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Coupling between spatial compartments integrates morphogenetic patterning in the organ of Corti [1]

['Anubhav Prakash', 'National Centre For Biological Sciences', 'Tata Institute Of Fundamental Research', 'Bangalore', 'Trivedi School Of Biosciences', 'Ashoka University', 'Plot No.', 'Rajiv Gandhi Education City', 'National Capital Region P.O. Rai', 'Sonipat Haryana']

Date: 2025-09 

The reproducibility and robustness of morphogenesis result from carefully implemented developmental instructions. Through a combination of signaling cues, gene networks, and self-organizing mechanisms, patterning divides an organogenic field into compartments [1,2]. While compartments allow the segregation of distinct cell behaviors, these behaviors must be spatiotemporally coordinated across compartments so that functional organs, with the correct shape and pattern, forms. The mouse auditory epithelia is an excellent system to investigate this unknown coordination.

The mouse auditory epithelium, called the organ of Corti (OC), is found within the cochlea. It is a spiral-shaped organ and is responsible for detecting and transducing sound across a wide spectrum of frequencies [3,4]. Sound is transduced by hair cells (HC) through an asymmetric hearing organelle, the hair bundle on their apical surface. HCs are of two types: inner HCs (IHC) arranged into a single row that transmit information to the brain; and outer HCs (OHC) arranged into three rows that amplify the mechanical input. Both these HCs are intercalated by supporting cells (SC) and together form the sensory domain of OC. This sensory domain is flanked by non-sensory compartments. On the medial side (inner edge of spiral) is Kölliker’s organ (KO) and on the lateral side (outer edge of spiral) is a lateral non-sensory compartment, which includes Hensen’s and Claudius’ cells (Figs 1A and S1A).

(A) Schematic of spiral-shaped mouse organ of Corti (OC) representing Kölliker’s organ (KO) (medial non-sensory domain), sensory domain, and lateral non-sensory domain. (B) Base part of OC from E15.5 and E18.5 stained for F-actin. Green overlay indicates KO domain, red overlay indicates sensory domain, and blue overlay indicates lateral non-sensory domain. Blue and orange arrowhead indicates medial and lateral sensory domain, respectively. Magenta arrowheads indicate Hensen’s cell and green arrowheads shows cells in KO domain. (C) Length of cochlea from E15.5 to post-natal day 2. N = 3 embryos for each stage. (D) Base of E18.5 OC from heterozygous (Vangl2 Lp/+ ) and homozygous (Vangl2 Lp/Lp ) looptail mutant stained for F-actin (gray). Overlay indicates domains as B. N = 8. (E) Base of E18.5 OC from heterozygous (Vangl2 Lp/+ ) and homozygous (Vangl2 Lp/Lp ) looptail mutant stained for F-actin (green) and Myosin 7a (magenta). N = 4. (F) Base of E18.5 OC from heterozygous (Vangl2 Lp/+ ) and homozygous (Vangl2 Lp/Lp ) looptail mutant stained for F-actin (green) and BLBP (magenta). N = 4. (G) Base of E18.5 OC from heterozygous (Vangl2 Lp/+ ) and homozygous (Vangl2 Lp/Lp ) looptail mutant stained for F-actin (green) and p75NTR (magenta). N = 4. Scale Bar: 5 µm in E–G, and 10 µm in B and D. Image orientation: Top is lateral, Right is Apex. Underlying data available in S1 Data .

The cochlea is initially apparent as a ventral out-pocketing of the otocyst. Radial patterning of the nascent cochlear duct by morphogen signaling establishes non-sensory and sensory domains of the OC [5,6]. The cells in the sensory domain become post-mitotic and, through juxtacrine signaling, differentiate into a mosaic of HC and SC [7,8]. As HC develop, they form asymmetrically localized hair bundles which align with the tissue axis, a process known as planar polarity. Both experimental approaches and mathematical modeling have shown that local coordination among HCs and SCs driven by differential junctional tension could drive the organization of HCs and align HC polarity to the tissue axis [9–12]. While cells locally coordinate in the sensory domain, large-scale convergence and extension (CE) movements together with growth and proliferation cause the OC to elongate contributing to morphogenesis and the spiraling of the OC [13–18]. Thus, local domain-specific processes that order HCs must integrate with large-scale tissue remodeling. How they integrate is not known.

The OC compartmentalizes into smaller domains that show a combinatorial expression of Cadherin 1, 2, and 4 [15,19,20]. Using mutants of fibroblast growth factor signaling and ex vivo cultures, we find that the adhesion code ensures compartment integrity during convergent and extension movements. Each compartment uses the planar cell polarity (PCP) molecule, Vangl2, to develop a distinct cellular organization. Using compartment-specific knockouts of Vangl2, we find that cellular organization within each compartment has a non-linear influence on the organization of another compartment, a novel phenomenon called compartment coupling. In mice mutant for the junctional force transmission component, Vinculin, we show that compartment coupling has a mechanical element. Our work suggests that compartment coupling underpins the integration of local cellular ordering with large-scale tissue remodeling. Given the widespread use of compartments, inter-compartment coupling is likely a fundamental feature of the morphogenesis of many developing tissues and organs.

Results

Domain organization is preserved during cochlear elongation. To investigate the mechanism that could integrate local organization with tissue-scale remodeling, we first asked how the organization in OC evolves from embryonic day (E)15.5, when the HCs are first apparent, to E18.5, when the HCs achieve their final organization. Immunostaining for a marker of HCs, Myosin 7a, shows the sensory domain is already established by E15.5 (S1B Fig). Positive immunostaining for p75NTR, a molecular marker for inner pillar cells, which segregates the IHCs from OHCs, shows the presence of medial and lateral sensory domains at this stage (S1C Fig). Further, the expression of brain lipid binding-protein (BLBP), a molecular marker for Hensen’s cells (HnC, a SC type lateral to OHC) and Inner Phalangeal cells (IPhC, a SC type intercalating IHC) shows that the sensory and the non-sensory domains are established by E15.5 (S1D Fig). This organization of OC into medial and lateral non-sensory and sensory domains suggests that by E15.5, the OC is radially patterned (Fig 1B). From E15.5 to E18.5, the cochlea elongates from 2,734 ± 33 µm to 4,702 ± 27 µm (Figs 1C and S1E). Previous studies have shown that cell growth, migration, intercalation, and tissue-scale convergent-extension movements drive this elongation [13–18]. Such movements are expected to disrupt the organization established at E15.5 [21]. However, they do not. Immunostaining of the E18.5 OC for Myosin 7a, BLBP, and p75NTR revealed that domain organization was maintained during CE-mediated cochlear elongation (Figs 1B and S1B–S1D). While previous studies have investigated the organization of the medial and lateral sensory domains when CE movements are perturbed, non-sensory domain organization is unclear. Mice mutant for the core PCP protein, Vangl2, show defects in HC PCP and convergence and extension (CE) movements [22–25]. We thus assessed the organization of non-sensory domains in these mutants. Homozygous looptail mutants of Vangl2 (referred to as Vangl2Lp/Lp) have cochlea 2/3rd the length of littermate controls (Vangl2Lp/+ 5,010 ± 41 µm and Vangl2Lp/Lp 3,238 ± 185 µm) (S2A and S2B Fig). Immunostaining for molecular markers for HCs, IPhCs, inner pillar cells, and the distinction in the morphological features of non-sensory cell types (Fig 1D–1G) revealed that the relative position of cell types and domain organization is maintained in the Vangl2Lp/Lp mutants with defects in convergent extension (S2C–S2E Fig). This suggests a mechanism to maintain the integrity of individual domains during cochlear elongation.

PCP regulated NMII-activity drives distinct cellular organization. To understand the mechanisms behind discrete cellular organization in compartments of the OC, we first looked at proliferation. We injected EdU into pregnant females at E13.5, E15.5, E16.5, and E18.5 and fixed embryos 6 h post-injection (S9A Fig). At E13.5 (+6 h), we observed EdU was incorporated into the entire OC (S9B Fig), similar to previous observations [18]. By E15.5, the number of EdU-positive cells at the base decreased to less than 10%. By E16.5, proliferation had ceased and remained so till E18.5 (S9B Fig). As the KO increased in length between E15.5 and E18.5, we concluded that there is a limited contribution by proliferation. We next asked if cellular rearrangements could contribute to compartment reorganization. In epithelia, cellular rearrangements result from neighbor exchange, with an obligatory intermediate step where 4 or more cells meet at a vertex. We thus assessed the number of 4-cell vertices between E15.5 and E18.5. At E15.5, 33% of all vertices in the sensory compartment have 4 or more cells. This decreases significantly such that by E18.5 only 20% of vertices are made up of 4 or more cells (S9D Fig). Similarly, in the KO domain, mKO showed 40% of vertices to have 4 or more cells, suggesting a higher rate of cellular reorganization. In the lKO, only 20% of the vertices showed 4 or more cells, suggesting a lower rate of reorganization compared to the mKO. The proportion of vertices with 4 or more cells decreased in the lKO by E18.5. The proportion of vertices with 4 or more cells in the mKO at E18.5 remained equivalent to the numbers observed at E15.5 (S9E Fig). This data suggests that cells in the KO domain undergo cell rearrangement, higher at the medial edge compared to the lateral edge. In the absence of cell division, we hypothesized that this cellular rearrangement drives cochlear morphogenesis. To test this, we sought to perturb the process of cellular reorganization by disrupting the activity of the acto-myosin complex, essential for cellular intercalations. Non-muscle myosin (NM) forms the motor component of the acto-myosin complex. The motor activity of NMII is regulated by the phosphorylation status of its regulatory light chain (RLC). Thus, we first immunostained OC for mono and di-phosphorylated forms of RLC. At E18.5, p-RLC is expressed on all junctions (S10A Fig). However, the pp-RLC is localized at the medial edge of OHC-DC junctions (S10B and S10B′ Fig) and IPhC-Pillar cells junctions. In the KO compartment, pp-RLC was localized along the junctions of the long axis of KO cells (S10B Fig). To test their role in morphogenesis of the cochlea, we used our ex vivo explant method to culture E16.5 cochlea for 8 h, in the presence or absence of Myosin Light Chain Kinase inhibitor, ML7 (which inhibits RLC phosphorylation) [41,42]. In MLCK-inhibited OC, we observed a decrease in apical surface area and circularity of OHC compared to the control samples (S10C–S10H Fig), suggesting a decrease in spatial organization within the sensory domain. Previous work on avian auditory epithelia has shown that the spatial organization of HC is coupled to the alignment of HC polarity to the tissue axis [12]. Similarly, we observed a decrease in the alignment of HC polarity for IHC and OHC in the MLCK-inhibited cochlea (S10E and S10F Fig). In addition, the difference in the apical surface area and the elongation axis of mKO and lKO cells was also reduced in the MLCK-inhibited OC (S10I–S10K Fig). This data suggested that NMII-driven neighbor-exchange drives reorganization of sensory and non-sensory domains during development. To further understand this organization, we decided to understand how NMII activity is regulated in each compartment. Previous studies, including our work on avian auditory epithelia, have shown that PCP cues through Vangl2 regulate RLC phosphorylation [12,43–45]. In mouse, the expression of Vangl2 largely overlapped with the expression of pp-RLC (S11A and S11B Fig). Hence, we used Vangl2Lp/Lp mutant, which, as previously reported, shows a reduction in alignment of HC polarity (S11C and S11D Fig) [16,22]. pp-RLC shows a down-regulation on the junctions of both sensory and non-sensory compartments in Vangl2Lp/Lp mutants, while the p-RLC was comparable to the littermate controls (S11E–S11H Fig). At the scale of the domain, the Vangl2Lp/Lp mutants showed a decrease in the width of both the KO and sensory domains at the base and middle turn of OC (Figs 3G and S11I–S11J). Further, at E18.5, Vangl2Lp/Lp mutants also showed a significant decrease in the number of 4 cell vertices in both mKO and sensory domain, suggesting a decrease in cell intercalation (Fig 3H). Interestingly, mutants showed a significant increase of four cell vertices in the lKO. The differences in the apical surface area and the preferential axis of elongation for mKO and lKO cells were also reduced in the mutants (Fig 3G, 3I, and 3J). The regulation of RLC phosphorylation by Vangl2 and the similarity of Vangl2Lp/Lp mutants with MLCK inhibited OC, suggests that Vangl2-regulated NMII activity may govern the organization of not only the sensory compartments of the OC but also the non-sensory compartments.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3003350

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