Excess Dally-like Induces Malformation of Drosophila Legs.

Glypicans are closely associated with organ development and tumorigenesis in animals. Dally-like (Dlp), a membrane-bound glypican, plays pivotal roles in various biological processes in Drosophila. In this study, we observed that an excess of Dlp led to the malformation of legs, particularly affecting the distal part. Accordingly, the leg disc was shrunken and frequently exhibited aberrant morphology. In addition, elevated Dlp levels induced ectopic cell death with no apparent cell proliferation changes. Furthermore, Dlp overexpression in the posterior compartment significantly altered Wingless (Wg) distribution. We observed a marked expansion of Wg distribution within the posterior compartment, accompanied by a corresponding decrease in the anterior compartment. It appears that excess Dlp guides Wg to diffuse to cells with higher Dlp levels. In addition, the distal-less (dll) gene, which is crucial for leg patterning, was up-regulated significantly. Notably, dachshund (dac) and homothorax (hth) expression, also essential for leg patterning and development, only appeared to be negligibly affected. Based on these findings, we speculate that excess Dlp may contribute to malformations of the distal leg region of Drosophila, possibly through its influence on Wg distribution, dll expression and induced cell death. Our research advances the understanding of Dlp function in Drosophila leg development.

Emergence of large-scale cell death through ferroptotic trigger waves.

Large-scale cell death is commonly observed during organismal development and in human pathologies1-5. These cell death events extend over great distances to eliminate large populations of cells, raising the question of how cell death can be coordinated in space and time. One mechanism that enables long-range signal transmission is trigger waves6, but how this mechanism might be used for death events in cell populations remains unclear. Here we demonstrate that ferroptosis, an iron- and lipid-peroxidation-dependent form of cell death, can propagate across human cells over long distances (≥5 mm) at constant speeds (around 5.5 µm min-1) through trigger waves of reactive oxygen species (ROS). Chemical and genetic perturbations indicate a primary role of ROS feedback loops (Fenton reaction, NADPH oxidase signalling and glutathione synthesis) in controlling the progression of ferroptotic trigger waves. We show that introducing ferroptotic stress through suppression of cystine uptake activates these ROS feedback loops, converting cellular redox systems from being monostable to being bistable and thereby priming cell populations to become bistable media over which ROS propagate. Furthermore, we demonstrate that ferroptosis and its propagation accompany the massive, yet spatially restricted, cell death events during muscle remodelling of the embryonic avian limb, substantiating its use as a tissue-sculpting strategy during embryogenesis. Our findings highlight the role of ferroptosis in coordinating global cell death events, providing a paradigm for investigating large-scale cell death in embryonic development and human pathologies.

Sall4 regulates downstream patterning genes during limb regeneration.

Many salamanders can completely regenerate a fully functional limb. Limb regeneration is a carefully coordinated process involving several defined stages. One key event during the regeneration process is the patterning of the blastema to inform cells of what they must differentiate into. Although it is known that many genes involved in the initial development of the limb are re-used during regeneration, the exact molecular circuitry involved in this process is not fully understood. Several large-scale transcriptional profiling studies of axolotl limb regeneration have identified many transcription factors that are up-regulated after limb amputation. Sall4 is a transcription factor that has been identified to play essential roles in maintaining cells in an undifferentiated state during development and also plays a unique role in limb development. Inactivation of Sall4 during limb bud development results in defects in anterior-posterior patterning of the limb. Sall4 has been found to be up-regulated during limb regeneration in both Xenopus and salamanders, but to date it function has been untested. We confirmed that Sall4 is up-regulated during limb regeneration in the axolotl using qRT-PCR and identified that it is present in the skin cells and also in cells within the blastema. Using CRISPR technology we microinjected gRNAs specific for Sall4 complexed with cas9 protein into the blastema to specifically knockout Sall4 in blastema cells only. This resulted in limb regenerate defects, including missing digits, fusion of digit elements, and defects in the radius and ulna. This suggests that during regeneration Sall4 may play a similar role in regulating the specification of anterior-proximal skeletal elements.

FOXC1 and FOXC2 regulate growth plate chondrocyte maturation towards hypertrophy in the embryonic mouse limb skeleton.

The Forkhead box transcription factors FOXC1 and FOXC2 are expressed in condensing mesenchyme cells at the onset of endochondral ossification. We used the Prx1-cre mouse to ablate Foxc1 and Foxc2 in limb skeletal progenitor cells. Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ limbs were shorter than controls, with worsening phenotypes in distal structures. Cartilage formation and mineralization was severely disrupted in the paws. The radius and tibia were malformed, whereas the fibula and ulna remained unmineralized. Chondrocyte maturation was delayed, with fewer Indian hedgehog-expressing, prehypertrophic chondrocytes forming and a smaller hypertrophic chondrocyte zone. Later, progression out of chondrocyte hypertrophy was slowed, leading to an accumulation of COLX-expressing hypertrophic chondrocytes and formation of a smaller primary ossification center with fewer osteoblast progenitor cells populating this region. Targeting Foxc1 and Foxc2 in hypertrophic chondrocytes with Col10a1-cre also resulted in an expanded hypertrophic chondrocyte zone and smaller primary ossification center. Our findings suggest that FOXC1 and FOXC2 direct chondrocyte maturation towards hypertrophic chondrocyte formation. At later stages, FOXC1 and FOXC2 regulate function in hypertrophic chondrocyte remodeling to allow primary ossification center formation and osteoblast recruitment.

Insights into digit evolution from a fate map study of the forearm using Chameleon, a new transgenic chicken line.

The cellular and genetic networks that contribute to the development of the zeugopod (radius and ulna of the forearm, tibia and fibula of the leg) are not well understood, although these bones are susceptible to loss in congenital human syndromes and to the action of teratogens such as thalidomide. Using a new fate-mapping approach with the Chameleon transgenic chicken line, we show that there is a small contribution of SHH-expressing cells to the posterior ulna, posterior carpals and digit 3. We establish that although the majority of the ulna develops in response to paracrine SHH signalling in both the chicken and mouse, there are differences in the contribution of SHH-expressing cells between mouse and chicken as well as between the chicken ulna and fibula. This is evidence that, although zeugopod bones are clearly homologous according to the fossil record, the gene regulatory networks that contribute to their development and evolution are not fixed.

Pre-hypertrophic chondrogenic enhancer landscape of limb and axial skeleton development.

Chondrocyte differentiation controls skeleton development and stature. Here we provide a comprehensive map of chondrocyte-specific enhancers and show that they provide a mechanistic framework through which non-coding genetic variants can influence skeletal development and human stature. Working with fetal chondrocytes isolated from mice bearing a Col2a1 fluorescent regulatory sensor, we identify 780 genes and 2'704 putative enhancers specifically active in chondrocytes using a combination of RNA-seq, ATAC-seq and H3K27ac ChIP-seq. Most of these enhancers (74%) show pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. Notably, genetic variations overlapping these enhancers better explain height differences than those overlapping non-chondrogenic enhancers. Finally, targeted deletions of identified enhancers at the Fgfr3, Col2a1, Hhip and, Nkx3-2 loci confirm their role in regulating cognate genes. This enhancer map provides a framework for understanding how genes and non-coding variations influence bone development and diseases.

Influence of DNA-methylation at multiple stages of limb chondrogenesis.

Precise regulation of gene expression is of utmost importance during cell fate specification. DNA methylation is a key epigenetic mechanism that plays a significant role in the regulation of cell fate by recruiting repression proteins or inhibiting the binding of transcription factors to DNA to regulate gene expression. Limb development is a well-established model for understanding cell fate decisions, and the formation of skeletal elements is coordinated through a sequence of events that control chondrogenesis spatiotemporally. It has been established that epigenetic control participates in cartilage maturation. However, further investigation is required to determine its role in the earliest stages of chondrocyte differentiation. This study investigates how the DNA methylation environment affects cell fate divergence during the early chondrogenic events. Our research has shown for the first time that inhibiting DNA methylation in interdigital tissue with 5-azacytidine results in the formation of an ectopic digit. This discovery suggested that DNA methylation dynamics could regulate the fate of cells between chondrogenesis and cell death during autopod development. Our in vitro findings indicate that DNA methylation at the early stages of chondrogenesis is integral in regulating condensation by controlling cell adhesion and proapoptotic genes. As a result, the dynamics of methylation and demethylation are crucial in governing chondrogenesis and cell death during different stages of limb chondrogenesis.

Affinity-optimizing enhancer variants disrupt development.

Enhancers control the location and timing of gene expression and contain the majority of variants associated with disease1-3. The ZRS is arguably the most well-studied vertebrate enhancer and mediates the expression of Shh in the developing limb4. Thirty-one human single-nucleotide variants (SNVs) within the ZRS are associated with polydactyly4-6. However, how this enhancer encodes tissue-specific activity, and the mechanisms by which SNVs alter the number of digits, are poorly understood. Here we show that the ETS sites within the ZRS are low affinity, and identify a functional ETS site, ETS-A, with extremely low affinity. Two human SNVs and a synthetic variant optimize the binding affinity of ETS-A subtly from 15% to around 25% relative to the strongest ETS binding sequence, and cause polydactyly with the same penetrance and severity. A greater increase in affinity results in phenotypes that are more penetrant and more severe. Affinity-optimizing SNVs in other ETS sites in the ZRS, as well as in ETS, interferon regulatory factor (IRF), HOX and activator protein 1 (AP-1) sites within a wide variety of enhancers, cause gain-of-function gene expression. The prevalence of binding sites with suboptimal affinity in enhancers creates a vulnerability in genomes whereby SNVs that optimize affinity, even slightly, can be pathogenic. Searching for affinity-optimizing SNVs in genomes could provide a mechanistic approach to identify causal variants that underlie enhanceropathies.

Distinct gene expression dynamics in developing and regenerating crustacean limbs.

Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether regeneration mirrors development is an open question in most regenerative species. Here, we take a transcriptomics approach to examine whether leg regeneration shows similar temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, the dynamics of gene expression during leg regeneration show a higher degree of variation related to the physiology of individual animals. A major driver of this variation is the molting cycle. We dissect the transcriptional signals of individual physiology and regeneration to obtain clearer temporal signals marking distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although the two processes use similar sets of genes, the temporal patterns in which these genes are deployed are different and cannot be systematically aligned.

The WNT7A/WNT7B/GPR124/RECK signaling module plays an essential role in mammalian limb development.

In central nervous system vascular endothelial cells, signaling via the partially redundant ligands WNT7A and WNT7B requires two co-activator proteins, GPR124 and RECK. WNT7A and RECK have been shown previously to play a role in limb development, but the mechanism of RECK action in this context is unknown. The roles of WNT7B and GPR124 in limb development have not been investigated. Using combinations of conventional and/or conditional loss-of-function alleles for mouse Wnt7a, Wnt7b, Gpr124 and Reck, including a Reck allele that codes for a protein that is specifically defective in WNT7A/WNT7B signaling, we show that reductions in ligand and/or co-activator function synergize to cause reduced and dysmorphic limb bone growth. Two additional limb phenotypes - loss of distal Lmx1b expression and ectopic growth of nail-like structures - occur with reduced Wnt7a/Wnt7b gene copy number and, respectively, with Reck mutations and with combined Reck and Gpr124 mutations. A third limb phenotype - bleeding into a digit - occurs with the most severe combinations of Wnt7a/Wnt7b, Reck and Gpr124 mutations. These data imply that the WNT7A/WNT7B-FRIZZLED-LRP5/LRP6-GPR124-RECK signaling system functions as an integral unit in limb development.

Normal skeletal pattern formation in chick limb bud with a mesenchymal hole is mediated by adjustment of cellular properties along the anterior-posterior axis in the limb bud.

The chick limb bud has plasticity to reconstruct a normal skeletal pattern after a part of mesenchymal mass is excised to make a hole in its early stage of development. To understand the details of hole closure and re-establishment of normal limb axes to reconstruct a normal limb skeleton, we focused on cellular and molecular changes during hole repair and limb restoration. We excised a cube-shaped mass of mesenchymal cells from the medial region of chick hindlimb bud (stage 23) and observed the following morphogenesis. The hole had closed by 15 â€‹h after excision, followed by restoration of the limb bud morphology, and the cartilage pattern was largely restored by 48 â€‹h. Lineage analysis of the mesenchymal cells showed that cells at the anterior and posterior margins of the hole were adjoined at the hole closure site, whereas cells at the proximal and distal margins were not. To investigate cell polarity during hole repair, we analyzed intracellular positioning of the Golgi apparatus relative to the nuclei. We found that the Golgi apparatus tended to be directed toward the hole among cells at the anterior and posterior margins but not among cells at identical positions in normal limb buds or cells at the proximal and distal hole margins. In the manipulated limb buds, the frequency of cell proliferation was maintained compared with the control side. Tbx3 expression, which was usually restricted to anterior and posterior margins of the limb bud, was temporarily expanded medially and then reverted to a normal pattern as limb reconstruction proceeded, with Tbx3 negative cells reappearing in the medial regions of the limb buds. Thus, mesenchymal hole closure and limb reconstruction are mainly mediated by cells at the anterior and posterior hole margins. These results suggest that adjustment of cellular properties along the anteroposterior axis is crucial to restore limb damage and reconstruct normal skeletal patterns.

Modeling uniquely human gene regulatory function via targeted humanization of the mouse genome.

The evolution of uniquely human traits likely entailed changes in developmental gene regulation. Human Accelerated Regions (HARs), which include transcriptional enhancers harboring a significant excess of human-specific sequence changes, are leading candidates for driving gene regulatory modifications in human development. However, insight into whether HARs alter the level, distribution, and timing of endogenous gene expression remains limited. We examined the role of the HAR HACNS1 (HAR2) in human evolution by interrogating its molecular functions in a genetically humanized mouse model. We find that HACNS1 maintains its human-specific enhancer activity in the mouse embryo and modifies expression of Gbx2, which encodes a transcription factor, during limb development. Using single-cell RNA-sequencing, we demonstrate that Gbx2 is upregulated in the limb chondrogenic mesenchyme of HACNS1 homozygous embryos, supporting that HACNS1 alters gene expression in cell types involved in skeletal patterning. Our findings illustrate that humanized mouse models provide mechanistic insight into how HARs modified gene expression in human evolution.

The T-box gene optomotor-blind organizes proximodistal leg patterning in the beetle Tribolium castaneum by repressing dorsal Dpp pathway activity.

Leg axis formation in Drosophila is organized by Wingless (Wg) and Decapentaplegic (Dpp) that control a number of downstream factors to pattern the dorsoventral (DV) and proximodistal (PD) axis. The T-box genes are important downstream factors mainly involved in dorsoventral leg axis formation. The ventral side is specified by H15 and midline, whereas optomotor-blind (omb) and Dorsocross (Doc1) are factors to specify dorsal cell fates. We show here that omb also organizes PD leg axis patterning in the beetle Tribolium castaneum. In the legs, Tc-omb is expressed along the dorsal side and represses ventral factors like wg and H15. Intriguingly, removing Tc-omb function leads to the activation of the Dpp pathway along the dorsal side of the legs, thus mimicking normal dpp expression in Drosophila. Dpp activity along the dorsal side leads to altered expression of proximal-distal patterning genes such as Distal-less (Dll) and dachshund (dac). Our results indicate a cell-autonomous activation of Dll and repression of dac by dpp. These findings are compatible with the cross-regulatory "cascade model" of proximal-distal leg imaginal disc patterning of Drosophila.

Dynamic Expression of Membrane Type 1-Matrix Metalloproteinase (Mt1-mmp/Mmp14) in the Mouse Embryo.

MT1-MMP/MMP14 belongs to a subgroup of the matrix metalloproteinases family that presents a transmembrane domain, with a cytosolic tail and the catalytic site exposed to the extracellular space. Deficient mice for this enzyme result in early postnatal death and display severe defects in skeletal, muscle and lung development. By using a transgenic line expressing the LacZ reporter under the control of the endogenous Mt1-mmp promoter, we reported a dynamic spatiotemporal expression pattern for Mt1-mmp from early embryonic to perinatal stages during cardiovascular development and brain formation. Thus, Mt1-mmp shows expression in the endocardium of the heart and the truncus arteriosus by E8.5, and is also strongly detected during vascular system development as well as in endothelial cells. In the brain, LacZ reporter expression was detected in the olfactory bulb, the rostral cerebral cortex and the caudal mesencephalic tectum. LacZ-positive cells were observed in neural progenitors of the spinal cord, neural crest cells and the intersomitic region. In the limb, Mt1-mmp expression was restricted to blood vessels, cartilage primordium and muscles. Detection of the enzyme was confirmed by Western blot and immunohistochemical analysis. We suggest novel functions for this metalloproteinase in angiogenesis, endocardial formation and vascularization during organogenesis. Moreover, Mt1-mmp expression revealed that the enzyme may contribute to heart, muscle and brain throughout development.

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and in vivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how in vivo tissue-specific phenotypes emerge in ribosomopathies.

Individual functions of the histone acetyl transferases CBP and p300 in regulating the inflammatory response of synovial fibroblasts.

Chromatin remodeling, and a persistent histone 3 lysine 27 acetylation (H3K27ac) in particular, are associated with a sustained inflammatory response of synovial fibroblasts (SF) in rheumatoid arthritis (RA). Here we investigated individual functions of the writers of H3K27ac marks, the homologues histone acetyl transferases (HAT) CBP and p300, in controlling the constitutive and inflammatory gene expression in RA SF. We applied a silencing strategy, followed by RNA-sequencing and pathway analysis, complemented with the treatment of SF with inhibitors targeting the HAT (C646) or bromo domains (I-CBP) of CBP and p300. We showed that CBP and p300 undertook overlapping and, in particular at gene levels, distinct regulatory functions in SF. p300 is the major HAT for H3K27ac in SF and regulated more diverse pathways than CBP. Whereas both factors regulated genes associated with extracellular matrix remodeling, adhesion and proliferation, p300 specifically controlled developmental genes associated with limb development. Silencing of CBP specifically down regulated the TNF-induced expression of interferon-signature genes. In contrast, silencing of p300 resulted in anti- and pro-inflammatory effects. Integration of data sets derived from RNA-sequencing and chromatin immunoprecipitation sequencing for H3K27ac revealed that changes in gene expression after CBP or p300 silencing could be only partially explained by changes in levels of H3K27ac. Inhibition of CBP/p300 using HAT and bromo domain inhibitors strongly mirrored effects obtained by silencing of p300, including anti- and pro-inflammatory effects, indicating that such inhibitors are not sufficient to be used as anti-inflammatory drugs.

Unexpected contribution of fibroblasts to muscle lineage as a mechanism for limb muscle patterning.

Positional information driving limb muscle patterning is contained in connective tissue fibroblasts but not in myogenic cells. Limb muscles originate from somites, while connective tissues originate from lateral plate mesoderm. With cell and genetic lineage tracing we challenge this model and identify an unexpected contribution of lateral plate-derived fibroblasts to the myogenic lineage, preferentially at the myotendinous junction. Analysis of single-cell RNA-sequencing data from whole limbs at successive developmental stages identifies a population displaying a dual muscle and connective tissue signature. BMP signalling is active in this dual population and at the tendon/muscle interface. In vivo and in vitro gain- and loss-of-function experiments show that BMP signalling regulates a fibroblast-to-myoblast conversion. These results suggest a scenario in which BMP signalling converts a subset of lateral plate mesoderm-derived cells to a myogenic fate in order to create a boundary of fibroblast-derived myonuclei at the myotendinous junction that controls limb muscle patterning.

Hox11 expression characterizes developing zeugopod synovial joints and is coupled to postnatal articular cartilage morphogenesis into functional zones in mice.

Previous studies on mouse embryo limbs have established that interzone mesenchymal progenitor cells emerging at each prescribed joint site give rise to joint tissues over fetal time. These incipient tissues undergo structural maturation and morphogenesis postnatally, but underlying mechanisms of regulation remain unknown. Hox11 genes dictate overall zeugopod musculoskeletal patterning and skeletal element identities during development. Here we asked where these master regulators are expressed in developing limb joints and whether they are maintained during postnatal zeugopod joint morphogenesis. We found that Hoxa11 was predominantly expressed and restricted to incipient wrist and ankle joints in E13.5 mouse embryos, and became apparent in medial and central regions of knees by E14.5, though remaining continuously dormant in elbow joints. Closer examination revealed that Hoxa11 initially characterized interzone and neighboring cells and was then restricted to nascent articular cartilage, intra joint ligaments and structures such as meniscal horns over prenatal time. Postnatally, articular cartilage progresses from a nondescript cell-rich, matrix-poor tissue to a highly structured, thick, zonal and mechanically competent tissue with chondrocyte columns over time, most evident at sites such as the tibial plateau. Indeed, Hox11 expression (primarily Hoxa11) was intimately coupled to such morphogenetic processes and, in particular, to the topographical rearrangement of chondrocytes into columns within the intermediate and deep zones of tibial plateau that normally endures maximal mechanical loads. Revealingly, these expression patterns were maintained even at 6 months of age. In sum, our data indicate that Hox11 genes remain engaged well beyond embryonic synovial joint patterning and are specifically tied to postnatal articular cartilage morphogenesis into a zonal and resilient tissue. The data demonstrate that Hox11 genes characterize adult, terminally differentiated, articular chondrocytes and maintain region-specificity established in the embryo.

Histone Epigenetic Signatures in Embryonic Limb Interdigital Cells Fated to Die.

During limb formation in vertebrates with free digits, the interdigital mesoderm is eliminated by a massive degeneration process that involves apoptosis and cell senescence. The degradation process is preceded by intense DNA damage in zones located close to methylated DNA, accompanied by the activation of the DNA repair response. In this study, we show that trimethylated histone 3 (H3K4me3, H3K9me3, and H3K27me3) overlaps with zones positive for 5mC in the nuclei of interdigital cells. This pattern contrasts with the widespread distribution of acetylated histones (H3K9ac and H4ac) and the histone variant H3.3 throughout the nucleoplasm. Consistent with the intense labeling of acetylated histones, the histone deacetylase genes Hdac1, Hdac2, Hdac3, and Hdac8, and at a more reduced level, Hdac10, are expressed in the interdigits. Furthermore, local treatments with the histone deacetylase inhibitor trichostatin A, which promotes an open chromatin state, induces massive cell death and transcriptional changes reminiscent of, but preceding, the physiological process of interdigit remodeling. Together, these findings suggest that the epigenetic profile of the interdigital mesoderm contributes to the sensitivity to DNA damage that precedes apoptosis during tissue regression.

Deletion of CTCF sites in the SHH locus alters enhancer-promoter interactions and leads to acheiropodia.

Acheiropodia, congenital limb truncation, is associated with homozygous deletions in the LMBR1 gene around ZRS, an enhancer regulating SHH during limb development. How these deletions lead to this phenotype is unknown. Using whole-genome sequencing, we fine-mapped the acheiropodia-associated region to 12 kb and show that it does not function as an enhancer. CTCF and RAD21 ChIP-seq together with 4C-seq and DNA FISH identify three CTCF sites within the acheiropodia-deleted region that mediate the interaction between the ZRS and the SHH promoter. This interaction is substituted with other CTCF sites centromeric to the ZRS in the disease state. Mouse knockouts of the orthologous 12 kb sequence have no apparent abnormalities, showcasing the challenges in modelling CTCF alterations in animal models due to inherent motif differences between species. Our results show that alterations in CTCF motifs can lead to a Mendelian condition due to altered enhancer-promoter interactions.