Ramray Bhat

Dynamical patterning modules: a "pattern language" for development and evolution of multicellular form

This article considers the role played by a core set of dynamical patterning modules (DPMs) in the origination, development and evolution of complex organisms. These consist of the products of a subset of the genes of what has come to be known as the developmental-genetic toolkit in association with physical processes they mobilize. The physical processes are those characteristic of chemically and mechanically excitable mesoscopic systems like cell aggregates: cohesion, viscoelasticity, diffusion, spatiotemporal heterogeneity based on activator-inhibitor interaction, and multistable and oscillatory dynamics. We focus on the emergence of the Metazoa, and show how toolkit gene products and pathways that pre-existed the metazoans acquired novel morphogenetic functions simply by virtue of the change in scale and context inherent to multicellularity. We propose that DPMs, acting singly and in combination with each other, constitute a pattern language capable of generating all metazoan body plans and organ forms. This concept implies that the multicellular organisms of the late Precambrian-early Cambrian were phenotypically plastic, fluently exploring morphospace in a fashion decoupled from both function-based selection and genotypic change. The relatively stable developmental trajectories and morphological phenotypes of modern organisms, then, are considered to be products of stabilizing selection. This perspective solves the apparent molecular homology-analogy paradox, whereby widely divergent modern animal types utilize the same molecular toolkit during development, but it does so by inverting the neo-Darwinian principle that phenotypic disparity was generated over long periods of time in concert with, and in proportion to genotypic change.

Reversible Aptamer-Au Plasmon Rulers for Secreted Single Molecules

Plasmon rulers, consisting of pairs of gold nanoparticles, allow single-molecule analysis without photobleaching or blinking; however, current plasmon rulers are irreversible, restricting detection to only single events. Here, we present a reversible plasmon ruler, comprised of coupled gold nanoparticles linked by a single aptamer, capable of binding individual secreted molecules with high specificity. We show that the binding of target secreted molecules to the reversible plasmon ruler is characterized by single-molecule sensitivity, high specificity, and reversibility. Such reversible plasmon rulers should enable dynamic and adaptive live-cell measurement of secreted single molecules in their local microenvironment.

Of plasticity and specificity: dialectics of the microenvironment and macroenvironment and the organ phenotype.

The study of biological form and how it arises is the domain of the developmental biologists; but once the form is achieved, the organ poses a fascinating conundrum for all the life scientists: how are form and function maintained in adult organs throughout most of the life of the organism? That they do appears to contradict the inherently plastic nature of organogenesis during development. How do cells with the same genetic information arrive at, and maintain such different architectures and functions, and how do they keep remembering that they are different from each other? It is now clear that narratives based solely on genes and an irreversible regulatory dynamics cannot answer these questions satisfactorily, and the concept of microenvironmental signaling needs to be added to the equation. During development, cells rearrange and differentiate in response to diffusive morphogens, juxtacrine signals, and the extracellular matrix (ECM). These components, which constitute the modular microenvironment, are sensitive to cues from other tissues and organs of the developing embryo as well as from the external macroenvironment. On the other hand, once the organ is formed, these modular constituents integrate and constrain the organ architecture, which ensures structural and functional homeostasis and therefore, organ specificity. We argue here that a corollary of the above is that once the organ architecture is compromised in adults by mutations or by changes in the microenvironment such as aging or inflammation, that organ becomes subjected to the developmental and embryonic circuits in search of a new identity. But since the microenvironment is no longer embryonic, the confusion leads to cancer: hence as we have argued, tumors become new evolutionary organs perhaps in search of an elusive homeostasis. WIREs Dev Biol 2014, 3:147–163. doi: 10.1002/wdev.130

SnapShot: Branching Morphogenesis.

Ectodermal appendages such as feathers, hair, mammary glands, salivary glands, and sweat glands form branches, allowing much-increased surface for functional differentiation and secretion. Here, the principles of branching morphogenesis are exemplified by the mammary gland and feathers.

Nuclear repartitioning of galectin-1 by an extracellular glycan switch regulates mammary morphogenesis

Significance Malignant cells of breast carcinoma and nonmalignant epithelia of branching mammary glands share the ability to migrate through their surroundings. To form the mammary tree-like architecture, nonmalignant epithelia must migrate in a controlled fashion, integrating cues from their microenvironment, notably, the glycan appendages on extracellular proteins and lipids. Here, we show that Galectin-1, a glycan-binding protein, is able to sense glycan signatures on mammary gland epithelia, transmit this information to epithelial nuclei by direct translocation, and drive branching migration. Nuclear galectin-1 is regulated by the relative levels of α2,6–sialic acids and N-acetyllactosamine on extracellular glycans. Similar lectin–glycan signatures were observed in malignant breast cells and suggest cancer cells use this pathway during their invasion. Branching morphogenesis in the mammary gland is achieved by the migration of epithelial cells through a microenvironment consisting of stromal cells and extracellular matrix (ECM). Here we show that galectin-1 (Gal-1), an endogenous lectin that recognizes glycans bearing N-acetyllactosamine (LacNAc) epitopes, induces branching migration of mammary epithelia in vivo, ex vivo, and in 3D organotypic cultures. Surprisingly, Gal-1’s effects on mammary patterning were independent of its glycan-binding ability and instead required localization within the nuclei of mammary epithelia. Nuclear translocation of Gal-1, in turn, was regulated by discrete cell-surface glycans restricted to the front of the mammary end buds. Specifically, α2,6–sialylation of terminal LacNAc residues in the end buds masked Gal-1 ligands, thereby liberating the protein for nuclear translocation. Within mammary epithelia, Gal-1 localized within nuclear Gemini bodies and drove epithelial invasiveness. Conversely, unsialylated LacNAc glycans, enriched in the epithelial ducts, sequestered Gal-1 in the extracellular environment, ultimately attenuating invasive potential. We also found that malignant breast cells possess higher levels of nuclear Gal-1 and α2,6–SA and lower levels of LacNAc than nonmalignant cells in culture and in vivo and that nuclear localization of Gal-1 promotes a transformed phenotype. Our findings suggest that differential glycosylation at the level of tissue microanatomy regulates the nuclear function of Gal-1 in the context of mammary gland morphogenesis and in cancer progression.

Laser Scanning–Based Tissue Autofluorescence/Fluorescence Imaging (LS-TAFI), a New Technique for Analysis of Microanatomy in Whole-Mount Tissues

Intact organ structure is essential in maintaining tissue specificity and cellular differentiation. Small physiological or genetic variations lead to changes in microanatomy that, if persistent, could have functional consequences and may easily be masked by the heterogeneity of tissue anatomy. Current imaging techniques rely on histological, two-dimensional sections requiring sample manipulation that are essentially two dimensional. We have developed a method for three-dimensional imaging of whole-mount, unsectioned mammalian tissues to elucidate subtle and detailed micro- and macroanatomies in adult organs and embryos. We analyzed intact or dissected organ whole mounts with laser scanning-based tissue autofluorescence/fluorescence imaging (LS-TAFI). We obtained clear visualization of microstructures within murine mammary glands and mammary tumors and other organs without the use of immunostaining and without probes or fluorescent reporter genes. Combining autofluorescence with reflected light signals from chromophore-stained tissues allowed identification of individual cells within three-dimensional structures of whole-mounted organs. This technique could be useful for rapid diagnosis of human clinical samples and possibly the effect of subtle variations such as low dose radiation.

Modeling the morphodynamic galectin patterning network of the developing avian limb skeleton

We present a mathematical model for the morphogenesis and patterning of the mesenchymal condensations that serve as primordia of the avian limb skeleton. The model is based on the experimentally established dynamics of a multiscale regulatory network consisting of two glycan-binding proteins expressed early in limb development: CG (chicken galectin)-1A, CG-8 and their counterreceptors that determine the formation, size, number and spacing of the protocondensations that give rise to the condensations and subsequently the cartilaginous elements that serve as the templates of the bones. The model, a system of partial differential and integro-differential equations containing a flux term to represent local adhesion gradients, is simulated in a full and a reduced form to confirm that the system has pattern-forming capabilities and to explore the nature of the patterning instability. The full model recapitulates qualitatively and quantitatively the experimental results of network perturbation and leads to new predictions, which are verified by further experimentation. The reduced model is used to demonstrate that the patterning process is inherently morphodynamic, with cell motility being intrinsic to it. Furthermore, subtle relationships between cell movement and the positive and negative interactions between the morphogens produce regular patterns without the requirement for activators and inhibitors with widely separated diffusion coefficients. The described mechanism thus represents an extension of the category of activator-inhibitor processes capable of generating biological patterns with repetitive elements beyond the morphostatic mechanisms of the Turing/Gierer-Meinhardt type.

Mammary Branching Morphogenesis Requires Reciprocal Signaling by Heparanase and MMP-14.

The development of the mammary gland involves formation of a branched arboreal structure resulting from the penetration and proliferation of epithelial cells into the fat pad. The mammary cells invade by remodeling their surrounding extracellular matrix (ECM), which are rich in proteins, and glycans such as heparan sulfate proteoglycans (HSPGs). There is increasing literature on how the interaction between signaling by ECM and matrix metalloproteinases (MMPs) is relevant to morphogenetic and physiological contexts. Here we sought to understand how heparanase, the sole mammalian heparan sulfate‐degrading endoglycosidase may regulate mammary gland development. We found a robust localization of heparanase within growing end buds during branching in vivo. Using three‐dimensional (3D) organotypic cultures, we showed that heparanase expression and activity are required for mammary epithelial invasion/branching within dense collagen I gels. Morphometric analysis of glands from both heparanase‐overexpressing and knockout mice showed a direct correlation between degree of branching and the heparanase levels, confirming our 3D organotypic culture observations. Finally, we uncovered a reciprocal association between levels of heparanase and MMP14, a membrane‐bound MMP, shedding further light on how branching occurs within developing mammary glands. J. Cell. Biochem. 116: 1668–1679, 2015.

Structural divergence in vertebrate phylogeny of a duplicated prototype galectin.

Prototype galectins, endogenously expressed animal lectins with a single carbohydrate recognition domain, are well-known regulators of tissue properties such as growth and adhesion. The earliest discovered and best studied of the prototype galectins is Galectin-1 (Gal-1). In the Gallus gallus (chicken) genome, Gal-1 is represented by two homologs: Gal-1A and Gal-1B, with distinct biochemical properties, tissue expression, and developmental functions. We investigated the origin of the Gal-1A/Gal-1B divergence to gain insight into when their developmental functions originated and how they could have contributed to vertebrate phenotypic evolution. Sequence alignment and phylogenetic tree construction showed that the Gal-1A/Gal-1B divergence can be traced back to the origin of the sauropsid lineage (consisting of extinct and extant reptiles and birds) although lineage-specific duplications also occurred in the amphibian and actinopterygian genomes. Gene synteny analysis showed that sauropsid gal-1b (the gene for Gal-1B) and its frog and actinopterygian gal-1 homologs share a similar chromosomal location, whereas sauropsid gal-1a has translocated to a new position. Surprisingly, we found that chicken Gal-1A, encoded by the translocated gal-1a, was more similar in its tertiary folding pattern than Gal-1B, encoded by the untranslocated gal-1b, to experimentally determined and predicted folds of nonsauropsid Gal-1s. This inference is consistent with our finding of a lower proportion of conserved residues in sauropsid Gal-1Bs, and evidence for positive selection of sauropsid gal-1b, but not gal-1a genes. We propose that the duplication and structural divergence of Gal-1B away from Gal-1A led to specialization in both expression and function in the sauropsid lineage.

Deep Phylogenomics of a Tandem-repeat Galectin Regulating Appendicular Skeletal Pattern Formation

BackgroundA multiscale network of two galectins Galectin-1 (Gal-1) and Galectin-8 (Gal-8) patterns the avian limb skeleton. Among vertebrates with paired appendages, chondrichthyan fins typically have one or more cartilage plates and many repeating parallel endoskeletal elements, actinopterygian fins have more varied patterns of nodules, bars and plates, while tetrapod limbs exhibit tandem arrays of few, proximodistally increasing numbers of elements. We applied a comparative genomic and protein evolution approach to understand the origin of the galectin patterning network. Having previously observed a phylogenetic constraint on Gal-1 structure across vertebrates, we asked whether evolutionary changes of Gal-8 could have critically contributed to the origin of the tetrapod pattern.ResultsTranslocations, duplications, and losses of Gal-8 genes in Actinopterygii established them in different genomic locations from those that the Sarcopterygii (including the tetrapods) share with chondrichthyans. The sarcopterygian Gal-8 genes acquired a potentially regulatory non-coding motif and underwent purifying selection. The actinopterygian Gal-8 genes, in contrast, did not acquire the non-coding motif and underwent positive selection.ConclusionThese observations interpreted through the lens of a reaction-diffusion-adhesion model based on avian experimental findings can account for the distinct endoskeletal patterns of cartilaginous, ray-finned, and lobe-finned fishes, and the stereotypical limb skeletons of tetrapods.