Cell behaviors that pattern developing tissues: the case of the vertebrate nervous system.

Morphogenesis from cells to tissue gives rise to the complex architectures that make our organs. How cells and their dynamic behavior are translated into functional spatial patterns is only starting to be understood. Recent advances in quantitative imaging revealed that, although highly heterogeneous, cellular behaviors make reproducible tissue patterns. Emerging evidence suggests that mechanisms of cellular coordination, intrinsic variability and plasticity are critical for robust pattern formation. While pattern development shows a high level of fidelity, tissue organization has undergone drastic changes throughout the course of evolution. In addition, alterations in cell behavior, if unregulated, can cause developmental malformations that disrupt function. Therefore, comparative studies of different species and of disease models offer a powerful approach for understanding how novel spatial configurations arise from variations in cell behavior and the fundamentals of successful pattern formation. In this chapter, I dive into the development of the vertebrate nervous system to explore efforts to dissect pattern formation beyond molecules, the emerging core principles and open questions.

Emergence of a left-right symmetric body plan in vertebrate embryos.

External bilateral symmetry is a prevalent feature in vertebrates, which emerges during early embryonic development. To begin with, vertebrate embryos are largely radially symmetric before transitioning to bilaterally symmetry, after which, morphogenesis of various bilateral tissues (e.g somites, otic vesicle, limb bud), and structures (e.g palate, jaw) ensue. While a significant amount of work has probed the mechanisms behind symmetry breaking in the left-right axis leading to asymmetric positioning of internal organs, little is known about how bilateral tissues emerge at the same time with the same shape and size and at the same position on the two sides of the embryo. By discussing emergence of symmetry in many bilateral tissues and structures across vertebrate model systems, we highlight that understanding symmetry establishment is largely an open field, which will provide deep insights into fundamental problems in developmental biology for decades to come.

Retinoic Acid-Mediated Control of Energy Metabolism Is Essential for Lung Branching Morphogenesis.

Lung branching morphogenesis relies on intricate epithelial-mesenchymal interactions and signaling networks. Still, the interplay between signaling and energy metabolism in shaping embryonic lung development remains unexplored. Retinoic acid (RA) signaling influences lung proximal-distal patterning and branching morphogenesis, but its role as a metabolic modulator is unknown. Hence, this study investigates how RA signaling affects the metabolic profile of lung branching. We performed ex vivo lung explant culture of embryonic chicken lungs treated with DMSO, 1 µM RA, or 10 µM BMS493. Extracellular metabolite consumption/production was evaluated by using 1H-NMR spectroscopy. Mitochondrial respiration and biogenesis were also analyzed. Proliferation was assessed using an EdU-based assay. The expression of crucial metabolic/signaling components was examined through Western blot, qPCR, and in situ hybridization. RA signaling stimulation redirects glucose towards pyruvate and succinate production rather than to alanine or lactate. Inhibition of RA signaling reduces lung branching, resulting in a cystic-like phenotype while promoting mitochondrial function. Here, RA signaling emerges as a regulator of tissue proliferation and lactate dehydrogenase expression. Furthermore, RA governs fatty acid metabolism through an AMPK-dependent mechanism. These findings underscore RA's pivotal role in shaping lung metabolism during branching morphogenesis, contributing to our understanding of lung development and cystic-related lung disorders.

Maternal Diet High in Linoleic Acid Alters Renal Branching Morphogenesis and mTOR/AKT Signalling Genes in Rat Fetal Kidneys.

Linoleic acid (LA), an n-6 polyunsaturated fatty acid (PUFA), is obtained from the maternal diet during pregnancy, and is essential for normal fetal growth and development. A maternal high-LA (HLA) diet alters maternal and offspring fatty acids, maternal leptin and male/female ratio at embryonic (E) day 20 (E20). We investigated the effects of an HLA diet on embryonic offspring renal branching morphogenesis, leptin signalling, megalin signalling and angiogenesis gene expression. Female Wistar Kyoto rats were fed low-LA (LLA; 1.44% energy from LA) or high-LA (HLA; 6.21% energy from LA) diets during pregnancy and gestation/lactation. Offspring were sacrificed and mRNA from kidneys was analysed by real-time PCR. Maternal HLA decreased the targets involved in branching morphogenesis Ret and Gdnf in offspring, independent of sex. Furthermore, downstream targets of megalin, namely mTOR, Akt3 and Prkab2, were reduced in offspring from mothers consuming an HLA diet, independent of sex. There was a trend of an increase in the branching morphogenesis target Gfra1 in females (p = 0.0517). These findings suggest that an HLA diet during pregnancy may lead to altered renal function in offspring. Future research should investigate the effects an HLA diet has on offspring kidney function in adolescence and adulthood.

Transitions in development - an interview with Thibaut Brunet.

Thibaut Brunet is a group leader at the Institut Pasteur in Paris, France, where he works on choanoflagellates (known as 'choanos' for short). These unicellular organisms are close relatives of animals that have the potential to form multicellular assemblies under certain conditions, and Thibaut's lab are leveraging them to gain insights into how animal morphogenesis evolved. We met with Thibaut over Zoom to discuss his career path so far, and learnt how an early interest in dinosaurs contributed to his life-long fascination with evolutionary biology.

Mechanochemical dynamics of collective cells and hierarchical topological defects in multicellular lumens.

Collective cell dynamics is essential for tissue morphogenesis and various biological functions. However, it remains incompletely understood how mechanical forces and chemical signaling are integrated to direct collective cell behaviors underlying tissue morphogenesis. Here, we propose a three-dimensional (3D) mechanochemical theory accounting for biochemical reaction-diffusion and cellular mechanotransduction to investigate the dynamics of multicellular lumens. We show that the interplay between biochemical signaling and mechanics can trigger either pitchfork or Hopf bifurcation to induce diverse static mechanochemical patterns or generate oscillations with multiple modes both involving marked mechanical deformations in lumens. We uncover the crucial role of mechanochemical feedback in emerging morphodynamics and identify the evolution and morphogenetic functions of hierarchical topological defects including cell-level hexatic defects and tissue-level orientational defects. Our theory captures the common mechanochemical traits of collective dynamics observed in experiments and could provide a mechanistic context for understanding morphological symmetry breaking in 3D lumen-like tissues.

Actin pushes open a leaky lumen.

Using a human stem cell-based model to understand how the human epiblast forms at the very beginning of implantation, Indana et al.1 establish a role for pushing forces that are generated by apical actin polymerization and reveal a two-stage, biomechanics-driven lumen growth process underlying epiblast cavity morphogenesis.

The enigma of cell intercalation.

Geometric criteria can be used to assess whether cell intercalation is active or passive during the convergent extension of tissue.

ΔNp63 regulates Sfrp1 expression to direct salivary gland branching morphogenesis.

Branching morphogenesis is a complex process shared by many organs including the lungs, kidney, prostate, as well as several exocrine organs including the salivary, mammary and lacrimal glands. This critical developmental program ensures the expansion of an organ's surface area thereby maximizing processes of cellular secretion or absorption. It is guided by reciprocal signaling from the epithelial and mesenchymal cells. While signaling pathways driving salivary gland branching morphogenesis have been relatively well-studied, our understanding of the underlying transcriptional regulatory mechanisms directing this program, is limited. Here, we performed in vivo and ex vivo studies of the embryonic mouse submandibular gland to determine the function of the transcription factor ΔNp63, in directing branching morphogenesis. Our studies show that loss of ΔNp63 results in alterations in the differentiation program of the ductal cells which is accompanied by a dramatic reduction in branching morphogenesis that is mediated by dysregulation of WNT signaling. We show that ΔNp63 modulates WNT signaling to promote branching morphogenesis by directly regulating Sfrp1 expression. Collectively, our findings have revealed a novel role for ΔNp63 in the regulation of this critical process and offers a better understanding of the transcriptional networks involved in branching morphogenesis.

Two distinct mechanisms of Plexin A function in Drosophila optic lobe lamination and morphogenesis.

Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that, in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In Plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of semaphorin function indicates that Semaphorin 1a, acting in a subset of medulla neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A has little effect on the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of Plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles.

Conserved physical mechanisms of cell and tissue elongation.

Living organisms have the ability to self-shape into complex structures appropriate for their function. The genetic and molecular mechanisms that enable cells to do this have been extensively studied in several model and non-model organisms. In contrast, the physical mechanisms that shape cells and tissues have only recently started to emerge, in part thanks to new quantitative in vivo measurements of the physical quantities guiding morphogenesis. These data, combined with indirect inferences of physical characteristics, are starting to reveal similarities in the physical mechanisms underlying morphogenesis across different organisms. Here, we review how physics contributes to shape cells and tissues in a simple, yet ubiquitous, morphogenetic transformation: elongation. Drawing from observed similarities across species, we propose the existence of conserved physical mechanisms of morphogenesis.

Mechanics of human embryo compaction.

The shaping of human embryos begins with compaction, during which cells come into close contact1,2. Assisted reproductive technology studies indicate that human embryos fail compaction primarily because of defective adhesion3,4. On the basis of our current understanding of animal morphogenesis5,6, other morphogenetic engines, such as cell contractility, could be involved in shaping human embryos. However, the molecular, cellular and physical mechanisms driving human embryo morphogenesis remain uncharacterized. Using micropipette aspiration on human embryos donated to research, we have mapped cell surface tensions during compaction. This shows a fourfold increase of tension at the cell-medium interface whereas cell-cell contacts keep a steady tension. Therefore, increased tension at the cell-medium interface drives human embryo compaction, which is qualitatively similar to compaction in mouse embryos7. Further comparison between human and mouse shows qualitatively similar but quantitively different mechanical strategies, with human embryos being mechanically least efficient. Inhibition of cell contractility and cell-cell adhesion in human embryos shows that, whereas both cellular processes are required for compaction, only contractility controls the surface tensions responsible for compaction. Cell contractility and cell-cell adhesion exhibit distinct mechanical signatures when faulty. Analysing the mechanical signature of naturally failing embryos, we find evidence that non-compacting or partially compacting embryos containing excluded cells have defective contractility. Together, our study shows that an evolutionarily conserved increase in cell contractility is required to generate the forces driving the first morphogenetic movement shaping the human body.

Coupling and uncoupling of midline morphogenesis and cell flow in amniote gastrulation.

Large-scale cell flow characterizes gastrulation in animal development. In amniote gastrulation, particularly in avian gastrula, a bilateral vortex-like counter-rotating cell flow, called 'polonaise movements', appears along the midline. Here, through experimental manipulations, we addressed relationships between the polonaise movements and morphogenesis of the primitive streak, the earliest midline structure in amniotes. Suppression of the Wnt/planar cell polarity (PCP) signaling pathway maintains the polonaise movements along a deformed primitive streak. Mitotic arrest leads to diminished extension and development of the primitive streak and maintains the early phase of the polonaise movements. Ectopically induced Vg1, an axis-inducing morphogen, generates the polonaise movements, aligned to the induced midline, but disturbs the stereotypical cell flow pattern at the authentic midline. Despite the altered cell flow, induction and extension of the primitive streak are preserved along both authentic and induced midlines. Finally, we show that ectopic axis-inducing morphogen, Vg1, is capable of initiating the polonaise movements without concomitant PS extension under mitotic arrest conditions. These results are consistent with a model wherein primitive streak morphogenesis is required for the maintenance of the polonaise movements, but the polonaise movements are not necessarily responsible for primitive streak morphogenesis. Our data describe a previously undefined relationship between the large-scale cell flow and midline morphogenesis in gastrulation.

Conserved regulatory switches for the transition from natal down to juvenile feather in birds.

The transition from natal downs for heat conservation to juvenile feathers for simple flight is a remarkable environmental adaptation process in avian evolution. However, the underlying epigenetic mechanism for this primary feather transition is mostly unknown. Here we conducted time-ordered gene co-expression network construction, epigenetic analysis, and functional perturbations in developing feather follicles to elucidate four downy-juvenile feather transition events. We report that extracellular matrix reorganization leads to peripheral pulp formation, which mediates epithelial-mesenchymal interactions for branching morphogenesis. α-SMA (ACTA2) compartmentalizes dermal papilla stem cells for feather renewal cycling. LEF1 works as a key hub of Wnt signaling to build rachis and converts radial downy to bilateral symmetry. Novel usage of scale keratins strengthens feather sheath with SOX14 as the epigenetic regulator. We show that this primary feather transition is largely conserved in chicken (precocial) and zebra finch (altricial) and discuss the possibility that this evolutionary adaptation process started in feathered dinosaurs.

egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis.

Biomechanical forces, and their molecular transducers, including key mechanosensitive transcription factor genes, such as KLF2, are required for cardiac valve morphogenesis. However, klf2 mutants fail to completely recapitulate the valveless phenotype observed under no-flow conditions. Here, we identify the transcription factor EGR3 as a conserved biomechanical force transducer critical for cardiac valve formation. We first show that egr3 null zebrafish display a complete and highly penetrant loss of valve leaflets, leading to severe blood regurgitation. Using tissue-specific loss- and gain-of-function tools, we find that during cardiac valve formation, Egr3 functions cell-autonomously in endothelial cells, and identify one of its effectors, the nuclear receptor Nr4a2b. We further find that mechanical forces up-regulate egr3/EGR3 expression in the developing zebrafish heart and in porcine valvular endothelial cells, as well as during human aortic valve remodeling. Altogether, these findings reveal that EGR3 is necessary to transduce the biomechanical cues required for zebrafish cardiac valve morphogenesis, and potentially for pathological aortic valve remodeling in humans.

Reverse engineering morphogenesis through Bayesian optimization of physics-based models.

Morphogenetic programs coordinate cell signaling and mechanical interactions to shape organs. In systems and synthetic biology, a key challenge is determining optimal cellular interactions for predicting organ shape, size, and function. Physics-based models defining the subcellular force distribution facilitate this, but it is challenging to calibrate parameters in these models from data. To solve this inverse problem, we created a Bayesian optimization framework to determine the optimal cellular force distribution such that the predicted organ shapes match the experimentally observed organ shapes. This integrative framework employs Gaussian Process Regression, a non-parametric kernel-based probabilistic machine learning modeling paradigm, to learn the mapping functions relating to the morphogenetic programs that maintain the final organ shape. We calibrated and tested the method on Drosophila wing imaginal discs to study mechanisms that regulate epithelial processes ranging from development to cancer. The parameter estimation framework successfully infers the underlying changes in core parameters needed to match simulation data with imaging data of wing discs perturbed with collagenase. The computational pipeline identifies distinct parameter sets mimicking wild-type shapes. It enables a global sensitivity analysis to support the regulation of actomyosin contractility and basal ECM stiffness to generate and maintain the curved shape of the wing imaginal disc. The optimization framework, combined with experimental imaging, identified that Piezo, a mechanosensitive ion channel, impacts fold formation by regulating the apical-basal balance of actomyosin contractility and elasticity of ECM. This workflow is extensible toward reverse-engineering morphogenesis across organ systems and for real-time control of complex multicellular systems.

ADA2b acts to positively regulate blue light-mediated photomorphogenesis in Arabidopsis.

Cryptochromes (CRYs) act as blue light photoreceptors to regulate various plant physiological processes including photomorphogenesis and repair of DNA double strand breaks (DSBs). ADA2b is a conserved transcription co-activator that is involved in multiple plant developmental processes. It is known that ADA2b interacts with CRYs to mediate blue light-promoted DSBs repair. Whether ADA2b may participate in CRYs-mediated photomorphogenesis is unknown. Here we show that ADA2b acts to inhibit hypocotyl elongation and hypocotyl cell elongation in blue light. We found that the SWIRM domain-containing C-terminus mediates the blue light-dependent interaction of ADA2b with CRYs in blue light. Moreover, ADA2b and CRYs act to co-regulate the expression of hypocotyl elongation-related genes in blue light. Based on previous studies and these results, we propose that ADA2b plays dual functions in blue light-mediated DNA damage repair and photomorphogenesis.

Dynamic microvilli sculpt bristles at nanometric scale.

Organisms generate shapes across size scales. Whereas patterning and morphogenesis of macroscopic tissues has been extensively studied, the principles underlying the formation of micrometric and submicrometric structures remain largely enigmatic. Individual cells of polychaete annelids, so-called chaetoblasts, are associated with the generation of chitinous bristles of highly stereotypic geometry. Here we show that bristle formation requires a chitin-producing enzyme specifically expressed in the chaetoblasts. Chaetoblasts exhibit dynamic cell surfaces with stereotypical patterns of actin-rich microvilli. These microvilli can be matched with internal and external structures of bristles reconstructed from serial block-face electron micrographs. Individual chitin teeth are deposited by microvilli in an extension-disassembly cycle resembling a biological 3D printer. Consistently, pharmacological interference with actin dynamics leads to defects in tooth formation. Our study reveals that both material and shape of bristles are encoded by the same cell, and that microvilli play a role in micro- to submicrometric sculpting of biomaterials.

The embryonic role of juvenile hormone in the firebrat, Thermobia domestica, reveals its function before its involvement in metamorphosis.

To gain insights into how juvenile hormone (JH) came to regulate insect metamorphosis, we studied its function in the ametabolous firebrat, Thermobia domestica. Highest levels of JH occur during late embryogenesis, with only low levels thereafter. Loss-of-function and gain-of-function experiments show that JH acts on embryonic tissues to suppress morphogenesis and cell determination and to promote their terminal differentiation. Similar embryonic actions of JH on hemimetabolous insects with short germ band embryos indicate that JH's embryonic role preceded its derived function as the postembryonic regulator of metamorphosis. The postembryonic expansion of JH function likely followed the evolution of flight. Archaic flying insects were considered to lack metamorphosis because tiny, movable wings were evident on the thoraces of young juveniles and their positive allometric growth eventually allowed them to support flight in late juveniles. Like in Thermobia, we assume that these juveniles lacked JH. However, a postembryonic reappearance of JH during wing morphogenesis in the young juvenile likely redirected wing development to make a wing pad rather than a wing. Maintenance of JH then allowed wing pad growth and its disappearance in the mature juvenile then allowed wing differentiation. Subsequent modification of JH action for hemi- and holometabolous lifestyles are discussed.

Basal actomyosin pulses expand epithelium coordinating cell flattening and tissue elongation.

Actomyosin networks constrict cell area and junctions to alter cell and tissue shape. However, during cell expansion under mechanical stress, actomyosin networks are strengthened and polarized to relax stress. Thus, cells face a conflicting situation between the enhanced actomyosin contractile properties and the expansion behaviour of the cell or tissue. To address this paradoxical situation, we study late Drosophila oogenesis and reveal an unusual epithelial expansion wave behaviour. Mechanistically, Rac1 and Rho1 integrate basal pulsatile actomyosin networks with ruffles and focal adhesions to increase and then stabilize basal area of epithelial cells allowing their flattening and elongation. This epithelial expansion behaviour bridges cell changes to oocyte growth and extension, while oocyte growth in turn deforms the epithelium to drive cell spreading. Basal pulsatile actomyosin networks exhibit non-contractile mechanics, non-linear structures and F-actin/Myosin-II spatiotemporal signal separation, implicating unreported expanding properties. Biophysical modelling incorporating these expanding properties well simulates epithelial cell expansion waves. Our work thus highlights actomyosin expanding properties as a key mechanism driving tissue morphogenesis.