Biology subjects

Smith, R. S.

Publications and source records attributed to Smith, R. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Molecular and functional profiling distinguishes PACS1 syndrome variant from PACS1 loss-of-function in iNeurons

PACS1 syndrome is a rare neurodevelopmental disorder caused by a recurrent de novo missense variant (p.R203W) in the PACS1 protein. However, it remains unclear whether the p.R203W variant acts through a loss-of-function or alternative mechanism. Here, we used isogenic iPSC-derived neurons (iNs) to directly compare the effects of PACS1 p.R203W to complete loss of PACS1 function. Using a combination of proteomic, biochemical and electrophysiological approaches, we identified molecular and functional phenotypes associated with each genotype. While PACS1(+/R203W) and PACS1(-/-) iNs shared phenotypic abnormalities, the overall molecular and functional consequences of the p.R203W variant were distinct from those caused by PACS1 deficiency. Notably, PACS1(+/R203W) presented with unique proteomic and kinase signaling signatures and a shift in stimulus dependent excitability. These findings demonstrate that PACS1 syndrome is not caused by a simple loss of function and instead support a non-loss-of-function mechanism. Lastly, our interactome analysis suggests that the p.R203W variant retains aspects of canonical PACS1 function while acquiring novel molecular interactions that could contribute to PACS1 syndrome pathogenesis. Altogether, these findings provide a framework for future mechanistic studies and therapeutic development in PACS1 syndrome. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/747101v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@d1522corg.highwire.dtl.DTLVardef@69e4dforg.highwire.dtl.DTLVardef@30eebcorg.highwire.dtl.DTLVardef@899b9d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

Maintenance of the leaf cell proliferation boundary by a cell size at division threshold mechanism.

The sizes and shapes of organs are established by the combined actions of cell proliferation and cell growth. In plants, development of the determinate planar leaf is initiated by primordia formation and establishment of abaxial/adaxial polarity [1,2,3]. Lamina outgrowth is driven by cell division and growth along proximo-distal (PD) and medio-lateral (ML) axes [4], established by mutually repressive PD gradients of miRNA and target transcription factors [5,6,7,8]. These gradients generate proximal regions of competence for cell division and increased growth, with distal regions of reduced growth, endoreduplication and differentiation. The transition from proliferation to growth and differentiation is marked by a cell cycle arrest front, which moves basipetally during leaf growth, progressively restricting proximal proliferative zones as the leaf grows [9,10,11]. Intersection of proximal proliferation-promoting gradients with distal differentiation-promoting gradients may delineate the arrest front, but its dynamics remain poorly understood. We reasoned that mutants affecting cell proliferation patterns may provide insights into the formation, maintenance and dissolution of the arrest front. Spatio-temporal modelling of live imaging data of loss of function mutants of the regulatory peptidase DA1 and its E3 ligase activator Big Brother (BB), which increase cell proliferation [12,13], showed that these proteins effectively establish a threshold cell size at division as a function of distance from the base of the growing leaf and the duration of growth. Loss of BB and DA1 activities increased the persistence of cell divisions and dissolved the arrest front. This suggested that the arrest front emerges from the interactions of threshold areas of cell division with the cessation of division over time, and not from an independently-specified boundary.

plant biology↗

bioRxiv2026

Evolution of moss leaf-like organs through variations in deeply conserved developmental principles

Leaves and leaf-like organs with laminar structures and determinate growth arose multiple times independently in land plants. The cellular basis of leaf development is well characterized in flowering plants, and molecular studies have shown that the plant hormone auxin plays a central role in this process, orchestrating cellular growth and differentiation. Auxin is also crucial for the formation of phyllids, the leaf-like organs of bryophytes, yet its precise role in morphogenesis remains unclear. More broadly, whether similar developmental principles are shared across distantly related evolutionary lineages is unknown. Here, we combine live-imaging, genetics, pharmacological treatments, and modeling to investigate the cellular and molecular basis of phyllid development in the model moss Physcomitrium patens. By tracking phyllid morphogenesis from a single initial cell to full maturity, we uncover the cellular growth dynamics underlying organ development. We demonstrate that auxin spatially inhibits cell divisions and promotes cellular elongation and differentiation. However, unlike in vascular plants, moss PIN transporters do not participate in polar auxin transport during phyllid development but mainly reduce intracellular auxin concentration. These findings indicate that while auxins role in organogenesis is conserved, its transport mechanisms have diverged across land plants. Overall, our study reveals shared principles of planar organ morphogenesis, highlighting how the repeated deployment of similar developmental strategies, with lineage-specific variations, drove the convergent evolution of leaves and leaf-like organs. One-Sentence SummaryAuxin modulates a conserved developmental program in moss leaf-like organs by spatiotemporally controlling cell division and growth.

plant biology↗

bioRxiv2026

Pannexin-1 channel activity regulates neurogenesis and cell survival in the developing cortex

Mutations in genes encoding a range of ion-conducting proteins disrupt development of the cerebral cortex in humans, often causing polymicrogyria (PMG); yet how ion conduction guides the development of cortical architecture is not clear. Here, we describe three individuals with brain malformations including PMG and microcephaly in whom de novo, missense mutations were identified in PANX1--encoding an ATP and ion conducting channel. We show that these PMG-associated PANX1 mutations (p.D14H, p.M37R, and p.N338T) disrupt normal glycosylation and confer gain-of-function with respect to ATP release and channel conductance. In vivo modeling of mutant PANX1 in cortical progenitor cells demonstrated disrupted cell migration and excess cell death in both mice and ferret models. In vitro modeling of the p.N338T allele in human induced pluripotent stem cell (hiPSC)-derived neurons further revealed changes in neuronal morphology and increased excitability, and dysregulation of key transcription factors critical for neurogenesis and cell fate. In addition, p.N338T iPSC-derived neural progenitor cells displayed increased PANX1 activation. Our results show that normal PANX1 function contributes to cortical structure through regulation of ion conductance and ATP release and provides insight into how these processes influence corticogenesis and cytoarchitecture more broadly.

neuroscience↗

bioRxiv2025

Outer epidermal edges mediate cell-cell adhesion for tissue integrity in plants

Cell-cell adhesion in plants is generally thought to be primarily mediated by the middle lamella, a supposedly thin adhesive layer of the cell wall. Here, we challenge this view. Through computational simulation we found that outer edges of cellular interfaces of the epidermis are hotspot for cell separating tensile stress. Characterization of the ultrastructure of those edges in planta revealed that they are locally thickened regions that harbor cellulose lamellae and pectin-based structural continuity across adjacent cells. We confirmed their dominant role for adhesion by studying mutants where those edges are defective and through direct mechanical testing. This reveals the key role of outer epidermal cell edges, rather than the middle lamella, in mediating cell-cell adhesion for tissue integrity in plants.

plant biology↗

bioRxiv2024

Rhamnogalacturonan-II dimerization deficiency impairs the coordination between growth and adhesion maintenance in plants

Cell adhesion is a fundamental feature of multicellular organisms. In plants, cell adhesion is mediated by the cell wall, but the control and maintenance of cell adhesion during growth and development remains poorly understood1. Here we uncover the role of a component of the cell wall, rhamnogalacturonan-II (RG-II) and its capacity to crosslink in the presence of Boron2, as a key regulator of plant cell adhesion maintenance. We show that RG-II dimerization deficiency leads to cell adhesion defects. Importantly, the analysis of mur1 mutants with RG-II dimerization defects uncovers a cell adhesion pathway that is distinct from that identified by the analysis of pectin deficient mutants3. We found that mutations in two cell wall integrity sensors, RESISTANCE TO FUSARIUM OXYSPORUM 1 and RECEPTOR-LIKE PROTEIN 44, as well as supplementation with the hormone brassinosteroid can partially rescue the adhesion defects associated with RG-II dimerization deficiency. We also show that adhesion defects associated with RG-II dimerization deficiency are related to increased epidermal tension as well as decreased homogalacturonan levels in the cell wall, which can also be rescued by supplementation with brassinosteroid. Overall, we propose that RG-II dimerization defects alter cell adhesion directly (reduced crosslinks) but also indirectly through cell wall integrity sensing, brassinosteroid signalling, cell wall remodelling and cell layer growth coordination. Thus, our results uncover the involvement of cell wall integrity sensors and hormonal signalling in the coordination between growth and adhesion maintenance in plants, which is a key feature for complex multicellularity. Highlights- RG-II dimerization is required for cell-cell adhesion in plants. - Cell wall integrity sensors and brassinosteroid signalling mediate cell adhesion downstream of RG-II dimerization. - Cell detachments due to defective RG-II dimerization are caused by weakened middle lamella and higher tissue tension.

plant biology↗

bioRxiv2024

Gene therapy for targeting a prenatally enriched potassium channel associated with severe childhood epilepsy and premature death

Dysfunction of the sodium-activated potassium channel KNa1.1 (encoded by KCNT1) is associated with a severe condition characterized by frequent seizures (up to hundreds per day) and is often fatal by age three years. We defined the early developmental onset of KNa1.1 channels in prenatal and neonatal brain tissue, establishing a timeline for pathophysiology and a window for therapeutic intervention. Using patch-clamp electrophysiology, we observed age-dependent increases in KNa1.1 K+conductance. In neurons derived from a child with a gain-of-function KCNT1 pathogenic variant (p.R474H), we detected abnormal excitability and action potential afterhyperpolarization kinetics. In a clinical trial, two individuals with the p.R474H variant showed dramatic reductions in seizure occurrence and severity with a first-in-human antisense oligonucleotide (ASO) RNA therapy. ASO-treated p.R474H neurons in vitro exhibited normalized spiking and burst properties. Finally, we demonstrated the feasibility of ASO knockdown of KNa1.1 in midgestation human neurons, suggesting potential for early therapeutic intervention before the onset of epileptic encephalopathy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/620125v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@eaf38corg.highwire.dtl.DTLVardef@11b7e3corg.highwire.dtl.DTLVardef@1fcce20org.highwire.dtl.DTLVardef@40d364_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

A myelinic channel system for organelle transport to the glial-axonal junction

Myelin sheaths comprise compacted layers of oligodendroglial membrane wrapped spirally around axons. Each sheath, if imagined unwrapped, has a cytoplasm-filled space at its perimeter, linking it to the oligodendrocyte soma via a short process. By electron microscopy (EM), this space, which we term the myelinic channel system contains microtubules and membranous organelles, but whether these are remnants of development or serve a function is unknown. Performing live imaging of myelinating oligodendrocytes expressing fluorescent reporters, we found that the myelinic channel system serves microtubule-dependent organelle transport. Further, the intra-myelinic movement of peroxisomes was modulated by neuronal electrical activity in these mixed neural cell cultures. Loss of oligodendroglial Kif21b or CNP in vivo led to apparent stasis of myelin organelles and secondary axon pathology. This suggests that oligodendrocytes require motor transport in myelin to maintain axonal integrity.

neuroscience↗

bioRxiv2024

Age-associated growth control modifies leaf proximodistal symmetry and enables leaf shape diversification

Biological shape diversity is often manifested in modulation of organ symmetry and modification of the patterned elaboration of repeated shape elements.1-5 Whether and how these two aspects of shape determination are coordinately regulated is unclear.5-7 Plant leaves provide an attractive system to investigate this problem, because they usually show asymmetries along their proximodistal axis, along which they can also produce repeated marginal outgrowths such as serrations or leaflets.1 One case of leaf shape diversity is heteroblasty, where the leaf form in a single genotype is modified with progressive plant age.8-11 In Arabidopsis thaliana, a plant with simple leaves, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9) controls heteroblasty by activating CyclinD3 expression, thereby sustaining proliferative growth and retarding differentiation in adult leaves.12 However, the precise significance of SPL9 action for leaf symmetry and marginal patterning is unknown. By combining genetics, quantitative shape analyses, and time-lapse imaging, we show that, in A. thaliana, proximodistal symmetry of the leaf blade decreases in response to an age-dependent SPL9 expression gradient, and that SPL9 action coordinately regulates the distribution and shape of marginal serrations and overall leaf form. Using comparative analyses, we demonstrate that heteroblastic growth reprogramming in Cardamine hirsuta, a complex-leafed relative of A. thaliana, also involves prolonging the duration of cell proliferation and delaying differentiation. We further provide evidence that SPL9 enables species-specific action of homeobox genes that promote leaf complexity. In conclusion, we identify an age-dependent layer of organ proximodistal growth regulation that modulates leaf symmetry and has enabled leaf shape diversification. In BriefAge-dependent regulation of proliferative growth and differentiation along the proximodistal axis of leaves modulates organ symmetry and marginal patterning in heteroblasty, and enabled the action of homeobox genes in complex leaf shape evolution. HighlightsO_LIAn age-dependent growth gradient underpins regulation of A. thaliana leaf symmetry C_LIO_LISPL9-mediated heteroblastic growth control potentiates leaf histogenic capacity C_LIO_LIA common growth framework underlies heteroblasty in complex and simple leaves C_LIO_LISPL9 enables the action of homeobox genes that promote leaf complexity C_LI

developmental biology↗

bioRxiv2024

SHOOTMERISTEMLESS autoactivation: A prerequisite for fruit metamorphosis

In animals and plants, organ shape is primarily determined during primordium development by carefully coordinated growth and cell division1-3. Rare examples of post-primordial change in morphology (reshaping) exist that offer tractable systems to study mechanisms required for organ-shape determination and diversification. One such example is the heart-shape formation of Capsella fruits that occurs by reshaping the ovate spheroid gynoecium upon fertilization4. Here we use whole-organ live-imaging to show that dynamic changes in growth and cell division coupled with local maintenance of meristematic identity drives Capsella fruit-shape formation. At the molecular level, we reveal an auxin-induced mechanism ultimately descending on a single cis regulatory element to mediate morphological alteration. This element resides in the promoter of the Capsella rubella SHOOTMERISTEMLESS5 (CrSTM) gene. The CrSTM meristem identity factor positively regulates its own expression through binding to this element thereby providing a feed-forward loop at the position and time when protrusions emerge to form the heart. Independent evolution of the STM-binding element in STM promoters across Brassicaceae species correlates with those undergoing a gynoecium-to-fruit metamorphosis. Accordingly, genetic and phenotypic studies showed that the STM-binding element is required to facilitate the shape transition and reveals a conserved molecular mechanism for organ morphogenesis.

plant biology↗

bioRxiv2024

A deep learning-based toolkit for 3D nuclei segmentation and quantitative analysis in cellular and tissue context

We present a new set of computational tools that enable accurate and widely applicable 3D segmentation of nuclei in various 3D digital organs. We developed a novel approach for ground truth generation and iterative training of 3D nuclear segmentation models, which we applied to popular CellPose, PlantSeg, and StarDist algorithms. We provide two high-quality models trained on plant nuclei that enable 3D segmentation of nuclei in datasets obtained from fixed or live samples, acquired from different plant and animal tissues, and stained with various nuclear stains or fluorescent protein-based nuclear reporters. We also share a diverse high-quality training dataset of about 10,000 nuclei. Furthermore, we advanced the MorphoGraphX analysis and visualization software by, among other things, providing a method for linking 3D segmented nuclei to their surrounding cells in 3D digital organs. We found that the nuclear-to-cell volume ratio varies between different ovule tissues and during the development of a tissue. Finally, we extended the PlantSeg 3D segmentation pipeline with a proofreading script that uses 3D segmented nuclei as seeds to correct cell segmentation errors in difficult-to-segment tissues. Summary StatementWe present computational tools that allow versatile and accurate 3D nuclear segmentation in plant organs, enable the analysis of cell-nucleus geometric relationships, and improve the accuracy of 3D cell segmentation.

plant biology↗

bioRxiv2023

Tradeoff Between Speed and Robustness in Primordium Initiation Mediated by Auxin-CUC1 Interaction

Robustness is the reproducible development of a phenotype despite stochastic noise. It often involves tradeoffs with other performance metrics, but the mechanisms underlying such tradeoffs were largely unknown. An Arabidopsis flower robustly develops four sepals from four precisely positioned auxin maxima. The development related myb-like 1 (drmy1) mutant generates noise in auxin signaling that disrupts robustness in sepal initiation. Here, we found that increased expression of CUP-SHAPED COTYLEDON1 (CUC1), a boundary specification transcription factor, in drmy1 underlies this loss of robustness. CUC1 surrounds and amplifies stochastic auxin noise in drmy1 to form variably positioned auxin maxima and sepal primordia. Removing CUC1 from drmy1 provides time for noisy auxin signaling to resolve into four precisely positioned auxin maxima, restoring robust sepal initiation. However, removing CUC1 decreases auxin maxima intensity and slows down sepal initiation. Thus, CUC1 increases morphogenesis speed but impairs robustness against auxin noise. Further, using a computational model, we found that the observed phenotype can be explained by the effect of CUC1 in repolarizing PIN FORMED1 (PIN1), a polar auxin transporter. Lastly, our model predicts that reducing global growth rate improves developmental robustness, which we validated experimentally. Thus, our study illustrates a tradeoff between speed and robustness during development.

developmental biology↗

bioRxiv2023

Transcriptional Control of Neocortical Size and Microcephaly

The mammalian neocortex differs vastly in size and complexity between mammalian species, yet the mechanisms that lead to an increase in brain size during evolution are not known. We show here that two transcription factors coordinate gene expression programs in progenitor cells of the neocortex to regulate their proliferative capacity and neuronal output in order to determine brain size. Comparative studies in mice, ferrets and macaques demonstrate an evolutionary conserved function for these transcription factors to regulate progenitor behaviors across the mammalian clade. Strikingly, the two transcriptional regulators control the expression of large numbers of genes linked to microcephaly suggesting that transcriptional deregulation as an important determinant of the molecular pathogenesis of microcephaly, which is consistent with the finding that genetic manipulation of the two transcription factors leads to severe microcephaly.

neuroscience↗

bioRxiv2023

Puzzle cell shape emerges from the interaction of growth with mechanical constraints

Puzzle-shaped epidermal cells not only reduce mechanical stress during organ growth but also record the expansion history of the tissue in their outlines. By combining mechanical simulations with time-lapse imaging, we show that transitions from directional to isotropic expansion induce new lobes to form along the previous growth axis, and that switching the order of anisotropic and isotropic phases yields hybrid shapes that reliably preserve those transitions. In maize, model predictions and live imaging coincide precisely, and in Ara-bidopsis, final lobe patterns correlate more with growth history than with cell size alone. Ge-netic or pharmacological disruption of lobe formation constrains leaf expansion or drives compensatory elongation, which underscores a mechanical function. A broad survey of living and fossil vascular plants reveals that the mechanism to make puzzle-shaped cells is both widespread and developmentally plastic, suggesting that single snapshots of leaves can give insight into their growth history. Together, these findings demonstrate that puzzle cells trans-form cell geometry into a living record of how tissues grow.

plant biology↗

bioRxiv2023

PRDM16 co-operates with LHX2 to shape the human brain

PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.

developmental biology↗

bioRxiv2023

Growth directions and stiffness across cell layers determine whether tissues stay smooth or buckle

From smooth shapes to buckles, nature exhibits organs of various shapes and forms. How cells grow to produce smooth shaped leaves and sepals remain unclear. Here, we show that growth along the longitudinal axis during early developmental stages and comparable stiffness across both epidermal layers of Arabidopsis sepals are essential for smoothness, as seen in the wild type. We identified a mutant (as2-7D) with ectopic expression of ASYMMETRIC LEAVES 2 (AS2) on the outer epidermis. Our analysis reveals that ectopic AS2 expression causes the outer epidermis of as2-7D sepals to buckle during early stages of sepal development. We show that buckling of the outer epidermis occurs due to conflicting cell growth directions and unequal tissue stiffness across the epidermal layers. Overexpression of cyclin-dependent kinase (CDK) inhibitor Kip-related protein 1 (KRP1) in as2-7D restores sepal smoothness by aligning the growth directions of the outer epidermal cells along the longitudinal axis, while also increasing the overall stiffness of the outer epidermis. Furthermore, buckling is associated with the convergence of auxin efflux transporter protein PIN-FORMED 1 (PIN1) to generate outgrowth in the sepals at later stages, suggesting that buckling can initiate outgrowths. Our findings suggest that in addition to molecular cues influencing tissue mechanics, tissue mechanics can also modulate molecular signals, giving rise to well-defined shapes. One-Sentence SummaryThe asymmetric leaves 2-7D mutant sepals buckle due to discoordination of growth between the two epidermal layers.

developmental biology↗

bioRxiv2023

Growth and tension in explosive fruit

Exploding seed pods of the common weed Cardamine hirsuta have the remarkable ability to launch seeds far from the plant. The energy for this explosion comes from tension that builds up in the fruit valves. Above a critical threshold, the fruit fractures along its dehiscence zone and the two valves coil explosively, ejecting the seeds. Tension is commonly generated as seed pods dry, causing fruit tissues to deform. However, this does not happen in C. hirsuta. Instead, tension is produced by active contraction of growing exocarp cells in the outer layer of the fruit valves. Exactly how growth leads to contraction in these cells is unknown. Here we show that microtubule dynamics in the exocarp cell cortex control the specific orientation of cellulose microfibrils in the cell wall, and the consequent cellular growth pattern, which together drive contraction. We used mechanical modeling and simulations to show how tension emerges through the general process of plant cell growth, due to the highly anisotropic orientation of load-bearing cellulose microfibrils and their effect on cell shape. By explicitly defining the cell wall as multi-layered in our model, we discovered that a cross-lamellate pattern of cellulose microfibrils further enhances the developing tension in growing cells. Therefore, the interplay of cell wall properties with turgor-driven growth enables the fruit exocarp layer to develop sufficient tension to explode.

plant biology↗

bioRxiv2023

Genetic control of cell layer interactions in plants via tissue mechanics

Plant development depends on coordination of growth between different cell layers. Coordination may be mediated by molecular signalling or mechanical connectivity between cells, but evidence for genetic control via direct mechanics has been lacking. We show that a brassinosteroid-deficient dwarf mutant of the aquatic plant Utricularia gibba has twisted internal tissue, likely caused by a mechanical constraint from a slow-growing epidermis creating tissue stresses. This conclusion is supported by showing that inhibition of brassinosteroid action in an Arabidopsis mutant compromised for cell adhesion, enhances epidermal crack formation, an indicator of increased tissue tension. Thus, genes driving brassinosteroid synthesis can promote growth of internal tissue by reducing mechanical epidermal constraint, showing that tissue mechanics plays a key role in coordinating growth between cell layers. One-Sentence SummaryInternal twists in a mutant carnivorous plant reveal how genes control growth via tissue mechanics.

plant biology↗