Biology subjects

Rubin, S.

Publications and source records attributed to Rubin, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Identification and inhibition of the Cyclin D Rb-docking interface that drives cell division

The animal cell division cycle is initiated by the cyclin-dependent kinases CDK4 and CDK6 in complex with D-type cyclins. Cyclin D-CDK4/6 complex formation is promoted by the assembly factors p21 and p27, which bind both subunits. p27 binds the hydrophobic patch on cyclin D that is similar to the patch used by other cell cycle cyclins to dock their substrates. This raised the question as to how cyclin D could find its substrates if its hydrophobic patch were already occupied? Here, we show that D-type cyclins use their A2 helix to dock the retinoblastoma protein Rb, a key substrate regulating cell cycle progression. The specific interface of cyclin Ds A2 helix is unique among cyclins and its mutation slows proliferation. Taken together, our work identifies a cyclin D-substrate docking mechanism that can be targeted by novel cancer therapeutics.

biochemistry↗

bioRxiv2025

A Fkh1/2 binding site array in the WHI5 promoter drives sub-scaling transcription

Cells typically regulate their size within a relatively tight range by coupling growth to the cell division cycle using a dedicated set of molecular mechanisms. In budding yeast, cells are born with a similar amount of the G1/S inhibitor protein Whi5 that is then diluted by growth throughout G1. As cells grow, Whi5 concentration decreases and cells become more likely to enter the cell cycle. Cells are born in G1 with similar amounts of Whi5 because of the size-independent (sub-scaling) expression of WHI5 mRNA during S/G2/M phases and the equal partitioning of Whi5 protein at division. While the latter is known to be achieved by association with chromatin before anaphase, the mechanism for the former is poorly understood. Through systematic mutations of the WHI5 promoter, we discovered that WHI5s core promoter region located -126 to -75 base pairs upstream of the start codon is responsible for sub-scaling expression. This sequence contains a repeating array of binding sites for the transcription factors Fkh1 and Fkh2. Mutation of any of these sites, deletion of either FKH1 or FKH2, or preventing Fkh1 or Fkh2 dimerization weakens the sub-scaling of WHI5 transcription. Taken together with structural predictions and a mathematical model of cooperative Fkh-DNA binding, we conclude that WHI5s sub-scaling transcription is regulated by a Fkh1/2 heteropolymer binding an array of sites in its core promoter.

cell biology↗

bioRxiv2025

E2F1 induces a G0-G1 reentry transcriptional program without changing chromatin accessibility

Quiescent cells actively repress cell-cycle genes via chromatin-based mechanisms to maintain a non-dividing state, yet remain poised to reenter upon stimulation. E2F1, a canonical activator of cell-cycle genes, is sufficient to induce reentry from quiescence, but how it overcomes chromatin-mediated repression remains unclear. Here, we show that inducible E2F1 expression triggers exit from quiescence and progression through the cycle without changes in chromatin accessibility, by harnessing regulatory elements with limited, pre-existing accessibility. Using time-resolved transcriptomics, we demonstrate that E2F1 induces an accelerated transcriptional program compared to serum. Unlike serum, which triggers broad chromatin remodeling, E2F1-induced activation occurs in a context of limited accessibility. ChIP-seq reveals that E2F1 directly binds target sites in quiescent cells to upregulate canonical genes. Biochemical reconstitution shows that E2F1 binds nucleosomes and accesses internal E2F sites within histone-wrapped DNA. These findings suggest that E2F1 can engage nucleosome-associated DNA and initiate transcription without major chromatin reorganization, redefining transcription factor-chromatin dynamics during cell fate transitions and establishing E2F1 as a potent regulator of cell-cycle reentry.

cell biology↗

bioRxiv2025

Tandem association of CLOCK:BMAL1 complexes on DNA enables recruitment of CBP/p300 through multivalent interactions

The basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) transcription factor CLOCK:BMAL1 interacts with E-box motifs in the context of nucleosomes to elicit a circadian pattern of gene expression that oscillates with approximately 24 hour periodicity. Core clock genes and other highly rhythmic targets of CLOCK:BMAL1 typically possess a tandem arrangement of E-boxes that is required for robust oscillations. Here, we show that the presence of tandem E-boxes enables CLOCK:BMAL1 to bind more internal sites on the nucleosome, leading to release of DNA from the histone core and the presentation of multiple coactivator binding motifs in close proximity to facilitate multivalent interactions with the coactivator CBP/p300. We show that the transactivation domain (TAD) of BMAL1, essential for CLOCK:BMAL1 activity, interacts with several modular domains of CBP. Deletion of these CBP domains or chemical inhibition of protein-protein interactions with CBP significantly reduces or eliminates CLOCK:BMAL1-driven activity. Altogether, this suggests that multivalent interactions with CBP may play a role in the ability of tandem CLOCK:BMAL1 heterodimers to recruit this limiting cofactor in cells.

biophysics↗

bioRxiv2025

Strain Matters: The 129S1/SvlmJ Mouse Model Reveals the Genetic and Inflammatory Susceptibility to Hypertensive Complications

BackgroundHypertension is a leading cause of microvascular injury, yet the genetic determinants of organ-specific vulnerability remain poorly understood. Yest, we need good mouse models to investigate the complication of hypertension. This study investigates the role of genetic background in shaping hypertensive complications by comparing two mouse strains with divergent inflammatory responses. MethodsThree-month-old 129S1/SvlmJ and C57BL/6J mice received 600 ng/kg/min of angiotensin II (AngII) or saline. We compared the consequences of ANG2-induced blood pressure elevation on kidney function, BBB intergrity and cardiac hypertropy. Blood pressure (BP) was assessed by telemetry. Vascular injury markers in the brain, heart, kidneys, and retinas were systematically evaluated. ResultsBoth strains developed similar moderate hypertension with AngII. Only 129S1/SvlmJ mice exhibited spatial learning and memory deficits, blood-brain barrier hyperpermeability, astrocyte activation, retinal artery damage, hypertrophic cardiomyopathy, and renal podocyte lesions with urinary albumin/creatinine ratio (UACR) after AngII treatment. Transcriptomic analysis of brain microvessels highlighted strain-specific differences in gene regulation, particularly in inflammatory pathways, which may explain the higher vulnerability of 129S1/SvlmJ mice to hypertensive organ damage. These findings were supported in vivo by increased resident and perivascular macrophage recruitment in the brain of C57BL6/J mice under AngII compared to the 129S1/SvlmJ strain. ConclusionOur findings highlight the critical role of genetic background in shaping hypertensive complications. The 129S1/SvlmJ strain serves as a valuable model for dissecting the molecular mechanisms of hypertensive organ damage, emphasizing neurovascular inflammation as a potential therapeutic target. Translational PerspectiveThis study highlights the 129/Sv mouse strain as a superior translational model compared to the widely used C57BL/6J strain, which, despite being a standard in cardiovascular research, fails to reliably reproduce severe hypertensive organ complications. The 129/Sv strain closely mimics human hypertensive damage, including cerebral small vessel disease, nephropathy, cardiomyopathy, and retinopathy. Transcriptomic analysis of cerebral microvessels identifies maladaptive inflammation as a critical mechanistic driver of susceptibility. These findings underline the clinical relevance of genetic predisposition, improving risk stratification and providing a robust preclinical platform to develop targeted anti-inflammatory therapies aimed at preventing hypertension-induced end-organ damage in patients.

pathology↗

bioRxiv2024

THE FAM53C/DYRK1A axis regulates the G1/S transition of the cell cycle

A growing number of therapies are being developed to target the cell cycle machinery for the treatment of cancer and other human diseases. Consequently, a greater understanding of the factors regulating cell cycle progression becomes essential to help enhance the response to these new therapies. Here, using data from the Cancer Dependency Map, we identified FAM53C as a new regulator of cell cycle progression. We found that FAM53C is critical for this cell cycle transition and that it acts upstream of the CyclinD-CDK4/6-RB axis and of p53 in the regulation of the G1/S transition. By mass spectrometry, biochemical, and cellular assays, we identified and validated DYRK1A as a cell cycle kinase that is inhibited by and directly interacts with FAM53C. Consistent with the role for FAM53C identified in cells in culture, FAM53C knockout human cortical organoids display increased cell cycle arrest and growth defects. Fam53C knockout mice show minor behavioral phenotypes. Because DYRK1A dysregulation contributes to developmental disorders such as Down syndrome as well as tumorigenesis, future strategies aiming at regulating FAM53C activity may benefit a broad range of patients.

cell biology↗

bioRxiv2024

Tuning the contractility and deformation modes of active actin-based assemblies in vitro: from 2D active networks to liquid crystal drops

Actin cytoskeleton-based materials are widely investigated as model cellular materials to elucidate physical mechanisms of cell mechanics, such as shape regulation and force production, as well as intriguing soft polymeric materials. In this method, we detail creating actin-based assemblies in vitro using purified protein for fluorescence microscopy studies. We polymerize long actin filaments in a sample chamber and use a polymer depletant to crowd filaments into a 2D-entangled network against a surface passivated with a surfactant layer. Adding skeletal muscle myosin II filaments in the presence of ATP induces contraction of the actin network. By bundling actin filaments with cross-linker, we tune the contractility of the assembly, transitioning from a material that buckles to a material that slides at the microscale. By reducing the length of the actin filaments through co-polymerizing actin in the presence of capping protein, we tune the material from being a 2D network to a liquid crystal. Cross-linking of dispersed short actin filaments results in 3D liquid crystal droplet formation.

biophysics↗

bioRxiv2024

Piezo2 in sensory neurons influences systemic and adipose tissue metabolism

Systemic metabolism ensures energy homeostasis through inter-organ crosstalk regulating thermogenic adipose tissue. Unlike the well-described inductive role of the sympathetic system, the inhibitory signal ensuring energy preservation remains poorly understood. Here, we show that, via the mechanosensor Piezo2, sensory neurons regulate morphological and physiological properties of brown and beige fat and prevent systemic hypermetabolism. Targeting Runx3/PV sensory neurons in independent genetic mouse models resulted in a systemic metabolic phenotype characterized by reduced body fat and increased insulin sensitivity and glucose tolerance. Deletion of Piezo2 in PV sensory neurons reproduced the phenotype, protected against high-fat diet-induced obesity and caused adipose tissue browning and beiging, likely driven by elevated norepinephrine levels. Finding that brown and beige fat are innervated by Runx3/PV sensory neurons expressing Piezo2, suggests a model where mechanical signals sensed by Piezo2 in sensory neurons protect energy storages and prevent a systemic metabolic phenotype. HighlightsLack of Runx3/PV sensory neurons reduces body fat and fasting glucose levels and increases glucose tolerance in mice Mechanosensitive ion channel PIEZO2 in PV sensory neurons plays an important role in systemic metabolism under physiological and pathological conditions PIEZO2 in PV sensory neurons regulates thermogenic programs and glucose uptake in brown and beige adipose tissues Brown and beige adipose tissues are innervated by Runx3/PV sensory neurons

physiology↗

bioRxiv2024

Cerebral Small Vessel Disease genetic determinant TRIM47 controls brain homeostasis via the NRF2 antioxidant system

Cerebral small vessel disease (cSVD) is a leading cause of stroke, cognitive decline and dementia, for which no specific mechanism-based treatments are available to date. Genome-wide and whole-exome association studies previously identified robust associations of common variants at chr17q25 with cSVD features on magnetic resonance imaging, with converging bioinformatic and experimental data for a causal involvement of TRIM47. Preliminary functional evaluation of TRIM47, an ubiquitin ligase enriched in brain endothelial cells (ECs), suggested its potential role in blood brain barrier (BBB) integrity. Here, we show that TRIM47 regulates brain EC resilience and adaptive responses to oxidative stress by binding to KEAP1, stabilizing NRF2 protein levels and promoting the NRF2 antioxidant signaling pathway. In vivo, Trim47-deficient mice exhibit downregulation of NRF2 target genes, BBB dysfunction, astrogliosis and cognitive impairments. Endothelial-specific deletion of Trim47 recapitulates these phenotypes. Treatment with the NRF2 activator tert- butylhydroquinone normalized BBB integrity and cognitive function in Trim47-deficient mice, highlighting the role of endothelial TRIM47 in driving brain homeostasis through NRF2 pathway activation. This work indicates that loss of the protective TRIM47/NRF2 axis may increase the susceptibility to developing human cSVD and that targeting the TRIM47/NRF2 axis could be a promising therapeutic approach for vascular cognitive impairment and dementia.

neuroscience↗

bioRxiv2023

Bone elongation in the embryo occurs without column formation in the growth plate

Chondrocyte columns, which are a hallmark of growth plate architecture, play a central role in bone elongation. Columns are formed by clonal expansion following rotation of the division plane, resulting in a stack of cells oriented parallel to the growth direction. However, despite decades of research, column structure has thus far been studied only in two dimensions. To fill this knowledge gap, we analyzed hundreds of Confetti multicolor clones in growth plates of mouse embryos using a pipeline comprising 3D imaging and algorithms for morphometric analysis. Surprisingly, analysis of the elevation angles between neighboring pairs of cells revealed that most cells did not display the typical stacking pattern associated with column formation, implying incomplete rotation of the division plane. Morphological analysis revealed that although embryonic clones were elongated, they formed clusters oriented perpendicular to the growth direction. Analysis of growth plates of postnatal mice revealed both complex columns, composed of both ordered and disordered cell stacks, and small, disorganized clusters located in the outer edges. Our finding that embryonic growth plates function without forming columns suggests that longitudinal bone growth is regulated by different cellular mechanisms during pre- and postnatal development. Moreover, the observed complex columnar and cluster arrangements may serve other, yet unknown morphogenetic functions. More broadly, our findings provide a new understanding of the cellular mechanisms underlying growth plate activity and bone elongation during development.

developmental biology↗

bioRxiv2023

Epidermal SIRT1 and BDNF modulate mechanical allodynia in mouse models of diabetic neuropathy

Diabetic neuropathy is a debilitating disorder characterized by spontaneous and mechanical pain. The role of skin mechanoreceptors in the development of mechanical pain (allodynia) is unclear. We discovered that mice with diabetic neuropathy had decreased sirtuin 1 (SIRT1) deacetylase activity in foot skin, leading to reduced expression of brain-derived neurotrophic factor (BDNF) and subsequent loss of innervation in Meissner corpuscles, a mechanoreceptor expressing the BDNF receptor TrkB. When SIRT1 was depleted from skin, the mechanical allodynia worsened in diabetic neuropathy mice, likely due to retrograde degeneration of the Meissner-corpuscle innervating A{beta} axons and aberrant formation of Meissner corpuscles which may have increased the mechanosensitivity. The same phenomenon was also noted in skin BDNF knockout mice. Furthermore, overexpression of SIRT1 in skin induced Meissner corpuscle reinnervation and regeneration, resulting in significant improvement of diabetic mechanical allodynia. Overall, the findings suggested that skin-derived SIRT1 and BDNF function in the same pathway in skin sensory apparatus regeneration and highlighted the potential of developing topical SIRT1-activating compounds as a novel treatment for diabetic mechanical allodynia.

neuroscience↗

bioRxiv2022

B-Myb association with DNA is mediated by its negative regulatory domain and Cdk phosphorylation

B-Myb is a highly conserved member of the vertebrate Myb family of transcription factors that plays a critical role in cell-cycle progression and proliferation. Myb proteins activate Myb-dependent promoters by interacting specifically with Myb binding site (MBS) sequences using their DNA binding domain (DBD). Transactivation of MBS promoters by B-Myb is repressed by its negative regulatory domain (NRD), and phosphorylation of the NRD by Cdk2-CyclinA relieves the repression to activate B-Myb dependent promoters. The structural mechanisms underlying autoinhibition and activation have been poorly characterized. We determined that a region in the B-Myb NRD (residues 510-600) directly associates with the DBD and inhibits DBD binding to the MBS DNA sequence. We demonstrate that phosphorylation of the NRD at T515, T518, and T520 is sufficient to disrupt the interaction between the NRD and the DBD, which results in increased affinity for MBS DNA and increased B-Myb-dependent promoter activation. Our biochemical characterization of B-Myb autoregulation and the activating effects of phosphorylation provides insight into how B-Myb functions as a site-specific transcription factor.

biochemistry↗

bioRxiv2022

Structural basis for tunable affinity and specificity of LxCxEdependentprotein interactions with the retinoblastoma protein family

The retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene expression during the cell cycle. Functional inactivation of Rb family proteins leads to loss of cell-cycle control and promotes genome instability and proliferation, which are hallmarks of cancer. Rb proteins share a structural domain, known as the "pocket domain", which mediates association with a large number of cellular proteins. A cleft in the pocket domain binds an LxCxE sequence motif in these proteins, many of which function with Rb proteins to co-regulate transcription during quiescence and G1. Proteins from oncogenic DNA viruses also bind this cleft to inactivate Rb family proteins, and the E7 protein from the human papillomavirus has been used as the primary model for understanding LxCxE motif interactions. Evidence presented previously and here demonstrates that the E7 sequence binds tighter and competes with cellular proteins for the LxCxE site, but the molecular basis for the relatively weak affinities of the cellular proteins has been poorly understood. Here, we present binding data and a series of crystal structures of the p107 pocket domain in complex with LxCxE peptides derived from the transcriptional co-repressor proteins HDAC1, ARI4DA, and EID1. The structures reveal determinants of protein binding beyond the L-C-E core that include the N and C-terminal sides of the LxCxE motif and the x (wild card) residues. Our results explain how differences in these interactions outside the L-C-E core result in weaker affinities of pocket protein for cellular LxCxE proteins compared to E7 and result in differences in Rb and p107 affinities for some LxCxE-containing sequences. Our study provides new insights into how Rb proteins bind their myriad of cell partners and lays the foundation for structure-based design of peptides that inhibit oncogenic viral protein interactions.

biochemistry↗

bioRxiv2022

The petrous bone contains high concentrations of osteocytes: one possible reason why ancient DNA is better preserved in this bone.

The characterization of ancient DNA in fossil bones is providing invaluable information on the genetics of past human and other animal populations. These studies have been aided enormously by the discovery that ancient DNA is relatively well preserved in the petrous bone compared to most other bones. The reasons for this better preservation are however not well understood. Here we examine the hypothesis that one reason for better DNA preservation in the petrous bone is that fresh petrous bone contains more DNA than other bones. We therefore determined the concentrations of osteocyte cells occluded inside lacunae within the petrous bone and compared these concentrations to other bones from the domestic pig using high resolution microCT. We show that the concentrations of osteocyte lacunae in the inner layer of the pig petrous bone adjacent to the otic chamber are about three times higher than in the temporal bone, as well as the cortical bone of the femur. The sizes and shapes of the lacuna in the inner layer of the petrous bone are similar to those in the femur. We also confirm that the petrous bone lacunae do contain osteocytes using a histological stain for DNA. We therefore conclude that one possible reason for better preservation of ancient DNA in the petrous bone is that this bone initially contains up to three times more DNA than other bones, and hence during diagenesis more DNA is likely to be preserved. We also note that the osteocytes in the inner layer of the petrous bone may have a function in hearing.

zoology↗

bioRxiv2021

Neonatal enthesis healing involves non-inflammatory formation of acellular scar through ECM secretion by resident cells

Wound healing is a well-orchestrated process that typically recruits the immune and vascular systems to restore the structure and function of the injured tissue. Injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, thereby severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Here, we established an injury model to the Achilles entheses of neonatal mice to study the possibility that at an early age, the enthesis can heal more effectively. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, neonatal enthesis healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including ER stress, autophagy and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and TUNEL assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. Collectively, these findings reveal a novel healing mechanism in neonatal entheses, whereby local ECM secretion by resident cells forms an acellular ECM deposit in the absence of inflammation markers, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries.

molecular biology↗

bioRxiv2020

3D MAPs discovers the morphological sequence chondrocytes undergo in the growth plate and the regulatory role of GDF5 in this process

The activity of the epiphyseal growth plates, which drive longitudinal growth of long bones, is dependent on the ability of chondrocytes to change their shape and size extensively as they differentiate. However, organ size, extracellular matrix density and cell number have hindered the study of chondrocyte morphology. Here, we describe a new pipeline called 3D Morphometric Analysis for Phenotypic significance (3D MAPs), which overcomes these obstacles. By using 3D MAPs, we have created an image database of hundreds of thousands of cells from orthologous long bones. Analysis of this database revealed the growth strategies that chondrocytes use during differentiation. We found that chondrocytes employed both allometric and isometric growth, and that allometric growth is achieved by changes either in volume or surface area along a specific cell axis in a zone-specific manner. Additionally, we discovered a new organization of chondrocytes within the growth plate, where cells are orientated such that their longest axis always aligns with the dorsal-ventral axis of the bone. To demonstrate the ability of 3D MAPs to explore mechanisms of growth plate regulation, we studied the abnormally short tibiae of Gdf5-null mice. 3D MAPs identified aberrant cellular growth behaviors which resulted in a 3-fold reduction in volumetric cell growth, as well as affected cell morphology and orientation, highlighting GDF5 as a new regulator of growth plate activity. Overall, our findings provide new insight into the morphological sequence that chondrocytes undergo during differentiation and highlight the ability of 3D MAPs to uncover molecular and cellular mechanisms regulating this process. More broadly, this work provides a new framework for studying growth plate biology.

developmental biology↗

bioRxiv2018

Multiscale analysis of 3D nuclear morphology reveals new insights into growth plate organization in mice

The shape of the nucleus is tightly associated with cell morphology, the mechanical environment, and differentiation and transcriptional states. Yet, imaging of nuclei in three dimensions while preserving the spatial context of the tissue has been highly challenging. Here, using the embryonic tibial growth plate as a model for cell differentiation, we study nuclear morphology by imaging cleared samples by light-sheet fluorescence microscopy. Next, we quickly segmented tens of thousands of nuclei using several open-source tools including machine learning. Finally, segmented nuclei underwent morphometric analysis and 3D spatial reconstruction using newly designed algorithms. Our method revealed differences in nuclear morphology between chondrocytes at different differentiation stages. Additionally, we identified different morphological patterns in opposing growth plates, such as gradients of volume and surface area, as well as features characteristic of specific growth plate zones, such as sphericity and orientation. Altogether, this work supports a link between nuclear morphology and cell differentiation. Moreover, it demonstrates the suitability of our approach for studying the relationships between nuclear morphology and organ development.\n\nAuthor summaryThere has been a growing interest in the relationship between nuclear morphology and its regulation of gene expression. However, to study global patterns of nuclear morphology within a tissue we must address the problem of acquiring and analyzing multiscale data, ranging from the tissue level through to subcellular resolution. We have established a new pipeline that enables acquisition and segmentation of hundreds of thousands of nuclei at a resolution that allows quantitative analysis. Moreover we have developed new algorithms that allow superimposing morphological aspects of hundreds of thousands of nuclei onto a visual representation of the entire tissue, allowing us to study nuclear morphology at an organ level. Using mouse growth plates as a model for the relationship between nuclear morphology and tissue differentiation, we show that nuclei change different aspects of their morphology during chondrocyte differentiation. Growth plates are usually described generically in the literature, suggesting they lack unique characteristics. We challenge this dogma by showing that morphological features such as volume distribute differently in opposing growth plates. Altogether, this work highlights the possible role of nuclear shape in the regulation of cell differentiation and demonstrates that our approach enables the study of nuclear morphology patterns within a tissue.

developmental biology↗

bioRxiv2018

Bone Morphology is Regulated Modularly by Global and Regional Genetic Programs

During skeletogenesis, a variety of protrusions of different shapes and sizes develop on the surfaces of long bones. These superstructures provide stable anchoring sites for ligaments and tendons during the assembly of the musculoskeletal system. Despite their importance, the mechanism by which superstructures are patterned and ultimately give rise to the unique morphology of each long bone is far from understood. In this work, we provide further evidence that long bones form modularly from Sox9+ cells, which contribute to their substructure, and from Sox9+/Scx+ progenitors that give rise to superstructures. Moreover, we identify components of the genetic program that controls the patterning of Sox9+/Scx+ progenitors and show that this program includes both global and regional regulatory modules.\n\nUsing light sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the Sox9+/Scx+ progenitors to the formation of bone superstructures. Additionally, by combining literature-based evidence and comparative transcriptomic analysis of different Sox9+/Scx+ progenitor populations, we identified genes potentially involved in patterning of bone superstructures. We present evidence indicating that Gli3 is a global regulator of superstructure patterning, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work coordinately. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning that further supports the notion that long bone development is a modular process.

developmental biology↗