regenerative therapy. For artificial heart remodeling, a mixture of heart constituent cells has been investigated for organ assembly, however, the cellular characteristics remain unclear. In this study, the glycan profiles of human cardiomyocytes (HCMs), human cardiac fibroblasts (HCFs), and human vascular endothelial cells (ECs) were analyzed using evanescent-field lectin microarray analysis, a tool of glycan profiling, to clarify the required cellular characteristics. We found that ECs had more “α1-2fucose” and “core α1-6fucose” residues than other cells, and that “α2-6sialic acid” residue was more abundant in ECs and HCMs than in HCFs. HCFs showed higher abundance of “β-galactose” and “β-N-acetylgalactosamine” residues on N-glycan and O-glycan, respectively, compared to other cells. Interestingly, cardiac glycan profiles were insignificantly changed with cellular senescence. The residues identified in this study may participate in organ maintenance by contributing to the preservation of glycan components. Therefore, future studies should investigate the roles of glycans in optimal tissue remodeling since identifying cellular characteristics is important for the development of regenerative therapies.

Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip1.

ABSTRACTOrgan morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveals distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tricellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics the junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function in junction formation and stabilization during sprouting angiogenesis.

Organoids by design.

Organoids are multicellular structures that can be derived from adult organs or pluripotent stem cells. Early versions of organoids range from simple epithelial structures to complex, disorganized tissues with large cellular diversity. The current challenge is to engineer cellular complexity into organoids in a controlled manner that results in organized assembly and acquisition of tissue function. These efforts have relied on studies of organ assembly during embryonic development and have resulted in the development of organoids with multilayer tissue complexity and higher-order functions. We discuss how the next generation of organoids can be designed by means of an engineering-based narrative design to control patterning, assembly, morphogenesis, growth, and function.

From morphogen to morphogenesis and back.

A long-term aim of the life sciences is to understand how organismal shape is encoded by the genome. An important challenge is to identify mechanistic links between the genes that control cell-fate decisions and the cellular machines that generate shape, therefore closing the gap between genotype and phenotype. The logic and mechanisms that integrate these different levels of shape control are beginning to be described, and recently discovered mechanisms of cross-talk and feedback are beginning to explain the remarkable robustness of organ assembly. The 'full-circle' understanding of morphogenesis that is emerging, besides solving a key puzzle in biology, provides a mechanistic framework for future approaches to tissue engineering.

The endoderm indirectly influences morphogenetic movements of the zebrafish head kidney through the posterior cardinal vein and VegfC.

Integration of blood vessels and organ primordia determines organ shape and function. The head kidney in the zebrafish interacts with the dorsal aorta (DA) and the posterior cardinal vein (PCV) to achieve glomerular filtration and definitive hematopoiesis, respectively. How the head kidney co-develops with both the axial artery and vein remains unclear. We found that in endodermless sox32-deficient embryos, the head kidney associated with the PCV but not the DA. Disrupted convergent migration of the PCV and the head kidney in sox32-deficient embryos was rescued in a highly coordinated fashion through the restoration of endodermal cells. Moreover, grafted endodermal cells abutted the host PCV endothelium in the transplantation assay. Interestingly, the severely-disrupted head kidney convergence in the sox32-deficient embryo was suppressed by both the cloche mutation and the knockdown of endothelial genes, indicating that an interaction between the endoderm and the PCV restricts the migration of the head kidney. Furthermore, knockdown of either vegfC or its receptor vegfr3 suppressed the head kidney convergence defect in endodermless embryos and perturbed the head kidney-PCV association in wild-type embryos. Our findings thus underscore a role for PCV and VegfC in patterning the head kidney prior to organ assembly and function.

Diversity in cell motility reveals the dynamic nature of the formation of zebrafish taste sensory organs

Taste buds are sensory organs in jawed vertebrates, composed of distinct cell types that detect and transduce specific taste qualities. Taste bud cells differentiate from oropharyngeal epithelial progenitors, which are localized mainly in proximity to the forming organs. Despite recent progress in elucidating the molecular interactions required for taste bud cell development and function, the cell behavior underlying the organ assembly is poorly defined. Here, we used time-lapse imaging to observe the formation of taste buds in live zebrafish larvae. We found that tg(fgf8a.dr17)-expressing cells form taste buds and get rearranged within the forming organs. In addition, differentiating cells move from the epithelium to the forming organs and can be displaced between developing organs. During organ formation, tg(fgf8a.dr17) and type II taste bud cells are displaced in random, directed or confined mode relative to the taste bud they join or by which they are maintained. Finally, ascl1a activity in the 5-HT/type III cell is required to direct and maintain tg(fgf8a.dr17)-expressing cells into the taste bud. We propose that diversity in displacement modes of differentiating cells acts as a key mechanism for the highly dynamic process of taste bud assembly.

Decellularization and recellularization technologies in tissue engineering.

Decellularization is the process by which cells are discharged from tissues/organs, but all of the essential cues for cell preservation and homeostasis are retained in a three-dimensional structure of the organ and its extracellular matrix components. During tissue decellularization, maintenance of the native ultrastructure and composition of the extracellular matrix (ECM) is extremely acceptable. For recellularization, the scaffold/matrix is seeded with cells, the final goal being to form a practical organ. In this review, we focus on the biological properties of the ECM that remains when a variety of decellularization methods are used, comparing recellularization technologies, including bioreactor expansion for perfusion-based bioartificial organs, and we discuss cell sources. In the future, decellularization-recellularization procedures may solve the problem of organ assembly on demand.

Evolutionarily conserved morphogenetic movements at the vertebrate head–trunk interface coordinate the transport and assembly of hypopharyngeal structures

The vertebrate head–trunk interface (occipital region) has been heavily remodelled during evolution, and its development is still poorly understood. In extant jawed vertebrates, this region provides muscle precursors for the throat and tongue (hypopharyngeal/hypobranchial/hypoglossal muscle precursors, HMP) that take a stereotype path rostrally along the pharynx and are thought to reach their target sites via active migration. Yet, this projection pattern emerged in jawless vertebrates before the evolution of migratory muscle precursors. This suggests that a so far elusive, more basic transport mechanism must have existed and may still be traceable today.Here we show for the first time that all occipital tissues participate in well-conserved cell movements. These cell movements are spearheaded by the occipital lateral mesoderm and ectoderm that split into two streams. The rostrally directed stream projects along the floor of the pharynx and reaches as far rostrally as the floor of the mandibular arch and outflow tract of the heart. Notably, this stream leads and engulfs the later emerging HMP, neural crest cells and hypoglossal nerve. When we (i) attempted to redirect hypobranchial/hypoglossal muscle precursors towards various attractants, (ii) placed non-migratory muscle precursors into the occipital environment or (iii) molecularly or (iv) genetically rendered muscle precursors non-migratory, they still followed the trajectory set by the occipital lateral mesoderm and ectoderm. Thus, we have discovered evolutionarily conserved morphogenetic movements, driven by the occipital lateral mesoderm and ectoderm, that ensure cell transport and organ assembly at the head–trunk interface.

Fibronectin mediates correct positioning of the interrenal organ in zebrafish

Fibronectin (Fn) forms a centripetal gradient during the fetal adrenal gland organogenesis, and modulates hormone responsiveness of adrenocortical cells in the primary culture. However, how Fn is involved in organ formation of the adrenal gland remains unclear. In this study, we found that Fn accumulates around migrating ff1b-expressing interrenal cells, which were marked by the ff1b promoter-driven transgenic fluorescence, during the course of interrenal organ assembly. The interrenal cells displaying the migratory phenotype were absent in the fn1 mutant, while specification and kidney association of the interrenal tissue remained normal. The Fn deposition in the interrenal microenvironment was severely reduced in the vessel-deficient ets1b morphant, implying its origin of synthesis from the peri-interrenal vasculature. In the fn1 mutant, early-migrating chromaffin cells were capable of interacting with steroidogenic interrenal cells, yet continuous migration and midline convergence of chromaffin cells were disrupted. Migration defects of both interrenal and chromaffin lineages, in the absence of Fn, thus led to incomplete interrenal organ assembly in aberrant positions. Our results indicate that Fn is essential for patterning interrenal organ formation, by modulating the migratory behavior of both steroidogenic interrenal and chromaffin cells.

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila

Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function.

Epithelial machines of morphogenesis and their potential application in organ assembly and tissue engineering

Sheets of embryonic epithelial cells coordinate their efforts to create diverse tissue structures such as pits, grooves, tubes, and capsules that lead to organ formation. Such cells can use a number of cell behaviors including contractility, proliferation, and directed movement to create these structures. By contrast, tissue engineers and researchers in regenerative medicine seeking to produce organs for repair or replacement therapy can combine cells with synthetic polymeric scaffolds. Tissue engineers try to achieve these goals by shaping scaffold geometry in such a way that cells embedded within these scaffold self-assemble to form a tissue, for instance aligning to synthetic fibers, and assembling native extracellular matrix to form the desired tissue-like structure. Although self-assembly is a dominant process that guides tissue assembly both within the embryo and within artificial tissue constructs, we know little about these critical processes. Here, we compare and contrast strategies of tissue assembly used by embryos to those used by engineers during epithelial morphogenesis and highlight opportunities for future applications of developmental biology in the field of tissue engineering.

Mammalian kidney development: principles, progress, and projections.

The mammalian kidney is a vital organ with considerable cellular complexity and functional diversity. Kidney development is notable for requiring distinct but coincident tubulogenic processes involving reciprocal inductive signals between mesenchymal and epithelial progenitor compartments. Key molecular pathways mediating these interactions have been identified. Further, advances in the analysis of gene expression and gene activity, coupled with a detailed knowledge of cell origins, are enhancing our understanding of kidney morphogenesis and unraveling the normal processes of postnatal repair and identifying disease-causing mechanisms. This article focuses on recent insights into central regulatory processes governing organ assembly and renal disease, and predicts future directions for the field.

Organ printing: computer-aided jet-based 3D tissue engineering

Tissue engineering technology promises to solve the organ transplantation crisis. However, assembly of vascularized 3D soft organs remains a big challenge. Organ printing, which we define as computer-aided, jet-based 3D tissue-engineering of living human organs, offers a possible solution. Organ printing involves three sequential steps: pre-processing or development of ‘blueprints’ for organs; processing or actual organ printing; and postprocessing or organ conditioning and accelerated organ maturation. A cell printer that can print gels, single cells and cell aggregates has been developed. Layer-by-layer sequentially placed and solidified thin layers of a thermo-reversible gel could serve as ‘printing paper’. Combination of an engineering approach with the developmental biology concept of embryonic tissue fluidity enables the creation of a new rapid prototyping 3D organ printing technology, which will dramatically accelerate and optimize tissue and organ assembly.

Regulation of dally, an Integral Membrane Proteoglycan, and Its Function during Adult Sensory Organ Formation of Drosophila

In Drosophila, imaginal wing discs, Wg and Dpp, play important roles in the development of sensory organs. These secreted growth factors govern the positions of sensory bristles by regulating the expression of achaete-scute (ac-sc), genes affecting neuronal precursor cell identity. Earlier studies have shown that Dally, an integral membrane, heparan sulfate-modified proteoglycan, affects both Wg and Dpp signaling in a tissue-specific manner. Here, we show that dally is required for the development of specific chemosensory and mechanosensory organs in the wing and notum. dally enhancer trap is expressed at the anteroposterior and dorsoventral boundaries of the wing pouch, under the control of hh and wg, respectively. dally affects the specification of proneural clusters for dally-sensitive bristles and shows genetic interactions with either wg or dpp signaling components for distinct sensory bristles. These findings suggest that dally can differentially regulate Wg- or Dpp-directed patterning during sensory organ assembly. We have also determined that, for pSA, a bristle on the lateral notum, dally shows genetic interactions with iroquois complex (IRO-C), a gene complex affecting ac-sc expression. Consistent with this interaction, dally mutants show markedly reduced expression of an iro::lacZ reporter. These findings establish dally as an important regulator of sensory organ formation via Wg- and Dpp-mediated specification of proneural clusters.

Simulated microgravity conditions enhance differentiation of cultured PC12 cells towards the neuroendocrine phenotype.

We are studying microenvironmental cues which contribute to neuroendocrine organ assembly and tissue-specific differentiation. As our in vitro model, we cultured rat adrenal medullary PC12 pheochromocytoma cells in a novel cell culture system, the NASA rotating wall vessel (RWV) bioreactors. This "simulated microgravity" environment in RWV bioreactors, characterized by randomizing gravitational vectors and minimizing shear stress, has been shown to favor macroscopic tissue assembly and to induce tissue-specific differentiation. We hypothesized that the unique culture conditions in the RWV bioreactors might enhance the in vitro formation of neuroendocrine organoids. To test our hypothesis, we evaluated the expression of several markers of neuroendocrine differentiation in cultures of PC12 cells maintained for up to 20 d in the slow turning lateral vessel (STLV) type RWV. PC12 cell differentiation was assessed by morphological, immunological, biochemical and molecular techniques. PC12 cells, cultured under "simulated microgravity" conditions, formed macroscopic, tissue-like organoids several millimeters in diameter. Concomitantly, the expression of phenylethanolamine-N-methyl transferase (PNMT), but not of other catecholamine synthesizing enzymes, was enhanced. Increased PNMT expression, as verified on both the gene and protein level, was accompanied by an increase in the specific activity of the enzyme. Furthermore, after 20 d in culture in the STLV, we observed altered patterns of protein tyrosine phosphorylation and prolonged activation of c-fos, a member of the AP-1 nuclear transcription factor complex. We conclude that culture conditions in the RWV appear to selectively activate signal transduction pathways leading to enhanced neuroendocrine differentiation of PC12 cells.

Patterning the heart's left-right axis: from zebrafish to man.

Normal left-right asymmetry is highly conserved among vertebrates. Errors in the proper patterning of this axis are believed to lead to congenital anomalies of the heart and abdominal viscera, often with profound clinical consequences. We review briefly the nature of potential signals and signaling sources that lead to the break in left-right symmetry. The evidence suggests that left-right reversal, or homogenization, of these signals may lead to different consequences, and we explain some malpositions and malalignments of the atria, ventricles, and/or outflow tract that are seen in a variety of congenital cardiac diseases. We speculate that there are units of organ assembly responsive to laterality signals, and these units may be driven independently. One crucial source of signals appears to be the notochord and floorplate. In order to examine the clinical relationship of these midline structures to putative disorders of laterality, we review all patients with disturbances of normal laterality seen at the Massachusetts General Hospital over the past 20 years. We find a significant association between laterality defects and anomalies of the spine and other midline structures.