Cytoskeletal Dynamics Research Articles

Regulation and functions of the DLC family of RhoGAP proteins: Implications for development and cancer.

The DLC (Deleted in Liver Cancer) family of RhoGAP (Rho GTPase-activating) proteins has been extensively studied since the identification of the first family member nearly 30 years ago. Rho GTPase signaling is essential for various cellular processes, including cytoskeletal dynamics, cell migration, and proliferation. Members of the DLC family are key regulators of this signaling pathway, with well-established roles in development and carcinogenesis. Here, we provide a comprehensive review of research into DLC regulation and cellular functions over the last three decades. In particular, we summarize control mechanisms of DLC gene expression at both the transcriptional and post-transcriptional level. Additionally, recent advances in understanding the post-translational regulation of DLC proteins that allow for tuning of protein activity and localization are highlighted. This detailed overview will serve as resource for future studies aimed at further elucidating the complex regulatory mechanisms of DLC family proteins and exploring their potential as targets for therapeutic applications.

STEM-03. TARGETING F-ACTIN DYNAMICS OF GLIOBLASTOMAS TO OVERCOME THERAPY RESISTANCE: IMPACTS ON DNA REPAIR AND GLIOMA STEM CELL PHENOTYPE

Abstract Glioblastoma (GBM) is a highly aggressive brain tumor with high recurrence and resistance to therapies. The F-actin cytoskeleton in GBM is related to cell invasion and the glioma stem cell (GSC) phenotype, affecting self-renewal and migration. Our research identifies a novel role for the F-actin pathway in DNA repair, impacting genomic stability and contributing to therapy resistance. Understanding F-actin’s role is crucial for developing targeted strategies to improve GBM treatment outcomes. This study examines the effects of pharmacological F-actin depolymerization on reversing resistance to ionizing radiation (IR) and temozolomide (TMZ) in GBM spheroids. We established resistant sublines of GBM U87-MG cells through IR and TMZ cycles, observing significant resistance increases via MTT assays. Also, resistant cells had enhanced spheroid formation capacity, with higher size and proliferation. Alterations in DNA repair and F-actin pathway were observed, with intensified and disorganized F-actin polymerization and efficient repair in resistant cells. Elevated expression of stemness markers (Nestin, CD133, and CD44) indicated a GSC-like phenotype in resistant cells, corroborated by increased tumorigenicity and de novo spheroid formation. These markers were further enhanced by DNA damage. Treating cells with actin polymerization inhibitors reduced resistance to TMZ and IR, partly due to impaired DNA repair capacity. Additionally, F-actin disassembly affected stemness marker expression, reinforcing the link between cytoskeletal dynamics and the GSC phenotype. Our findings suggest that F-actin dynamic is crucial for resistance in GBM. Targeting F-actin could resensitize recurrent GBM tumours by diminishing DNA repair capabilities and affecting the GSC-like phenotype, offering new therapeutic avenues.

Implementation of actin polymerization and depolymerization in a two-dimensional cell migration model and its implications on mammalian cell morphology and velocity

Cell migration, a pivotal process in wound healing, immune response, and even cancer metastasis, manifests through intricate interplay between morphology, speed, and cytoskeletal dynamics. Mathematical modeling emerges as a powerful tool to dissect these complex interactions. This work presents a two-dimensional immersed boundary model for mammalian cell migration, incorporating both filamentous actin (F-actin) and monomeric actin (G-actin) to explicitly capture polymerization and depolymerization. This model builds upon our previous one-dimensional efforts, now enabling us to explore the impact of G-actin on not just cell velocity but also morphology. We compare predictions from both models, revealing that while the one-dimensional model captures core dynamics along the cell’s axis, the two-dimensional model excels in portraying cell shape evolution and transverse variations in actin concentration and velocity. Our findings highlight the crucial role of including G-actin in shaping cell morphology. Actin velocity aligned with migration direction elongates the cell, while velocity normal to the membrane promotes spreading. Importantly, the model establishes a link between these microscopic aspects and macroscopic observables like cell shape, offering a deeper understanding of cell migration dynamics. This work not only provides a more comprehensive picture of cell migration but also paves the way for future studies exploring the interplay of actin dynamics, cell morphology, and biophysical parameters in diverse biological contexts.

Abl kinases regulate FGF signaling independent of Crk phosphorylation to prevent Peters anomaly.

Peters anomaly, the most common cause of congenital corneal opacity, stems from corneal-lenticular adhesion. Despite numerous identified mutations, a cohesive molecular framework of the disease's etiology remains elusive. Here, we identified Abl kinases as pivotal regulators of FGF signaling, as genetic ablation of Abl kinases restores lens induction even in the absence of FGF signaling. Intriguingly, both Abl kinase deficiency and increased FGF-Ras activity result in Peters anomaly independent of ERK signaling, which can be rescued by allelic deletion of Abl substrate, Crk. However, contrary to the prevailing belief that Abl kinases regulate Crk proteins by direct phosphorylation, mutations at Abl kinase phosphorylation sites on Crk and CrkL did not yield any observable effects. Instead, our findings reveal that Abl kinases phosphorylate Ptpn12, which in turn inhibits p130Cas phosphorylation and Crk recruitment, crucial for Rho GTPases activation and cytoskeletal dynamics. Consequently, Abl kinase deficiency reduces actomyosin contractility within the lens vesicle and genetically interacts with RhoA inhibition. Conversely, Rac1 deletion mitigates Peters anomaly in models with aberrant FGF, Abl kinase and RhoA signaling. Our results demonstrate that Abl kinases regulate FGF signaling to balance RhoA and Rac1 activity via the Ptpn12-p130Cas pathway, suggesting that targeting tension-mediated lens vesicle separation could be a therapeutic strategy for Peters anomaly.

A Novel Single-Color FRET Sensor for Rho-Kinase Reveals Calcium-Dependent Activation of RhoA and ROCK.

Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to PKA activity. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity and depleting intracellular calcium with EGTA decreased RhoKAR activity. We also investigated the signaling intermediates in this process. Blocking calmodulin or CaMKII prevented calcium-dependent activation of ROCK. These results indicate that ROCK activity is increased by calcium in fibroblasts and that this activation occurs downstream of CaM/CaMKII.

RHO subfamily of small GTPases in the development and function of hematopoietic cells.

RHOA, RHOB, and RHOC comprise a subfamily of RHO GTPase proteins famed for controlling cytoskeletal dynamics. RHO proteins operate downstream of multiple signals emerging from the microenvironment, leading to diverse cell responses, such as proliferation, adhesion, and migration. Therefore, RHO signaling has been centrally placed in the regulation of blood cells. Despite their high homology, unique roles of RHOA, RHOB, and RHOC have been described in hematopoietic cells. In this article, we overview the contribution of RHO proteins in the development and function of each blood cell lineage. Additionally, we highlight the aberrations of the RHO signaling pathways found in hematological malignancies, providing clues for the identification of new therapeutic targets.

Assessing sarcoma cell cytoskeleton remodeling in response to varying collagen concentration

Sarcomas, rare malignant tumors of mesenchymal origin, are often underdiagnosed and have face diagnostic ambiguities and limited treatment options. The main objective of this study was to define the nanomechanical and biophysical properties of sarcoma cells, particularly examining how the cytoskeleton's remodeling and related cellular processes such as cell migration and invasion in response to environmental stimuli due to collagen content. Utilizing one murine fibrosarcoma and one osteosarcoma cell line we employed atomic force microscopy, immunostaining, advanced image processing, in vitro cellular assays, and molecular techniques to investigate cells' cytoskeleton remodeling in response to varying collagen concentration. Our study focused on how alterations in collagen content affects the cytoskeletal dynamics and correlate with changes in gene expression profiles relevant to metastasis and an aggressive cancer phenotypes. Our findings indicate that despite their shared classification, fibrosarcoma and osteosarcoma cells display distinct biophysical properties and respond differently to mechanical forces. Notably, this difference in cellular behavior renders mechanical properties a potent novel biomarkers. Furthermore, the metastasis-related identified genes related to metastatic capability, could be potential therapeutic targets. This study highlights the significance of understanding the unique traits of sarcoma cells to improve diagnostic precision and expand therapeutic strategies, for this rare type of cancer.

How Parkinson's Disease-Linked LRRK2 Mutations Affect Different CNS Cell Types.

LRRK2 is a relatively common genetic risk factor for Parkinson's disease (PD), with six coding variants known to cause familial PD. Non-coding variation at the same locus is also associated with sporadic PD. LRRK2 plays a role in many different intracellular signaling cascades including those involved in endolysosomal function, cytoskeletal dynamics, and Ca2+ homeostasis. PD-causing LRRK2 mutations cause hyperactive LRRK2 kinase activity, resulting in altered cellular signaling. Importantly, LRRK2 is lowly expressed in neurons and prominently expressed in non-neuronal cells in the brain. In this review, we will summarize recent and novel findings on the effects of PD-causing LRRK2 mutations in different nervous system cell types. This review will also provide novel insight into future areas of research at the intersection of LRRK2 cell biology, cell type specificity, and PD.

Mechanism and regulation of kinesin motors.

Kinesins are a diverse superfamily of microtubule-based motors that perform fundamental roles in intracellular transport, cytoskeletal dynamics and cell division. These motors share a characteristic motor domain that powers unidirectional motility and force generation along microtubules, and they possess unique tail domains that recruit accessory proteins and facilitate oligomerization, regulation and cargo recognition. The location, direction and timing of kinesin-driven processes are tightly regulated by various cofactors, adaptors, microtubule tracks and microtubule-associated proteins. This Review focuses on recent structural and functional studies that reveal how members of the kinesin superfamily use the energy of ATP hydrolysis to transport cargoes, depolymerize microtubules and regulate microtubule dynamics. I also survey how accessory proteins and post-translational modifications regulate the autoinhibition, cargo binding and motility of some of the best-studied kinesins. Despite much progress, the mechanism and regulation of kinesins are still emerging, and unresolved questions can now be tackled using newly developed approaches in biophysics and structural biology.

Characterization and Physiological Differences of Two Primary Cultures of Human Normal and Hypertrophic Scar Dermal Fibroblasts: A Pilot Study.

Background/Objectives: Dermal fibroblasts (DFs) are key participants in skin hypertrophic scarring, and their properties are being studied to identify the molecular and cellular mechanisms underlying the pathogenesis of skin scarring. Methods: In the present work, we performed a comparative analysis of DFs isolated from normal skin (normal dermal fibroblasts, NDFs), and hypertrophic scar skin (hypertrophic scar fibroblasts, HTSFs). The fibroblasts were karyotyped and phenotyped, and experiments on growth rate, wound healing, and single-cell motility were conducted. Results: Comparative analysis revealed a minor karyotype difference between cells. However, HTSFs are characterized by higher proliferation level and motility compared to NDFs. These significant differences may be associated with quantitative and qualitative differences in the cell secretome. A proteomic comparison of NDF and HTSF found that differences were associated with metabolic proteins reflecting physiological differences between the two cells lines. Numerous unique proteins were found only in the vesicular phase of vHTSFs. Some proteins involved in cell proliferation (protein-glutamine gamma-glutamyltransferase K) and cell motility (catenin delta-1), which regulate gene transcription and the activity of Rho family GTPases and downstream cytoskeletal dynamics, were identified. A number of proteins which potentially play a role in fibrosis and inflammation (mucin-5B, CD97, adhesion G protein-coupled receptor E2, antileukoproteinase, protein S100-A8 and S100-A9, protein caspase recruitment domain-containing protein 14) were detected in vHTSFs. Conclusions: A comparative analysis of primary cell cultures revealed their various properties, especially in the cell secretome. These proteins may be considered promising target molecules for developing treatment or prevention strategies for pathological skin scarring.

Naa80 is required for actin N-terminal acetylation and normal hearing in zebrafish.

Actin is a critical component of the eukaryotic cytoskeleton. In animals, actins undergo unique N-terminal processing by dedicated enzymes resulting in mature acidic and acetylated forms. The final step, N-terminal acetylation, is catalyzed by NAA80 in humans. N-terminal acetylation of actin is crucial for maintaining normal cytoskeletal dynamics and cell motility in human cell lines. However, the physiological impact of actin N-terminal acetylation remains to be fully understood. We developed a zebrafish naa80 knockout model and demonstrated that Naa80 acetylates both muscle and non-muscle actins in vivo. Assays with purified Naa80 revealed a preference for acetylating actin N-termini. Zebrafish lacking actin N-terminal acetylation exhibited normal development, morphology, and behavior. In contrast, humans with pathogenic actin variants can present with hypotonia and hearing impairment. Whereas zebrafish lacking naa80 showed no obvious muscle defects or abnormalities, we observed abnormal inner ear development, small otoliths, and impaired response to sound. In conclusion, we have established that zebrafish Naa80 N-terminally acetylates actins in vitro and in vivo, and that actin N-terminal acetylation is essential for normal hearing.

BONCAT-iTRAQ Labelling Reveals Molecular Markers of Adaptive Responses in Toxoplasma gondii to Pyrimethamine Treatment.

We employed a BONCAT-iTRAQ labelling approach to investigate newly synthesised proteins (NSPs) in Toxoplasma gondii subjected to varying concentrations of the antifolate drug pyrimethamine. Our results reveal that numerous NSPs exhibited altered expression levels in response to the drug, with significant upregulation observed at higher concentrations. Key proteins involved in protein synthesis, stress responses, energy metabolism, and cytoskeletal dynamics were identified, indicating that T. gondii undergoes complex adaptive responses to pyrimethamine treatment. While some of the identified pathways reflect a generic stress response, this study provides important molecular markers and mechanistic insights specific to the parasite's adaptation strategies. These findings contribute to understanding how T. gondii modulates its proteome in response to drug-induced stress and lay the groundwork for further investigations into potential therapeutic targets.

MiRNA expression profiling reveals a potential role of microRNA-148b-3p in cerebral vasospasm in subarachnoid hemorrhage

Cerebral vasospasm (CVS) is an important contributor to delayed cerebral ischemia following aneurysmal subarachnoid hemorrhage (aSAH), leading to high morbidity and long-term disability. While several microRNAs (miRNAs) have been implicated in vasospasm, the underlying mechanisms for CVS remain poorly understood. Our study aims to identify miRNAs that may contribute to the development of CVS. Whole-blood samples were obtained during or outside of vasospasm from aSAH patients whose maximal vasospasm was moderate or severe. MiRNAs were isolated from serial whole-blood samples, and miRNA sequencing was performed. Differentially expressed miRNAs were identified and the expression levels in patients’ samples were verified using real-time qPCR. The biological functions of identified miRNA were evaluated in human brain endothelial cells (HBECs). MiRNA profiling revealed significant upregulation of miR-148b-3p in patients during CVS. We demonstrated that miR-148b-3p directly targeted and decreased the expression of ROCK1, affecting cell proliferation, migration, and invasion of HBECs through the ROCK-LIMK-Cofilin pathway. We propose that the upregulation of miRNA-148b-3p plays a role in the development of CVS by regulating actin cytoskeletal dynamics in HBECs, which is crucial for vascular function. Our study highlights miR-148b-3p as a potential diagnostic marker as well as therapeutic target for CVS following aSAH.

Septins: Structural Insights, Functional Dynamics, and Implications in Health and Disease.

Septins are a class of proteins with diverse and vital roles in cell biology. Structurally, they form hetero-oligomeric complexes and assemble into filaments, contributing to the organization of cells. These filaments act as scaffolds, aiding in processes like membrane remodeling, cytokinesis, and cell motility. Functionally, septins are essential to cell division, playing essential roles in cytokinetic furrow formation and maintaining the structural integrity of the contractile ring. They also regulate membrane trafficking and help organize intracellular organelles. In terms of physiology, septins facilitate cell migration, phagocytosis, and immune responses by maintaining membrane integrity and influencing cytoskeletal dynamics. Septin dysfunction is associated with pathophysiological conditions. Mutations in septin genes have been linked to neurodegenerative diseases, such as hereditary spastic paraplegias, underscoring their significance in neuronal function. Septins also play a role in cancer and infectious diseases, making them potential targets for therapeutic interventions. Septins serve as pivotal components of intracellular signaling networks, engaging with diverse proteins like kinases and phosphatases. By modulating the activity of these molecules, septins regulate vital cellular pathways. This integral role in signaling makes septins central to orchestrating cellular responses to environmental stimuli. This review mainly focuses on the human septins, their structural composition, regulatory functions, and implication in pathophysiological conditions underscores their importance in fundamental cellular biology. Moreover, their potential as therapeutic targets across various diseases further emphasizes their significance.

Arp2/3 complex activity enables nuclear YAP for naïve pluripotency of human embryonic stem cells.

Our understanding of the transitions of human embryonic stem cells between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of human embryonic stem cells (hESCs) as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for the actin ring, to establish uniform cell mechanics within naïve colonies, promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restores the actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

Role of Receptor for Advanced Glycation End-Products in Endometrial Cancer: A Review.

Endometrial cancer (EC) is the most common gynecological malignancy. EC is associated with metabolic disorders that may promote non-enzymatic glycation and activate the receptor for advanced glycation end-products (RAGE) signaling pathways. Thus, we assumed that RAGE and its ligands may contribute to EC. Of particular interest is the interaction between diaphanous-related formin 1 (Diaph1) and RAGE during the progression of human cancers. Diaph1 is engaged in the proper organization of actin cytoskeletal dynamics, which is crucial in cancer invasion, metastasis, angiogenesis, and axonogenesis. However, the detailed molecular role of RAGE in EC remains uncertain. In this review, we discuss epigenetic factors that may play a key role in the RAGE-dependent endometrial pathology. We propose that DNA methylation may regulate the activity of the RAGE pathway in the uterus. The accumulation of negative external factors, such as hyperglycemia, inflammation, and oxidative stress, may interfere with the DNA methylation process. Therefore, further research should take into account the role of epigenetic mechanisms in EC progression.

Effects of zinc in podocytes and cortical collecting duct in vitro and Dahl salt-sensitive rats in vivo

Zinc is one of the essential divalent cations in the human body and a fundamental microelement involved in the regulation of many cellular and subcellular functions. Experimental studies reported that zinc deficiency is associated with renal damage and could increase blood pressure. It was proposed that zinc dietary supplementation plays a renoprotective role. Our study aimed to investigate the effects of zinc on intracellular signaling in renal cells and explore the correlation between dietary zinc and the progression of salt-induced hypertension. The impact of extracellular zinc concentrations on two different kidney epithelial cell types - podocytes and principal cells of the cortical collecting duct (CCD), was tested. In podocytes, a rise in extracellular zinc promotes TRPC6 channel-mediated calcium entry but not altered intracellular zinc levels. However, we observe the opposite effect in CCD cells with no alteration in calcium levels and steady-state elevation in intracellular zinc. Moreover, prolonged extracellular zinc exposure leads to cytotoxic insults in CCD cells but not in podocytes, characterized by increased cell death and disrupted cytoskeletal organization. Next, we tested if dietary zinc plays a role in the development of hypertension in Dahl salt-sensitive rats. Neither zinc-rich nor deficient diets impact the regular development of salt-sensitive hypertension. These results suggest specialized roles for zinc in renal function, implicating its involvement in proliferation and apoptosis in CCD cells and calcium signaling and cytoskeletal dynamics modulation in podocytes. Further research is required to elucidate the detailed mechanisms of zinc action and its implications in renal health and disease.

IL-2 delivery to CD8+ T cells during infection requires MRTF/SRF-dependent gene expression and cytoskeletal dynamics

Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8+ T cell proliferation, but is required for sustained IL-2 dependent CD8+ effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8+ T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8+ T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8+ T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling.

New targets and designed inhibitors of ASAP Arf-GAPs derived from structural characterization of the ASAP1/440-kD ankyrin-B interaction

ASAP1 and its paralog ASAP2 belong to a PI4,5P2-dependent Arf GTPase-activating protein (Arf-GAP) family capable of modulating membrane and cytoskeletal dynamics. ASAPs regulate cell adhesive structures such as invadosomes and focal adhesions during cell attachment and migration. Malfunctioning of ASAP1 has been implicated in the malignant phenotypes of various cancers. Here, we discovered that the SH3 domain of ASAP1 or ASAP2 specifically binds to a 12-residue, positively charged peptide fragment from the 440 kDa giant ankyrin-B, a neuronal axon specific scaffold protein. The high-resolution structure of the ASAP1-SH3 domain in complex with the gAnkB peptide revealed a non-canonical SH3-ligand binding mode with high affinity and specificity. Structural analysis of the complex readily uncovered a consensus ASAP1-SH3 binding motif, which allowed the discovery of a number of previously unknown binding partners of ASAP1-SH3 including Clasp1/Clasp2, ALS2, β-Pix, DAPK3, PHIP, and Limk1. Fittingly, these newly identified ASAP1 binding partners are primarily key modulators of the cytoskeletons. Finally, we designed a cell-penetrating, highly potent ASAP1 SH3 domain binding peptide with a Kd ∼7 nM as a tool for studying the roles of ASAPs in different cellular processes.

Comprehensive profiling of migratory primordial germ cells reveals niche-specific differences in non-canonical Wnt and Nodal-Lefty signaling in anterior vs posterior migrants.

Mammalian primordial germ cells (PGCs) migrate asynchronously through the embryonic hindgut and dorsal mesentery to reach the gonads. We previously found that interaction with different somatic niches regulates PGC proliferation along the migration route. To characterize transcriptional heterogeneity of migrating PGCs and their niches, we performed single-cell RNA sequencing of 13,262 mouse PGCs and 7,868 surrounding somatic cells during migration (E9.5, E10.5, E11.5) and in anterior versus posterior locations to enrich for leading and lagging migrants. Analysis of PGCs by position revealed dynamic gene expression changes between faster or earlier migrants in the anterior and slower or later migrants in the posterior at E9.5; these differences include migration-associated actin polymerization machinery and epigenetic reprogramming-associated genes. We furthermore identified changes in signaling with various somatic niches, notably strengthened interactions with hindgut epithelium via non-canonical WNT (ncWNT) in posterior PGCs compared to anterior. Reanalysis of a previously published dataset suggests that ncWNT signaling from the hindgut epithelium to early migratory PGCs is conserved in humans. Trajectory inference methods identified putative differentiation trajectories linking cell states across timepoints and from posterior to anterior in our mouse dataset. At E9.5, we mainly observed differences in cell adhesion and actin cytoskeletal dynamics between E9.5 posterior and anterior migrants. At E10.5, we observed divergent gene expression patterns between putative differentiation trajectories from posterior to anterior including Nodal signaling response genes Lefty1, Lefty2, and Pycr2 and reprogramming factors Dnmt1, Prc1, and Tet1. At E10.5, we experimentally validated anterior migrant-specific Lefty1/2 upregulation via whole-mount immunofluorescence staining for LEFTY1/2 proteins, suggesting that elevated autocrine Nodal signaling accompanies the late stages of PGC migration. Together, this positional and temporal atlas of mouse PGCs supports the idea that niche interactions along the migratory route elicit changes in proliferation, actin dynamics, pluripotency, and epigenetic reprogramming.