@article {160482, title = {Coupling between spatial compartments integrates morphogenetic patterning in the organ of Corti.}, journal = {PLoS Biol}, volume = {23}, year = {2025}, month = {2025 Sep}, pages = {e3003350}, abstract = {

Morphogenetic information arises from a combination of genetically encoded cellular properties and emergent cellular behaviors. The spatio-temporal implementation of this information is critical to ensure robust, reproducible tissue shapes, yet the principles underlying its organization remain unknown. We investigated this principle using the mouse auditory epithelium, the organ of Corti (OC). OC consists of a sensory domain, which transduces sound through polar mechanosensory hair cells (HC), part of a mosaic with supporting cells (SC). On either side of the sensory domain are non-sensory domains. These domains undergo cellular rearrangements, which, together, lead to a spiral cochlea that contains planar polarized HCs. This makes the mammalian cochlea a compelling system to understand coordination across spatial scales. Using genetic and ex vivo approaches, we found patterning of OC into sensory and non-sensory domains is associated with a combinatorial expression of adhesion molecules, which underpins OC into spatially defined compartments, enabling planar cell polarity (PCP) cues to regulate compartment-specific organization. Through compartment-specific knockouts of the PCP protein, Vangl2, we find evidence of compartment coupling, a non-linear influence on the organization within one compartment when cellular organization is disrupted in another. In the OC, compartment coupling originates from vinculin-dependent junctional mechanics, coordinating cellular dynamics across spatial scales.

}, keywords = {Animals, Body Patterning, Cell Polarity, Hair Cells, Auditory, Mice, Mice, Knockout, Morphogenesis, Nerve Tissue Proteins, Organ of Corti}, issn = {1545-7885}, doi = {10.1371/journal.pbio.3003350}, author = {Prakash, Anubhav and Raman, Sukanya and Kaushik, Raman and Manchanda, Pallavi and Iyer, Anton S and Ladher, Raj K} } @article {160483, title = {Epithelial fusion is mediated by a partial epithelial-mesenchymal transition.}, journal = {Biol Open}, volume = {14}, year = {2025}, month = {2025 Sep 15}, abstract = {

Epithelial fusion is a fundamental morphogenetic process critical for the closure and compartmentalisation of developing organs. While widely studied in systems such as neural tube and palatal closure, the cellular transitions that enable fusion remain poorly understood. Here, we investigate epithelial fusion during chick otic vesicle closure and identify a transient population of cells at the epithelial interface that mediate this process. These otic epithelial edge (OE) cells exhibit distinct morphology, reduced apicobasal polarity, and dynamic junctional remodelling, including altered distribution of ZO-1, CDH1 and RAC1. Notably, OE cells lack basal contact and display high sphericity, consistent with a partial epithelial-to-mesenchymal transition (EMT) phenotype. Transcriptomic profiling of microdissected tissues reveals that OE cells constitute a transcriptionally distinct population, enriched for EMT regulators, extracellular matrix remodelling genes, and WNT pathway components. Among these, the transcription factors Grhl2 and Sp8 were specifically expressed at the OE and exhibited opposing roles in epithelial identity. CRISPR-Cas9-mediated knockdown of either gene led to disrupted CDH1 localisation, loss of OE cell morphology and failure in epithelial segregation. These results suggest that epithelial fusion requires a regulated, hybrid EMT state that balances junctional plasticity with tissue cohesion. Our findings demonstrate that fusion-competent epithelial cells are not merely passive participants but actively modulate their shape, polarity, adhesion and genetic identity to enable morphogenesis.

}, keywords = {Animals, Chick Embryo, Epithelial Cells, Epithelial-Mesenchymal Transition, Gene Expression Profiling, Gene Expression Regulation, Developmental, Morphogenesis, Transcription Factors}, issn = {2046-6390}, doi = {10.1242/bio.062213}, author = {Tamilkumar, Varsha N and Purushothama, Harsha and Ladher, Raj K} } @article {156898, title = {Junctional force patterning drives both positional order and planar polarity in the auditory epithelia.}, journal = {Nat Commun}, volume = {16}, year = {2025}, month = {2025 Apr 26}, pages = {3927}, abstract = {

Tissue function depends on the precise organisation of the constituent cells. In the cochlea, the fidelity of hearing depends on mechanosensory hair cells being consistently surrounded by supporting cells. In addition to this positional order, auditory sensitivity depends crucially on planar cell polarity. This is characterised by the alignment of the orientation of eccentrically placed hair bundles on each hair cell. These two levels of order emerge simultaneously despite the cellular fluxes that occur during cochlear development. However, the link between tissue-scale cellular rearrangements and intrinsic cellular mechanisms remains unknown. By combining experimental and theoretical approaches, we find a precise force patterning underpinning positional order and planar cell polarity. This occurs through the modulation of the levels and phospho-type of the regulatory light chain of non-muscle myosin II at specific cell-cell junctions of the auditory epithelium. We propose that the control of junctional mechanics is vital for the organisation of multi-cell-type epithelia.

}, keywords = {Animals, Cell Polarity, Cochlea, Epithelium, Hair Cells, Auditory, Intercellular Junctions, Mice, Myosin Light Chains, Myosin Type II}, issn = {2041-1723}, doi = {10.1038/s41467-025-58557-0}, url = {https://www.nature.com/articles/s41467-025-58557-0}, author = {Prakash, Anubhav and Weninger, Julian and Singh, Nishant and Raman, Sukanya and Rao, Madan and Kruse, Karsten and Ladher, Raj K} } @article {161134, title = {The patterning and proliferation roles of Shh are partitioned on distinct exosomes.}, journal = {Dev Biol}, volume = {528}, year = {2025}, month = {2025 Dec}, pages = {239-254}, abstract = {

Sonic hedgehog (Shh) is a pivotal morphogen in spinal cord development, orchestrating both ventral neural patterning and progenitor proliferation. How these distinct outcomes are specified has remained elusive. Here, we uncover that Shh is secreted via two biochemically and functionally distinct exosomal pools. A dense vesicle fraction, Shh-P150, drives Smoothened-Gli1 signalling to establish ventral progenitor identities, while a lighter pool, Shh-P450, activates a Smoothened-Gαi-dependent pathway that enhances progenitor proliferation without inducing ventral fate. We identify Rab7, a late endosomal regulator, as essential for Shh-P150 biogenesis and for notochord-mediated ventral neural patterning. Loss of Rab7 biases secretion toward the proliferative Shh-P450 pool and disrupts morphogenetic signalling. These findings establish exosomal packaging as a molecular switch that toggles Shh between its mitogenic and morphogenetic roles. By linking exosome biogenesis to developmental outcomes, our work reveals a novel mechanism that safeguards the balance between pattern formation and progenitor expansion during neural tube development, with implications for both developmental disorders and disease contexts where Shh signalling is misregulated.

}, keywords = {Animals, Body Patterning, Cell Proliferation, Exosomes, Gene Expression Regulation, Developmental, Hedgehog Proteins, Mice, Neural Tube, Notochord, rab GTP-Binding Proteins, rab7 GTP-Binding Proteins, Signal Transduction, Smoothened Receptor, Spinal Cord, Zinc Finger Protein GLI1}, issn = {1095-564X}, doi = {10.1016/j.ydbio.2025.09.017}, author = {Walvekar, Ankita and Pandey, Shivangi and Kamat, Siddhesh S and Ladher, Raj K and Vyas, Neha} } @article {159724, title = {Role of Pcdh15 in the development of intrinsic polarity of inner ear hair cells.}, journal = {PLoS Genet}, volume = {21}, year = {2025}, month = {2025 Aug}, pages = {e1011825}, abstract = {

In vertebrates, auditory information is transduced in the cochlea by mechanosensory hair cells (HC) through an eccentrically organised structure known as the hair bundle. This consists of a true cilium, known as the kinocilium, and modified microvilli, known as stereocilia. The hair bundle has a distinct structure with stereocilia organised in graded rows, with the longest abutting the kinocilium. The hair bundles of all HC are aligned to the tissue axis and are planar polarised. Important in the development and physiology of HC are protein bridges consisting of cadherin-23 (CDH23) and protocadherin-15 (PCDH15). These link the tips of stereocilia, where they play a role in mechanotransduction, and between the kinocilia and the stereocilia, where they are involved in development. Both Cdh23 and Pcdh15 mutations result in defects in planar polarity; however, the mechanism through which this defect arises is unclear. Using a novel mutant for the Pcdh15-CD2 isoform, we show that while the initial deflection of the kinocilium occurs, its peripheral migration to register with Gαi is perturbed. Pcdh15-CD2 genetically interacts with Gpsm2, perturbing vestibular function. We find that the earliest expression of PCDH15-CD2 is at the base of the kinocilia, and the defects in morphogenesis occur before the formation of kinocilial links. By re-introducing functional PCDH15-CD2, we show that polarity can be restored. Our data suggest that, in addition to its adhesive role, PCDH15-CD2 has an early role in intrinsic hair cell polarity through a mechanism independent of kinocilial links.

}, keywords = {Animals, Basal Bodies, Cadherin Related Proteins, Cell Polarity, Ear, Inner, Gene Knock-In Techniques, Hair Cells, Auditory, Mice, Mutation, Phosphorylation, Protein Domains, Protein Precursors, Tyrosine}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1011825}, author = {Kaushik, Raman and Pandey, Shivangi and Prakash, Anubhav and Ganatra, Fenil and Abe, Takaya and Kiyonari, Hiroshi and Ladher, Raj K} } @article {161981, title = {Timing of Delamination of Inner Ear Neurons Specifies Their Topography and Target Innervation.}, journal = {J Comp Neurol}, volume = {533}, year = {2025}, month = {2025 Nov}, pages = {e70103}, abstract = {

The neurons of the inner ear delaminate from a sensorineurogenic epithelium in the ventral part of the otocyst. Delaminated neuroblasts then condense to form the acoustico-vestibular ganglion (AVG). As they differentiate, the neurons connect mechanosensory hair cells (HCs) of the inner ear with their targets in the hindbrain in a precise topographical manner. However, it is unclear how or when positional identities of neurons are specified within the AVG, such that topographical information from HCs is maintained into the auditory centers of the brain. Here, we find that the time of delamination from the otocyst correlates with neuroblast position in the ganglion. Using markers for neuronal differentiation, we find that the ganglion differentiates from a dorsal to ventral wave. Neurons that differentiate first innervate the vestibular apparatus, including the lagena and the proximal regions of the BP. Using sequential somatic cell labeling, we find that the central projection also follows a similar dependency on delamination order. Our studies show that the time of delamination of otic neuroblasts presages their target innervation choice and fiber positions within the developing eighth cranial nerve. We suggest that temporal information specifies the spatial identities during early inner ear neuron development.

}, keywords = {Animals, Cell Differentiation, Chick Embryo, Ear, Inner, Neurogenesis, Neurons, Time Factors}, issn = {1096-9861}, doi = {10.1002/cne.70103}, author = {Saini, Surjit Singh and Ladher, Raj K} } @article {113534, title = {In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development.}, journal = {J Vis Exp}, year = {2022}, month = {2022 Jun 16}, abstract = {

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

}, keywords = {Animals, Chickens, Cochlea, Hair Cells, Auditory, Mammals, Mice, Organ of Corti, Vestibule, Labyrinth}, issn = {1940-087X}, doi = {10.3791/64172}, author = {Singh, Nishant and Prakash, Anubhav and Chakravarthy, Suraj Ranganath and Kaushik, Raman and Ladher, Raj K} } @article {43856, title = {FGFR1-mediated protocadherin-15 loading mediates cargo specificity during intraflagellar transport in inner ear hair-cell kinocilia.}, journal = {Proc Natl Acad Sci U S A}, volume = {115}, year = {2018}, month = {2018 08 14}, pages = {8388-8393}, abstract = {

The mechanosensory hair cells of the inner ear are required for hearing and balance and have a distinctive apical structure, the hair bundle, that converts mechanical stimuli into electrical signals. This structure comprises a single cilium, the kinocilium, lying adjacent to an ensemble of actin-based projections known as stereocilia. Hair bundle polarity depends on kinociliary protocadherin-15 (Pcdh15) localization. Protocadherin-15 is found only in hair-cell kinocilia, and is not localized to the primary cilia of adjacent supporting cells. Thus, Pcdh15 must be specifically targeted and trafficked into the hair-cell kinocilium. Here we show that kinocilial Pcdh15 trafficking relies on cell type-specific coupling to the generic intraflagellar transport (IFT) transport mechanism. We uncover a role for fibroblast growth factor receptor 1 (FGFR1) in loading Pcdh15 onto kinociliary transport particles in hair cells. We find that on activation, FGFR1 binds and phosphorylates Pcdh15. Moreover, we find a previously uncharacterized role for clathrin in coupling this kinocilia-specific cargo with the anterograde IFT-B complex through the adaptor, DAB2. Our results identify a modified ciliary transport pathway used for Pcdh15 transport into the cilium of the inner ear hair cell and coordinated by FGFR1 activity.

}, keywords = {Adaptor Proteins, Vesicular Transport, Animals, Cadherins, Chick Embryo, Clathrin, Flagella, Hair Cells, Auditory, Inner, Mice, Phosphorylation, Protein Precursors, Protein Transport, Receptor, Fibroblast Growth Factor, Type 1}, issn = {1091-6490}, doi = {10.1073/pnas.1719861115}, author = {Honda, Akira and Kita, Tomoko and Seshadri, Shri Vidhya and Misaki, Kazuyo and Ahmed, Zamal and Ladbury, John E and Richardson, Guy P and Yonemura, Shigenobu and Ladher, Raj K} }