Functional analysis of a first hindlimb positioning enhancer via Gdf11 expression.

During the early development of tetrapods, including humans, the embryonic body elongates caudally once the anterior-posterior axis is established. During this process, region-specific vertebral morphogenesis occurs, with the determination of limb positioning along the anterior-posterior axis. We previously reported that Gdf11 functions as an anatomical integration system that determines the positioning of hindlimbs and sacral vertebrae where Gdf11 is expressed. However, the molecular mechanisms underlying induction of Gdf11 expression remain unclear. In this study, we searched for non-coding regions near the Gdf11 locus that were conserved across species to elucidate the regulatory mechanisms of Gdf11 expression. We identified an enhancer of the Gdf11 gene in intron 1 and named it highly conserved region (HCR). In HCR knockout mice, the expression level of endogenous Gdf11 was decreased, and the position of the sacral-hindlimb unit was shifted posteriorly. We also searched for factors upstream of Gdf11 based on the predicted transcription factor binding sites within the HCR. We found that inhibition of FGF signaling increased endogenous Gdf11 expression, suggesting that FGF signaling negatively regulates Gdf11 expression. However, FGF signaling does not regulate HCR activity. Our results suggest that there are species-specific Gdf11 enhancers other than HCR and that FGF signaling regulates Gdf11 expression independent of HCR.

Collective effects of cell cleavage dynamics.

A conserved process of early embryonic development in metazoans is the reductive cell divisions following oocyte fertilization, termed cell cleavages. Cell cleavage cycles usually start synchronously, lengthen differentially between the embryonic cells becoming asynchronous, and cease before major morphogenetic events, such as germ layer formation and gastrulation. Despite exhibiting species-specific characteristics, the regulation of cell cleavage dynamics comes down to common controllers acting mostly at the single cell/nucleus level, such as nucleus-to-cytoplasmic ratio and zygotic genome activation. Remarkably, recent work has linked cell cleavage dynamics to the emergence of collective behavior during embryogenesis, including pattern formation and changes in embryo-scale mechanics, raising the question how single-cell controllers coordinate embryo-scale processes. In this review, we summarize studies across species where an association between cell cleavages and collective behavior was made, discuss the underlying mechanisms, and propose that cell-to-cell variability in cell cleavage dynamics can serve as a mechanism of long-range coordination in developing embryos.

Molecular control of cellulosic fin morphogenesis in ascidians.

BACKGROUND: The tunicates form a group of filter-feeding marine animals closely related to vertebrates. They share with them a number of features such as a notochord and a dorsal neural tube in the tadpole larvae of ascidians, one of the three groups that make tunicates. However, a number of typical chordate characters have been lost in different branches of tunicates, a diverse and fast-evolving phylum. Consequently, the tunic, a sort of exoskeleton made of extracellular material including cellulose secreted by the epidermis, is the unifying character defining the tunicate phylum. In the larva of ascidians, the tunic differentiates in the tail into a median fin (with dorsal and ventral extended blades) and a caudal fin. RESULTS: Here we have performed experiments in the ascidian Phallusia mammillata to address the molecular control of tunic 3D morphogenesis. We have demonstrated that the tail epidermis medio-lateral patterning essential for peripheral nervous system specification also controls tunic elongation into fins. More specifically, when tail epidermis midline identity was abolished by BMP signaling inhibition, or CRISPR/Cas9 inactivation of the transcription factor coding genes Msx or Klf1/2/4/17, median fin did not form. We postulated that this genetic program should regulate effectors of tunic secretion. We thus analyzed the expression and regulation in different ascidian species of two genes acquired by horizontal gene transfer (HGT) from bacteria, CesA coding for a cellulose synthase and Gh6 coding for a cellulase. We have uncovered an unexpected dynamic history of these genes in tunicates and high levels of variability in gene expression and regulation among ascidians. Although, in Phallusia, Gh6 has a regionalized expression in the epidermis compatible with an involvement in fin elongation, our functional studies indicate a minor function during caudal fin formation only. CONCLUSIONS: Our study constitutes an important step in the study of the integration of HGT-acquired genes into developmental networks and a cellulose-based morphogenesis of extracellular material in animals.

Reconstructing axial progenitor field dynamics in mouse stem cell-derived embryoids.

Embryogenesis requires substantial coordination to translate genetic programs to the collective behavior of differentiating cells, but understanding how cellular decisions control tissue morphology remains conceptually and technically challenging. Here, we combine continuous Cas9-based molecular recording with a mouse embryonic stem cell-based model of the embryonic trunk to build single-cell phylogenies that describe the behavior of transient, multipotent neuro-mesodermal progenitors (NMPs) as they commit into neural and somitic cell types. We find that NMPs show subtle transcriptional signatures related to their recent differentiation and contribute to downstream lineages through a surprisingly broad distribution of individual fate outcomes. Although decision-making can be heavily influenced by environmental cues to induce morphological phenotypes, axial progenitors intrinsically mature over developmental time to favor the neural lineage. Using these data, we present an experimental and analytical framework for exploring the non-homeostatic dynamics of transient progenitor populations as they shape complex tissues during critical developmental windows.

Basal delamination during mouse gastrulation primes pluripotent cells for differentiation.

The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak, and their priming toward primitive streak fates. How these different events are coordinated remains unknown. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that captures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this system in combination with microfabrication and in vivo experiments, we found that proliferation-induced crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that the right cell types become specified at the right place during embryonic development.

ASH2L mediates epidermal differentiation and hair follicle morphogenesis via H3K4me3 modification.

The processes of epidermal development in mammals are regulated by complex molecular mechanisms, such as histone modifications. Histone H3 lysine K4 (H3K4) methylation mediated by COMPASS methyltransferase is associated with gene activation, but its effect on epidermal lineage development remains unclear. Therefore, we constructed a mouse model of specific ASH2L (COMPASS methyltransferase core subunit) deletion in epidermal progenitor cells and investigated its effect on the development of mouse epidermal lineage. Furthermore, downstream target genes regulated by H3K4me3 were screened using RNA-sequencing combined with Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing. Deletion of ASH2L in epidermal progenitor cells caused thinning of the suprabasal layer of the epidermis and delayed hair follicle morphogenesis in newborn mice. These phenotypes may be related to the reduced proliferative capacity of epidermal and hair follicle progenitor cells. ASH2L depletion may also lead to depletion of the epidermal stem cell pools in late mouse development. Finally, genes related to hair follicle development (Shh, Edar and Fzd6), Notch signaling pathway (Notch2, Notch3, Hes5 and Nrarp) and ΔNp63 were identified as downstream target genes regulated by H3K4me3. Collectively, ASH2L-dependent H3K4me3 modification served as an upstream epigenetic regulator in epidermal differentiation and hair follicle morphogenesis in mice.

Comprehensive deciphering the alternative splicing patterns involved in leaf morphogenesis of Liriodendron chinense.

Alternative splicing (AS), a pivotal post-transcriptional regulatory mechanism, profoundly amplifies diversity and complexity of transcriptome and proteome. Liriodendron chinense (Hemsl.) Sarg., an excellent ornamental tree species renowned for its distinctive leaf shape, which resembles the mandarin jacket. Despite the documented potential genes related to leaf development of L. chinense, the underlying post-transcriptional regulatory mechanisms remain veiled. Here, we conducted a comprehensive analysis of the transcriptome to clarify the genome-wide landscape of the AS pattern and the spectrum of spliced isoforms during leaf developmental stages in L. chinense. Our investigation unveiled 50,259 AS events, involving 10,685 genes (32.9%), with intron retention as the most prevalent events. Notably, the initial stage of leaf development witnessed the detection of 804 differentially AS events affiliated with 548 genes. Although both differentially alternative splicing genes (DASGs) and differentially expressed genes (DEGs) were enriched into morphogenetic related pathways during the transition from fishhook (P2) to lobed (P7) leaves, there was only a modest degree of overlap between DASGs and DEGs. Furthermore, we conducted a comprehensively AS analysis on homologous genes involved in leaf morphogenesis, and most of which are subject to post-transcriptional regulation of AS. Among them, the AINTEGUMENTA-LIKE transcript factor LcAIL5 was characterization in detailed, which experiences skipping exon (SE), and two transcripts displayed disparate expression patterns across multiple stages. Overall, these findings yield a comprehensive understanding of leaf development regulation via AS, offering a novel perspective for further deciphering the mechanism of plant leaf morphogenesis.

In vivo activities of heparan sulfate differentially modified by NDSTs during development.

Heparan sulfate proteoglycans (HSPGs) serve as co-receptors for growth factor signaling during development. It is well known that the level and patterns of sulfate groups of heparan sulfate (HS) chains, or HS fine structures, have a major impact on HSPG function. On the other hand, the physiological significance of other structural features of HS, including NS/NA domain organization, remains to be elucidated. A blueprint of the HS domain structures is mainly controlled by HS N-deacetylase/N-sulfotransferases (NDSTs). To analyze in vivo activities of differentially modified HS, we established two knock-in (KI) Drosophila strains with the insertion of mouse Ndst1 (mNdst1) or Ndst2 (mNdst2) in the locus of sulfateless (sfl), the only Drosophila NDST. In these KI lines, mNDSTs are expressed from the sfl locus, in the level and patterns identical to the endogenous sfl gene. Thus, phenotypes of Ndst1 KI and Ndst2KI animals reflect the ability of HS structures made by these enzymes to rescue sfl mutation. Remarkably, we found that mNdst1 completely rescued the loss of sfl. mNdst2 showed a limited rescue ability, despite a higher level of HS sulfation compared to HS in mNdst1 KI. Our study suggests that independent of sulfation levels, additional HS structural features controlled by NDSTs play key roles during tissue patterning.

OsMADS6-OsMADS32 and REP1 control palea cellular heterogeneity and morphogenesis in rice.

Precise regulation of cell proliferation and differentiation is vital for organ morphology. Rice palea, serving as sepal, comprises two distinct regions: the marginal region (MRP) and body of palea (BOP), housing heterogeneous cell populations, which makes it an ideal system for studying organ morphogenesis. We report that the transcription factor (TF) REP1 promotes epidermal cell proliferation and differentiation in the BOP, resulting in hard silicified protrusion cells, by regulating the cyclin-dependent kinase gene, OsCDKB1;1. Conversely, TFs OsMADS6 and OsMADS32 are expressed exclusively in the MRP, where they limit cell division rates by inhibiting OsCDKB2;1 expression and promote endoreduplication, yielding elongated epidermal cells. Furthermore, reciprocal inhibition between the OsMADS6-OsMADS32 complex and REP1 fine-tunes the balance between cell division and differentiation during palea morphogenesis. We further show the functional conservation of these organ identity genes in heterogeneous cell growth in Arabidopsis, emphasizing a critical framework for controlling cellular heterogeneity in organ morphogenesis.

Aging as a loss of morphostatic information: a developmental bioelectricity perspective.

Maintaining order at the tissue level is crucial throughout the lifespan, as failure can lead to cancer and an accumulation of molecular and cellular disorders. Perhaps, the most consistent and pervasive result of these failures is aging, which is characterized by the progressive loss of function and decline in the ability to maintain anatomical homeostasis and reproduce. This leads to organ malfunction, diseases, and ultimately death. The traditional understanding of aging is that it is caused by the accumulation of molecular and cellular damage. In this article, we propose a complementary view of aging from the perspective of endogenous bioelectricity which has not yet been integrated into aging research. We propose a view of aging as a morphostasis defect, a loss of biophysical prepattern information, encoding anatomical setpoints used for dynamic tissue and organ homeostasis. We hypothesize that this is specifically driven by abrogation of the endogenous bioelectric signaling that normally harnesses individual cell behaviors toward the creation and upkeep of complex multicellular structures in vivo. Herein, we first describe bioelectricity as the physiological software of life, and then identify and discuss the links between bioelectricity and life extension strategies and age-related diseases. We develop a bridge between aging and regeneration via bioelectric signaling that suggests a research program for healthful longevity via morphoceuticals. Finally, we discuss the broader implications of the homologies between development, aging, cancer and regeneration and how morphoceuticals can be developed for aging.

Mechanical stress combines with planar polarised patterning during metaphase to orient embryonic epithelial cell divisions.

The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. We ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing cell elongation, despite mitotic rounding and over-riding interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, there is a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias over-rides mechanical cues in mildly elongated cells but the spindle is constrained to the high stress axis in strongly elongated cells.

Pioneering a chick embryo model to explore the intrauterine etiology of developmental dysplasia of the hip in oligohydramnios conditions.

OBJECTIVE: To explore the impact of oligohydramnios on fetal movement and hip development, given its association with developmental dysplasia of the hip (DDH) but unclear mechanisms. METHODS: Chick embryos were divided into four groups based on the severity of oligohydramnios induced by amniotic fluid aspiration (control, 0.2 mL, 0.4 mL, 0.6 mL). Fetal movement was assessed by detection of movement and quantification of residual amniotic fluid volume. Hip joint development was assessed by gross anatomic analysis, micro-computed tomography (micro-CT) for cartilage assessment, and histologic observation at multiple time points. In addition, a subset of embryos from the 0.4 mL aspirated group underwent saline reinfusion and subsequent evaluation. RESULTS: Increasing volumes of aspirated amniotic fluid resulted in worsening of fetal movement restrictions (e.g., 0.4 mL aspirated and control group at E10: frequency difference -7.765 [95% CI: -9.125, -6.404]; amplitude difference -0.343 [95% CI: -0.588, -0.097]). The 0.4 mL aspirated group had significantly smaller hip measurements compared to controls, with reduced acetabular length (-0.418 [95% CI: -0.575, -0.261]) and width (-0.304 [95% CI: -0.491, -0.117]) at day E14.5. Histological analysis revealed a smaller femoral head (1.084 ±â€¯0.264 cm) and shallower acetabulum (0.380 ±â€¯0.106 cm) in the 0.4 mL group. Micro-CT showed cartilage matrix degeneration (13.6% [95% CI: 0.6%, 26.7%], P = 0.043 on E14.5). Saline reinfusion resulted in significant improvements in the femoral head to greater trochanter (0.578 [95% CI: 0.323, 0.833], P = 0.001). CONCLUSIONS: Oligohydramnios can cause DDH by restricting fetal movement and disrupting hip morphogenesis in a time-dependent manner. Timely reversal of oligohydramnios during the fetal period may prevent DDH.

Pten, PI3K, and PtdIns(3,4,5)P3 dynamics control pulsatile actin branching in Drosophila retina morphogenesis.

Epithelial remodeling of the Drosophila retina depends on the pulsatile contraction and expansion of apical contacts between the cells that form its hexagonal lattice. Phosphoinositide PI(3,4,5)P3 (PIP3) accumulates around tricellular adherens junctions (tAJs) during contact expansion and dissipates during contraction, but with unknown function. Here, we found that manipulations of Pten or PI3-kinase (PI3K) that either decreased or increased PIP3 resulted in shortened contacts and a disordered lattice, indicating a requirement for PIP3 dynamics and turnover. These phenotypes are caused by a loss of branched actin, resulting from impaired activity of the Rac1 Rho GTPase and the WAVE regulatory complex (WRC). We additionally found that during contact expansion, PI3K moves into tAJs to promote the cyclical increase of PIP3 in a spatially and temporally precise manner. Thus, dynamic control of PIP3 by Pten and PI3K governs the protrusive phase of junctional remodeling, which is essential for planar epithelial morphogenesis.

Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium.

Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes adopting a shape named "scutoid" that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here we use live-imaging of the sea star embryo coupled with deep learning-based segmentation, to dissect the relative contributions of cell density, tissue compaction, and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing just after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

Morphogenetic theory of mental and cognitive disorders: the role of neurotrophic and guidance molecules.

Mental illness and cognitive disorders represent a serious problem for the modern society. Many studies indicate that mental disorders are polygenic and that impaired brain development may lay the ground for their manifestation. Neural tissue development is a complex and multistage process that involves a large number of distant and contact molecules. In this review, we have considered the key steps of brain morphogenesis, and the major molecule families involved in these process. The review provides many indications of the important contribution of the brain development process and correct functioning of certain genes to human mental health. To our knowledge, this comprehensive review is one of the first in this field. We suppose that this review may be useful to novice researchers and clinicians wishing to navigate the field.

Identifying Causal Genes for Thermo-Responsive Rosette Growth in Arabidopsis by Genome-Wide Association Study.

Temperature responsive plant growth, such as thermo-morphogenesis, varied greatly among Arabidopsis natural accessions. Here we describe a procedure to identify causal genes for thermo-morphogenesis variation by a genome-wide association study (GWAS). It includes methods of measuring thermo-responsive rosette growth architecture, using GWA-Portal webtool for GWAS, analyzing haplotypes, and validating functional natural variations. In addition, we discuss key factors that affect the success of identifying causal genes from GWAS.

Abnormal H3K4 enzyme catalytic activity and neuronal morphology caused by ASH1L mutations in individuals with Tourette syndrome.

ASH1L potentially contributes to Tourette syndrome (TS) and other neuropsychiatric disorders, as our previous studies have shown. It regulates essential developmental genes by counteracting polycomb-mediated transcriptional repression, which restricts chromatin accessibility at target genes. ASH1L is highly expressed in the adult brain, playing a crucial role in the early stage. However, it remains unclear how ASH1L mutations carried by patients with TS participate in regulating neuronal growth processes leading to TS traits. Five TS families recruited in our study underwent comprehensive physical examinations and questionnaires to record clinical phenotypes and environmental impact factors. We validated the variants via Sanger sequencing and constructed two mutants near the catalytic domain of ASH1L. We conducted molecular modeling, in vitro assays, and primary neuron cultures to find the role of ASH1L in neuronal development and its correlation with TS. In this study, we validated five pathogenic ASH1L rare variants and observed symptoms in patients with simple tics and behavioral comorbidities. Mutations near the catalytic domain of TS patients cause mental state abnormalities and disrupt ASH1L function by destabilizing its spatial conformation, leading to decreased activity of catalytic H3K4, thereby affecting the neurite growth. We need to conduct larger-scale studies on TS patients and perform additional neurological evaluations on mature neurons. We first reported the effects of ASH1L mutations in TS patients, including phenotypic heterogeneity, protein function, and neurological growth. This information contributes to understanding the neurodevelopmental pathogenesis of TS in patients with ASH1L mutations.

Substrate evaporation drives collective construction in termites.

Termites build complex nests which are an impressive example of self-organization. We know that the coordinated actions involved in the construction of these nests by multiple individuals are primarily mediated by signals and cues embedded in the structure of the nest itself. However, to date there is still no scientific consensus about the nature of the stimuli that guide termite construction, and how they are sensed by termites. In order to address these questions, we studied the early building behavior of Coptotermes gestroi termites in artificial arenas, decorated with topographic cues to stimulate construction. Pellet collections were evenly distributed across the experimental setup, compatible with a collection mechanism that is not affected by local topography, but only by the distribution of termite occupancy (termites pick pellets at the positions where they are). Conversely, pellet depositions were concentrated at locations of high surface curvature and at the boundaries between different types of substrate. The single feature shared by all pellet deposition regions was that they correspond to local maxima in the evaporation flux. We can show analytically and we confirm experimentally that evaporation flux is directly proportional to the local curvature of nest surfaces. Taken together, our results indicate that surface curvature is sufficient to organize termite building activity and that termites likely sense curvature indirectly through substrate evaporation. Our findings reconcile the apparently discordant results of previous studies.

Modeling the extracellular matrix in cell migration and morphogenesis: a guide for the curious biologist.

The extracellular matrix (ECM) is a highly complex structure through which biochemical and mechanical signals are transmitted. In processes of cell migration, the ECM also acts as a scaffold, providing structural support to cells as well as points of potential attachment. Although the ECM is a well-studied structure, its role in many biological processes remains difficult to investigate comprehensively due to its complexity and structural variation within an organism. In tandem with experiments, mathematical models are helpful in refining and testing hypotheses, generating predictions, and exploring conditions outside the scope of experiments. Such models can be combined and calibrated with in vivo and in vitro data to identify critical cell-ECM interactions that drive developmental and homeostatic processes, or the progression of diseases. In this review, we focus on mathematical and computational models of the ECM in processes such as cell migration including cancer metastasis, and in tissue structure and morphogenesis. By highlighting the predictive power of these models, we aim to help bridge the gap between experimental and computational approaches to studying the ECM and to provide guidance on selecting an appropriate model framework to complement corresponding experimental studies.