The developmental mechanics of divergent buckling patterns in the chick gut.

Tissue buckling is an increasingly appreciated mode of morphogenesis in the embryo, but it is often unclear how geometric and material parameters are molecularly determined in native developmental contexts to generate diverse functional patterns. Here, we study the link between differential mechanical properties and the morphogenesis of distinct anteroposterior compartments in the intestinal tract-the esophagus, small intestine, and large intestine. These regions originate from a simple, common tube but adopt unique forms. Using measured data from the developing chick gut coupled with a minimal theory and simulations of differential growth, we investigate divergent lumen morphologies along the entire early gut and demonstrate that spatiotemporal geometries, moduli, and growth rates control the segment-specific patterns of mucosal buckling. Primary buckling into wrinkles, folds, and creases along the gut, as well as secondary buckling phenomena, including period-doubling in the foregut and multiscale creasing-wrinkling in the hindgut, are captured and well explained by mechanical models. This study advances our existing knowledge of how identity leads to form in these regions, laying the foundation for future work uncovering the relationship between molecules and mechanics in gut morphological regionalization.

Selection, growth and form. Turing's two biological paths towards intelligent machinery.

We inquire into the role of Turing's biological thought in the development of his concept of intelligent machinery. We trace the possible relations between his proto-connectionist notion of 'organising' machines in Turing (1948) on the one hand and his mathematical theory of morphogenesis in developmental biology (1952) on the other. These works were concerned with distinct fields of inquiry and followed distinct paradigms of biological theory, respectively postulating analogues of Darwinian selection in learning and mathematical laws of form in organic pattern formation. Still, these strands of Turing's work are related, first, in terms of being amenable in principle to his (1936) computational method of modelling. Second, they are connected by Turing's scattered speculations about the possible bearing of learning processes on the anatomy of the brain. We argue that these two theories form an unequal couple that, from different angles and in partial fashion, point towards cognition as a biological and embodied phenomenon while, for reasons inherent to Turing's computational approach to modelling, not being capable of directly addressing it as such. We explore ways in which these two distinct-but-related theories could be more explicitly and systematically connected, using von Neumann's contemporaneous and related work on Cellular Automata and more recent biomimetic approaches as a foil. We conclude that the nature of 'initiative' and the mode of material realisation are the key issues that decide on the possibility of intelligent machinery in Turing.

Lattice light sheet microscopy reveals 4D force propagation dynamics and leading-edge behaviors in an embryonic epithelium in Drosophila.

How pulsed contractile dynamics drive the remodeling of cell and tissue topologies in epithelial sheets has been a key question in development and disease. Due to constraints in imaging and analysis technologies, studies that have described the in vivo mechanisms underlying changes in cell and neighbor relationships have largely been confined to analyses of planar apical regions. Thus, how the volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface in three dimensions, including especially the apical-basal axis, is unclear. Here, we perform lattice light sheet microscopy (LLSM)-based analysis to determine how far and fast forces propagate across different apical-basal layers, as well as where topological changes initiate from in a columnar epithelium. These datasets are highly time- and depth-resolved and reveal that topology-changing forces are spatially entangled, with contractile force generation occurring across the observed apical-basal axis in a pulsed fashion, while the conservation of cell volumes constrains instantaneous cell deformations. Leading layer behaviors occur opportunistically in response to favorable phasic conditions, with lagging layers "zippering" to catch up as new contractile pulses propel further changes in cell topologies. These results argue against specific zones of topological initiation and demonstrate the importance of systematic 4D-based analysis in understanding how forces and deformations in cell dimensions propagate in a three-dimensional environment.

The spliceosome impacts morphogenesis in the human fungal pathogen Candida albicans.

At human body temperature, the fungal pathogen Candida albicans can transition from yeast to filamentous morphologies in response to host-relevant cues. Additionally, elevated temperatures encountered during febrile episodes can independently induce C. albicans filamentation. However, the underlying genetic pathways governing this developmental transition in response to elevated temperatures remain largely unexplored. Here, we conducted a functional genomic screen to unravel the genetic mechanisms orchestrating C. albicans filamentation specifically in response to elevated temperature, implicating 45% of genes associated with the spliceosome or pre-mRNA splicing in this process. Employing RNA-Seq to elucidate the relationship between mRNA splicing and filamentation, we identified greater levels of intron retention in filaments compared to yeast, which correlated with reduced expression of the affected genes. Intriguingly, homozygous deletion of a gene encoding a spliceosome component important for filamentation (PRP19) caused even greater levels of intron retention compared with wild type and displayed globally dysregulated gene expression. This suggests that intron retention is a mechanism for fine-tuning gene expression during filamentation, with perturbations of the spliceosome exacerbating this process and blocking filamentation. Overall, this study unveils a novel biological process governing C. albicans filamentation, providing new insights into the complex regulation of this key virulence trait.IMPORTANCEFungal pathogens such as Candida albicans can cause serious infections with high mortality rates in immunocompromised individuals. When C. albicans is grown at temperatures encountered during human febrile episodes, yeast cells undergo a transition to filamentous cells, and this process is key to its virulence. Here, we expanded our understanding of how C. albicans undergoes filamentation in response to elevated temperature and identified many genes involved in mRNA splicing that positively regulate filamentation. Through transcriptome analyses, we found that intron retention is a mechanism for fine-tuning gene expression in filaments, and perturbation of the spliceosome exacerbates intron retention and alters gene expression substantially, causing a block in filamentation. This work adds to the growing body of knowledge on the role of introns in fungi and provides new insights into the cellular processes that regulate a key virulence trait in C. albicans.

Soft Polyethylene Glycol Hydrogels Support Human PSC Pluripotency and Morphogenesis.

Lumenogenesis within the epiblast represents a critical step in early human development, priming the embryo for future specification and patterning events. However, little is known about the specific mechanisms that drive this process due to the inability to study the early embryo in vivo. While human pluripotent stem cell (hPSC)-based models recapitulate many aspects of the human epiblast, most approaches for generating these 3D structures rely on ill-defined, reconstituted basement membrane matrices. Here, we designed synthetic, nonadhesive polyethylene glycol (PEG) hydrogel matrices to better understand the role of matrix mechanical cues in iPSC morphogenesis, specifically elastic modulus. First, we identified a narrow range of hydrogel moduli that were conducive to the hPSC viability, pluripotency, and differentiation. We then used this platform to investigate the effects of the hydrogel modulus on lumenogenesis, finding that matrices of intermediate stiffness yielded the most epiblast-like aggregates. Conversely, stiffer matrices impeded lumen formation and apico-basal polarization, while the softest matrices yielded polarized but aberrant structures. Our approach offers a simple, modular platform for modeling the human epiblast and investigating the role of matrix cues in its morphogenesis.

Modeling the mechanosensitive collective migration of cells on the surface and the interior of morphing soft tissues.

Cellular contractility, migration, and extracellular matrix (ECM) mechanics are critical for a wide range of biological processes including embryonic development, wound healing, tissue morphogenesis, and regeneration. Even though the distinct response of cells near the tissue periphery has been previously observed in cell-laden microtissues, including faster kinetics and more prominent cell-ECM interactions, there are currently no models that can fully combine coupled surface and bulk mechanics and kinetics to recapitulate the morphogenic response of these constructs. Mailand et al. (Biophys J 117(5):975-986, 2019) had shown the importance of active elastocapillarity in cell-laden microtissues, but modeling the distinct mechanosensitive migration of cells on the periphery and the interior of highly deforming tissues has not been possible thus far, especially in the presence of active elastocapillary effects. This paper presents a framework for understanding the interplay between cellular contractility, migration, and ECM mechanics in dynamically morphing soft tissues accounting for distinct cellular responses in the bulk and the surface of tissues. The major novelty of this approach is that it enables modeling the distinct migratory and contractile response of cells residing on the tissue surface and the bulk, where concurrently the morphing soft tissues undergo large deformations driven by cell contractility. Additionally, the simulation results capture the changes in shape and cell concentration for wounded and intact microtissues, enabling the interpretation of experimental data. The numerical procedure that accounts for mechanosensitive stress generation, large deformations, diffusive migration in the bulk and a distinct mechanism for diffusive migration on deforming surfaces is inspired from recent work on bulk and surface poroelasticity of hydrogels involving elastocapillary effects, but in this work, a two-field weak form is proposed and is able to alleviate numerical instabilities that were observed in the original method that utilized a three-field mixed finite element formulation.

The Differential Developmental Neurotoxicity of Valproic Acid on Anterior and Posterior Neural Induction of Human Pluripotent Stem Cells.

Valproic acid (VPA), widely used as an antiepileptic drug, exhibits developmental neurotoxicity when exposure occurs during early or late pregnancy, resulting in various conditions ranging from neural tube defects to autism spectrum disorders. However, toxicity during the very early stages of neural development has not been addressed. Therefore, we investigated the effects of VPA in a model where human pluripotent stem cells differentiate into anterior or posterior neural tissues. Exposure to VPA during the induction of neural stem cells induced different developmental toxic effects in a dose-dependent manner. For instance, VPA induced cell death more profoundly during anteriorly guided neural progenitor induction, while inhibition of cell proliferation and enhanced differentiation were observed during posteriorly guided neural induction. Furthermore, acute exposure to VPA during the posterior induction step also retarded the subsequent neurulation-like tube morphogenesis process in neural organoid culture. These results suggest that VPA exposure during very early embryonic development might exhibit cytotoxicity and subsequently disrupt neural differentiation and morphogenesis processes.

Molecular Regulation of Cardiac Conduction System Development.

PURPOSE OF REVIEW: The cardiac conduction system, composed of pacemaker cells and conducting cardiomyocytes, orchestrates the propagation of electrical activity to synchronize heartbeats. The conduction system plays a crucial role in the development of cardiac arrhythmias. In the embryo, the cells of the conduction system derive from the same cardiac progenitors as the contractile cardiomyocytes and and the key question is how this choice is made during development. RECENT FINDINGS: This review focuses on recent advances in developmental biology using the mouse as animal model to better understand the cellular origin and molecular regulations that control morphogenesis of the cardiac conduction system, including the latest findings in single-cell transcriptomics. The conducting cell fate is acquired during development starting with pacemaking activity and last with the formation of a complex fast-conducting network. Cardiac conduction system morphogenesis is controlled by complex transcriptional and gene regulatory networks that differ in the components of the cardiac conduction system.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Passiflora spp. (Passifloraceae).

In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20 years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

Keratan sulfate proteoglycan: putative template for neuroblast migratory and axonal fascicular pathways and fetal expression in globus pallidus, thalamus, and olfactory bulb.

Keratan sulfate (KS) is a proteoglycan secreted in the fetal brain astrocytes and radial glia into extracellular parenchyma as granulofilamentous deposits. KS surrounds neurons except dendritic spines, repelling glutamatergic and facilitating GABAergic axons. The same genes are expressed in both neuroblast migration and axonal growth. This study examines timing of KS during morphogenesis of some normally developing human fetal forebrain structures. Twenty normal human fetal brains from 9-41 weeks gestational age were studied at autopsy. KS was examined by immunoreactivity in formalin-fixed paraffin sections, plus other markers including synaptophysin, S-100ß protein, vimentin and nestin. Radial and tangential neuroblast migratory pathways from subventricular zone to cortical plate were marked by KS deposits as early as 9wk GA, shortly after neuroblast migration initiated. During later gestation this reactivity gradually diminished and disappeared by term. Long axonal fascicles of the internal capsule and short fascicles of intrinsic bundles of globus pallidus and corpus striatum also appeared as early as 9-12wk, as fascicular sleeves before axons even entered. Intense KS occurs in astrocytic cytoplasm and extracellular parenchyma at 9wk in globus pallidus, 15wk thalamus, 18wk corpus striatum, 22wk cortical plate, and hippocampus postnatally. Corpus callosum and anterior commissure do not exhibit KS at any age. Optic chiasm shows reactivity at the periphery but not around intrinsic subfasciculi. We postulate that KS forms a chemical template for many long and short axonal fascicles before axons enter and neuroblast migratory pathways at initiation of migration. Cross-immunoreactivity with aggrecan may render difficult molecular distinction.

Hierarchical global and local auxin signals coordinate cellular interdigitation in Arabidopsis.

The development of multicellular tissues requires both local and global coordination of cell polarization, however, the mechanisms underlying their interplay are poorly understood. In Arabidopsis, leaf epidermal pavement cells (PC) develop a puzzle-piece shape locally coordinated through apoplastic auxin signaling. Here we show auxin also globally coordinates interdigitation by activating the TIR1/AFB-dependent nuclear signaling pathway. This pathway promotes a transient maximum of auxin at the cotyledon tip, which then moves across the leaf activating local PC polarization, as demonstrated by locally uncaged auxin globally rescuing defects in tir1;afb1;afb2;afb4;afb5 mutant but not in tmk1;tmk2;tmk3;tmk4 mutants. Our findings show that hierarchically integrated global and local auxin signaling systems, which respectively depend on TIR1/AFB-dependent gene transcription in the nucleus and TMK-mediated rapid activation of ROP GTPases at the cell surface, control PC interdigitation patterns in Arabidopsis cotyledons, revealing a mechanism for coordinating a local cellular process with the development of whole tissues.

Biomechanical properties of the capsule and extracellular matrix play a major role during the Wolffian/epididymal duct development.

BACKGROUND: The epididymis is important for sperm maturation and without its proper development, male infertility will result. Biomechanical properties of tissues/organs play key roles during their morphogenesis, including the Wolffian duct. It is hypothesized that structural/bulk stiffness of the capsule and mesenchyme/extracellular matrix that surround the duct is a major biomechanical property that regulates Wolffian duct morphogenesis. These data will provide key information as to the mechanisms that regulate the development of this important organ. OBJECTIVES: To measure the structural/bulk stiffness in Pascals (force/area) of the capsule and the capsule and mesenchyme together that surrounds the Wolffian duct during the development. To examine the relative membrane tension of mesenchymal cells during the Wolffian duct development. Since Ptk7 was previously shown to regulate ECM integrity and Wolffian duct elongation and coiling, the hypothesis that Ptk7 regulates structural/bulk stiffness and mesenchymal cell membrane tension was tested. MATERIALS AND METHODS: Atomic force microscopy and a microsquisher compression apparatus were used to measure the structural stiffness. Biomechanical properties within the membranes of cells within the capsule and mesenchyme were examined using a membrane-tension fluorescent probe. RESULTS AND DISCUSSION: The structural stiffness (Pascals) of the capsule and underlying mesenchyme was relatively constant during development, with a significant increase in the capsule at the later stages. However, this increase may reflect the ECM and associated mesenchyme being close to the capsule because the coiling of the duct pushed or compressed them into that space. Keeping the capsule and mesenchyme/ECM at constant stiffness would ensure that the duct will continue to coil under similar biomechanical forces throughout the development. Cells within the capsule and mesenchyme at different Wolffian duct regions during the development had varying degrees of membrane lipid tension. It is hypothesized that the dynamic changes ensure the duct is kept at a constant stiffness regardless of any external forces. Loss of Ptk7 resulted in an increase in stiffness at E18.5, which was presumable due to the loss of integrity of the ECM within the mesenchyme. CONCLUSION: Biomechanical properties of the capsule and the mesenchyme/extracellular matrix that surround the Wolffian duct play an important role toward Wolffian duct morphogenesis, thereby allowing for the proper development of the epididymis and subsequent male fertility.

ß-H-Spectrin is a key component of an apical-medial hub of proteins during cell wedging in tube morphogenesis.

Coordinated cell shape changes are a major driver of tissue morphogenesis, with apical constriction of epithelial cells leading to tissue bending. During the tube budding of the salivary glands in the Drosophila embryo we previously identified a key interplay between the apical-medial actomyosin, driving apical constriction, with the underlying longitudinal microtubule array. At this microtubule-actomyosin-interface a hub of proteins accumulates: as shown before, the microtubule-actin-crosslinker Shot and the minus-end-binder Patronin, and now identified two actin-crosslinkers, ß-H-Spectrin and Filamin, and the multi-PDZ-protein Big-bang. We show that tissue-specific-degradation of ß-H-Spectrin led to reduction of apical-medial F-actin, Shot, Patronin and Big-bang and concomitant defects in apical constriction, but residual Patronin was still sufficient to assist microtubule reorganisation. Contrary to Patronin and Shot, neither ß-H-Spectrin nor Big bang required microtubules for their localisation. ß-H-Spectrin was instead recruited via binding to apical-medial phosphoinositides. Overexpression of ß-H-33 containing the PH domain displaced endogenous ß-H-Spectrin and led to strong morphogenetic defects. This protein hub therefore required the synergy and coincidence of membrane- and microtubule-associated components for its assembly and function in sustaining the apical constriction during tubulogenesis.

Increased Netrin downstream of overactive Hedgehog signaling disrupts optic fissure formation.

Background: Uveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye and vasculature enters. The Hedgehog (Hh) signaling pathway regulates optic fissure development: loss-of-function mutations in the Hh receptor ptch2 produce overactive Hh signaling and can result in coloboma. We previously proposed a model where overactive Hh signaling disrupts optic fissure formation by upregulating transcriptional targets acting both cell- and non-cell-autonomously. Here, we examine the Netrin family of secreted ligands as candidate Hh target genes. Results: We find multiple Netrin ligands upregulated in the zebrafish ptch2 mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find that netrin1a or netrin1b overexpression is sufficient to cause coloboma and disrupt wild-type optic fissure formation. We used loss-of-function alleles, CRISPR/Cas9 mutagenesis, and morpholino knockdown to test if loss of Netrin can rescue coloboma in the ptch2 mutant: loss of netrin genes does not rescue the ptch2 mutant phenotype. Conclusion: These results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in the ptch2 mutant.

Contrasting philosophical and scientific views in the long history of studying the generation of form in development.

Focusing on the opposing ways of thinking of philosophers and scientists to explain the generation of form in biological development, I show that today's controversies over explanations of early development bear fundamental similarities to the dichotomy of preformation theory versus epigenesis in Greek antiquity. They are related to the acceptance or rejection of the idea of a physical form of what today would be called information for the generating of the embryo as a necessary pre-requisite for specific development and heredity. As a recent example, I scrutinize the dichotomy of genomic causality versus self-organization in 20th and 21st century theories of the generation of form. On the one hand, the generation of patterns and form, as well as the constant outcome in development, are proposed to be causally related to something that is "preformed" in the germ cells, the nucleus of germ cells, or the genome. On the other hand, it is proposed that there is no pre-existing form or information, and development is seen as a process where genuinely new characters emerge from formless matter, either by immaterial "forces of life," or by physical-chemical processes of self-organization. I also argue that these different ways of thinking and the research practices associated with them are not equivalent, and maintain that it is impossible to explain the generation of form and constant outcome of development without the assumption of the transmission of pre-existing information in the form of DNA sequences in the genome. Only in this framework of "preformed" information can "epigenesis" in the form of physical and chemical processes of self-organization play an important role.

The small GTPase Cdc42 regulates shell field morphogenesis in a gastropod mollusk.

In most mollusks (conchiferans), the early tissue responsible for shell development, namely, the shell field, shows a common process of invagination during morphogenesis. Moreover, lines of evidence indicated that shell field invagination is not an independent event, but an integrated output reflecting the overall state of shell field morphogenesis. Nevertheless, the underlying mechanisms of this conserved process remain largely unknown. We previously found that actomyosin networks (regularly organized filamentous actin (F-actin) and myosin) may play essential roles in this process by revealing the evident aggregation of F-actin in the invaginated region and demonstrating that nonmuscle myosin II (NM II) is required for invagination in the gastropod Lottia peitaihoensis (= Lottia goshimai). Here, we investigated the roles of the Rho family of small GTPases (RhoA, Rac1, and Cdc42) to explore the upstream regulators of actomyosin networks. Functional assays using small molecule inhibitors suggested that Cdc42 modulates key events of shell field morphogenesis, including invagination and cell rearrangements, while the roles of RhoA and Rac1 may be nonspecific or negligible. Further investigations revealed that the Cdc42 protein was concentrated on the apical side of shell field cells and colocalized with F-actin aggregation. The aggregation of these two molecules could be prevented by treatment with Cdc42 inhibitors. These findings suggest a possible regulatory cascade of shell field morphogenesis in which Cdc42 recruits F-actin (actomyosin networks) on the apical side of shell field cells, which then generates resultant mechanical forces that mediate correct shell field morphogenesis (cell shape changes, invagination and cell rearrangement). Our results emphasize the roles of the cytoskeleton in early shell development and provide new insights into molluscan shell evolution.

AdamTS proteases control basement membrane heterogeneity and organ shape in Drosophila.

The basement membrane (BM) is an extracellular matrix that plays important roles in animal development. A spatial heterogeneity in composition and structural properties of the BM provide cells with vital cues for morphogenetic processes such as cell migration or cell polarization. Here, using the Drosophila egg chamber as a model system, we show that the BM becomes heterogeneous during development, with a reduction in Collagen IV density at the posterior pole and differences in the micropattern of aligned fiber-like structures. We identified two AdamTS matrix proteases required for the proper elongated shape of the egg chamber, yet the molecular mechanisms by which they act are different. Stall is required to establish BM heterogeneity by locally limiting Collagen IV protein density, whereas AdamTS-A alters the micropattern of fiber-like structures within the BM at the posterior pole. Our results suggest that AdamTS proteases control BM heterogeneity required for organ shape.

Perspectives in collective cell migration - moving forward.

Collective cell migration, where cells move as a cohesive unit, is a vital process underlying morphogenesis and cancer metastasis. Thanks to recent advances in imaging and modelling, we are beginning to understand the intricate relationship between a cell and its microenvironment and how this shapes cell polarity, metabolism and modes of migration. The use of biophysical and mathematical models offers a fresh perspective on how cells migrate collectively, either flowing in a fluid-like state or transitioning to more static states. Continuing to unite researchers in biology, physics and mathematics will enable us to decode more complex biological behaviours that underly collective cell migration; only then can we understand how this coordinated movement of cells influences the formation and organisation of tissues and directs the spread of metastatic cancer. In this Perspective, we highlight exciting discoveries, emerging themes and common challenges that have arisen in recent years, and possible ways forward to bridge the gaps in our current understanding of collective cell migration.

Self-Improvising Memory: A Perspective on Memories as Agential, Dynamically Reinterpreting Cognitive Glue.

Many studies on memory emphasize the material substrate and mechanisms by which data can be stored and reliably read out. Here, I focus on complementary aspects: the need for agents to dynamically reinterpret and modify memories to suit their ever-changing selves and environment. Using examples from developmental biology, evolution, and synthetic bioengineering, in addition to neuroscience, I propose that a perspective on memory as preserving salience, not fidelity, is applicable to many phenomena on scales from cells to societies. Continuous commitment to creative, adaptive confabulation, from the molecular to the behavioral levels, is the answer to the persistence paradox as it applies to individuals and whole lineages. I also speculate that a substrate-independent, processual view of life and mind suggests that memories, as patterns in the excitable medium of cognitive systems, could be seen as active agents in the sense-making process. I explore a view of life as a diverse set of embodied perspectives-nested agents who interpret each other's and their own past messages and actions as best as they can (polycomputation). This synthesis suggests unifying symmetries across scales and disciplines, which is of relevance to research programs in Diverse Intelligence and the engineering of novel embodied minds.