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Abstract Background Considerable progress has been made in our understanding of sex determination and dosage compensation mechanisms in model organisms such as C. elegans, Drosophila and M. musculus. Strikingly, the mechanism involved in sex determination and dosage compensation are very different among these three model organisms. Birds present yet another situation where the heterogametic sex is the female. Sex determination is still poorly understood in birds and few key determinants have so far been identified. In contrast to most other species, dosage compensation of bird sex chromosomal genes appears rather ineffective. Results By comparing microarrays from microdissected primitive streak from single chicken embryos, we identified a large number of genes differentially expressed between male and female embryos at a very early stage (Hamburger and Hamilton stage 4), long before any sexual differentiation occurs. Most of these genes are located on the Z chromosome, which indicates that dosage compensation is ineffective in early chicken embryos. Gene ontology analyses, using an enhanced annotation tool for Affymetrix probesets of the chicken genome developed in our laboratory (called Manteia), show that among these male-biased genes found on the Z chromosome, more than 20 genes play a role in sex differentiation. Conclusions These results corroborate previous studies demonstrating the rather inefficient dosage compensation for Z chromosome in birds and show that this sexual dimorphism in gene regulation is observed long before the onset of sexual differentiation. These data also suggest a potential role of non-compensated Z-linked genes in somatic sex differentiation in birds.

The vertebrate bauplan is primarily established via the formation of embryonic tissues in a head-to-tail progression. The mechanics of this elongation, which requires the presomitic mesoderm (PSM), remain poorly understood. Here, we find that avian PSM explants can elongate autonomously when physically confined in vitro, producing a pushing force promoting posterior elongation of the embryo. This tissue elongation is caused by volumetric expansion, which results from an increase in the extracellular fraction accompanied by graded cellular motility. We show that fibroblast growth factor (FGF) signaling promotes glycolysis-dependent production of hyaluronic acid (HA), which is required for expansion of the posterior PSM. Our findings link body axis elongation to tissue expansion through the metabolic control of extracellular matrix production downstream of FGF signaling., Competing Interests: Declaration of interests A.R.H. is listed as inventor on a patent describing nanopore analysis of HA., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Left-right asymmetry of the human brain is widespread through its anatomy and function. However, limited microscopic understanding of it exists, particularly for anatomical asymmetry where there are few well-established animal models. In humans, most brain regions show subtle, population-average regional asymmetries in thickness or surface area, alongside a macro-scale twisting called the cerebral petalia in which the right hemisphere protrudes past the left. Here, we ask whether neuroanatomical asymmetries can be observed in mice, leveraging 6 mouse neuroimaging cohorts from 5 different research groups (∼3,500 animals). We found an anterior-posterior pattern of volume asymmetry with anterior regions larger on the right and posterior regions larger on the left. This pattern appears driven by similar trends in surface area and positional asymmetries, with the results together indicating a small brain-wide twisting pattern, similar to the human cerebral petalia. Furthermore, the results show no apparent relationship to known functional asymmetries in mice, emphasizing the complexity of the structure-function relationship in brain asymmetry. Our results recapitulate and extend previous patterns of asymmetry from two published studies as well as capture well-established, bilateral male-female differences in the mouse brain as a positive control. By establishing a signature of anatomical brain asymmetry in mice, we aim to provide a foundation for future studies to probe the mechanistic underpinnings of brain asymmetry seen in humans - a feature of the brain with extremely limited understanding., Competing Interests: Declaration of competing interest These authors declare no competing interests., (Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.)

Engineering biomimetic tissue implants with human induced pluripotent stem cells (hiPSCs) holds promise for repairing volumetric tissue loss. However, these implants face challenges in regenerative capability, survival, and geometric scalability at large-scale injury sites. Here, we present scalable vessel-integrated muscle-like lattices (VMLs), containing dense and aligned hiPSC-derived myofibers alongside passively perfusable vessel-like microchannels inside an endomysium-like supporting matrix using an embedded multimaterial bioprinting technology. The contractile and millimeter-long myofibers are created in mechanically tailored and nanofibrous extracellular matrix-based hydrogels. Incorporating vessel-like lattice enhances myofiber maturation in vitro and guides host vessel invasion in vivo, improving implant integration. Consequently, we demonstrate successful de novo muscle formation and muscle function restoration through a combinatorial effect between improved graft-host integration and its increased release of paracrine factors within volumetric muscle loss injury models. The proposed modular bioprinting technology enables scaling up to centimeter-sized prevascularized hiPSC-derived muscle tissues with custom geometries for next-generation muscle regenerative therapies., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated biochemical and biomechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Herein, we develop a human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on the PSM tissues cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and the PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the biochemical and biomechanical events that guide somite formation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Segmentation is a fundamental feature of the vertebrate body plan. This metameric organization is first implemented by somitogenesis in the early embryo, when paired epithelial blocks called somites are rhythmically formed to flank the neural tube. Recent advances in in vitro models have offered new opportunities to elucidate the mechanisms that underlie somitogenesis. Notably, models derived from human pluripotent stem cells introduced an efficient proxy for studying this process during human development. In this Review, we summarize the current understanding of somitogenesis gained from both in vivo studies and in vitro studies. We deconstruct the spatiotemporal dynamics of somitogenesis into four distinct modules: dynamic events in the presomitic mesoderm, segmental determination, somite anteroposterior polarity patterning, and epithelial morphogenesis. We first focus on the segmentation clock, as well as signalling and metabolic gradients along the tissue, before discussing the clock and wavefront and other models that account for segmental determination. We then detail the molecular and cellular mechanisms of anteroposterior polarity patterning and somite epithelialization., (© 2024. Springer Nature Limited.)

In amniotes, embryonic tissues originate from multipotent epiblast cells, arranged in a mosaic of presumptive territories. How these domains fated to specific lineages become segregated during body formation remains poorly understood. Using single cell RNA sequencing analysis and lineage tracing in the chicken embryo, we identify epiblast cells contributing descendants to the neural tube, somites and lateral plate after completion of gastrulation. We show that intercalation after cell division generates important movements of epiblast cells which lead to their relocation to different presumptive territories, explaining this broad spectrum of fates. This tissue remodeling phase is transient, being soon followed by the establishment of boundaries restricting cell movements therefore defining the presumptive territories of the epiblast. Finally, we find that the epiblast faces distinct mechanical constraints along the antero-posterior axis, leading to cell fate alterations when challenged. Together, we demonstrate the critical role of mechanical cues in epiblast fate determination.

In this issue of Developmental Cell, Bolondi et al. systematically assesses neuro-mesodermal progenitor (NMP) dynamics by combining a mouse stem-cell-based embryo model with molecular recording of single cells, shedding light on the dynamics of neural tube and paraxial mesoderm formation during mammalian development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Skeletal muscle connective tissue (MCT) surrounds myofiber bundles to provide structural support, produce force transduction from tendons, and regulate satellite cell differentiation during muscle regeneration. Engineered muscle tissue composed of myofibers layered within MCT has not yet been developed. Herein, a bioengineering strategy to create MCT-layered myofibers through the development of stem cell fate-controlling biomaterials that achieve both myogenesis and fibroblast differentiation in a locally controlled manner at the single construct is introduced. The reciprocal role of transforming growth factor-beta 1 (TGF- β 1) and its inhibitor as well as 3D matrix stiffness to achieve co-differentiation of MCT fibroblasts and myofibers from a human-induced pluripotent stem cell (hiPSC)-derived paraxial mesoderm is studied. To avoid myogenic inhibition, TGF- β 1 is conjugated on the gelatin-based hydrogel to control the fibroblasts' populations locally; the TGF- β 1 degrades after 2 weeks, resulting in increased MCT-specific extracellular matrix (ECM) production. The locations of myofibers and fibroblasts are precisely controlled by using photolithography and co-axial wet spinning techniques, which results in the formation of MCT-layered functional myofibers in 3D constructs. This advanced engineering strategy is envisioned as a possible method for obtaining biomimetic human muscle grafts for various biomedical applications., Competing Interests: Conflict of Interest The authors declare no conflict of interest.

Paraxial mesoderm in the early embryo is segmented into epithelial blocks called somites that establish the metameric organization of the vertebrate body plan. Somites are sequentially formed from head to tail in a rhythmic manner controlled by an oscillating gene regulatory network known as the segmentation clock. We know very little about this important process during human development due to limited access to human embryos and ethical concerns. To bypass these difficulties, model systems derived from human pluripotent stem cells have been established. Here, we detail three protocols modeling different aspects of human paraxial mesoderm development in vitro: a 2D cell monolayer system recapitulating dynamics of the human segmentation clock, a 3D organoid system called "somitoid" supporting the simultaneous formation of somite-like structures, and another organoid system called "segmentoid" reconstituting in vivo-like hallmarks of somitogenesis. Together, these complementary model systems provide an excellent platform to decode somitogenesis and advance human developmental biology., (© 2023. Springer Science+Business Media, LLC.)

Organismal development requires the reproducible unfolding of an ordered sequence of discrete steps (cell fate determination, migration, tissue folding, etc.) in both time and space. Here, we review the mechanisms that grant temporal specificity to developmental steps, including molecular clocks and timers. Individual timing mechanisms must be coordinated with each other to maintain the overall developmental sequence. However, phenotypic novelties can also arise through the modification of temporal patterns over the course of evolution. Two main types of variation in temporal patterning characterize interspecies differences in developmental time: allochrony, where the overall developmental sequence is either accelerated or slowed down while maintaining the relative duration of individual steps, and heterochrony, where the duration of specific developmental steps is altered relative to the rest. New advances in in vitro modeling of mammalian development using stem cells have recently enabled the revival of mechanistic studies of allochrony and heterochrony. In both cases, differences in the rate of basic cellular functions such as splicing, translation, protein degradation, and metabolism seem to underlie differences in developmental time. In the coming years, these studies should identify the genetic differences that drive divergence in developmental time between species.

Brown adipocytes (BAs) represent a specialized cell type that is able to uncouple nutrient catabolism from ATP generation to dissipate energy as heat. In humans, the brown fat tissue is composed of discrete depots found throughout the neck and trunk region. BAs originate from a precursor common to skeletal muscle, but their developmental trajectory remains poorly understood. Here, we used single-cell RNA sequencing to characterize the development of interscapular brown fat in mice. Our analysis identified a transient stage of BA differentiation characterized by the expression of the transcription factor GATA6. We show that recapitulating the sequence of signaling cues identified in mice can lead to efficient differentiation of BAs in vitro from human pluripotent stem cells. These precursors can in turn be efficiently converted into functional BAs that can respond to signals mimicking adrenergic stimuli by increasing their metabolism, resulting in heat production., Competing Interests: Declaration of interests J.R. and O.P. have filed a patent related to this work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated bio-chemical and -mechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Here we report a new human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on PSM cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the bio-chemical and -mechanical events that guide somite formation.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is a powerful analytical technique that provides spatially preserved detection and quantification of analytes in tissue specimens. However, clinical translation still requires improved throughput, precision, and accuracy. To accomplish this, we created "Chemical QuantArray", a gelatin tissue microarray (TMA) mold filled with serial dilutions of isotopically labeled endogenous metabolite standards. The mold is then cryo-sectioned onto a tissue homogenate to produce calibration curves. To improve precision and accuracy, we automatically remove pixels outside of each TMA well and investigated several intensity normalizations, including the utilization of a second stable isotope internal standard (IS). Chemical QuantArray enables the quantification of several endogenous metabolites over a wide dynamic range and significantly improve over current approaches. The technique reduces the space needed on the MALDI slides for calibration standards by approximately 80%. Furthermore, removal of empty pixels and normalization to an internal standard or matrix peak provided precision (<20% RSD) and accuracy (<20% DEV). Finally, we demonstrate the applicability of Chemical QuantArray by quantifying multiple purine metabolites in 14 clinical tumor specimens using a single MALDI slide. Chemical QuantArray improves the analytical characteristics and practical feasibility of MALDI-MSI metabolite quantification in clinical and translational applications.

Developmental morphogenesis is driven by tissue stresses acting on tissue rheology. Direct measurements of forces in small tissues (100 µm-1 mm) in situ, such as in early embryos, require high spatial precision and minimal invasiveness. Here, we introduce a control-based approach, tissue force microscopy (TiFM), that integrates a mechanical cantilever probe and live imaging with closed-loop feedback control of mechanical loading in early chicken embryos. By testing previously qualitatively characterized force-producing tissues in the elongating body axis, we show that TiFM quantitatively captures stress dynamics with high sensitivity. TiFM also provides the means to apply stable, minimally invasive and physiologically relevant loads to drive tissue deformation and to follow the resulting morphogenetic progression associated with large-scale cell movements. Together, TiFM allows us to control tissue force measurement and manipulation in small developing embryos, and promises to contribute to the quantitative understanding of complex multi-tissue mechanics during development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)