Charged multivesicular body protein 4b forms complexes with gap junction proteins during lens fiber cell differentiation.

Charged multivesicular body protein 4b (CHMP4B) is a core sub-unit of the endosomal sorting complex required for transport III (ESCRT-III) machinery that serves myriad remodeling and scission processes of biological membranes. Mutation of the human CHMP4B gene underlies rare forms of early-onset lens opacities or cataracts, and CHMP4B is required for lens growth and differentiation in mice. Here, we determine the sub-cellular distribution of CHMP4B in the lens and uncover a novel association with gap junction alpha-3 protein (GJA3) or connexin 46 (Cx46) and GJA8 or Cx50. Immunofluorescence confocal microscopy revealed that CHMP4B localized to cell membranes of elongated fiber cells in the outer cortex of the lens-where large gap junction plaques begin to form-particularly, on the broad faces of these flattened hexagon-like cells in cross-section. Dual immunofluorescence imaging showed that CHMP4B co-localized with gap junction plaques containing Cx46 and/or Cx50. When combined with the in situ proximity ligation assay, immunofluorescence confocal imaging indicated that CHMP4B lay in close physical proximity to Cx46 and Cx50. In Cx46-knockout (Cx46-KO) lenses, CHMP4B-membrane distribution was similar to that of wild-type, whereas, in Cx50-KO lenses, CHMP4B localization to fiber cell membranes was lost. Immunoprecipitation and immunoblotting analyses revealed that CHMP4B formed complexes with Cx46 and Cx50 in vitro. Collectively, our data suggest that CHMP4B forms plasma membrane complexes, either directly and/or indirectly, with gap junction proteins Cx46 and Cx50 that are often associated with "ball-and-socket" double-membrane junctions during lens fiber cell differentiation.

Recent Advances in Rib Plating.

Rib fractures are a common injury in blunt trauma and are associated with high morbidity and mortality. Recent advances in surgical stabilization of rib fractures (SSRF) have led to better patient outcomes for those with highly unstable complex rib fractures, as well as those with less severe injuries. This result has been due in part to the expansion of indications for repair, as well as the development of new hardware systems to address a variety of fracture patterns and injuries. This joint advancement of operator techniques, outcomes research, and industry development has brought SSRF to the forefront of rib fracture management and challenged non-operative paradigms. The future of repair is now shifting focus, as surgeons develop minimally invasive approaches and challenge manufacturers to develop new systems, instruments, and materials to address increasingly complex fracture patterns. These expansions promise to make SSRF an increasingly effective form of management for traumatic rib fractures.

Increased Hemichannel Activity Displayed by a Connexin43 Mutation Causing a Familial Connexinopathy Exhibiting Hypotrichosis with Follicular Keratosis and Hyperostosis.

Mutations in the GJA1 gene that encodes connexin43 (Cx43) cause several rare genetic disorders, including diseases affecting the epidermis. Here, we examined the in vitro functional consequences of a Cx43 mutation, Cx43-G38E, linked to a novel human phenotype of hypotrichosis, follicular keratosis and hyperostosis. We found that Cx43-G38E was efficiently translated in Xenopus oocytes and localized to gap junction plaques in transfected HeLa cells. Cx43-G38E formed functional gap junction channels with the same efficiency as wild-type Cx43 in Xenopus oocytes, although voltage gating of the gap junction channels was altered. Notably, Cx43-G38E significantly increased membrane current flow through the formation of active hemichannels when compared to wild-type Cx43. These data demonstrate the association of increased hemichannel activity to a connexin mutation linked to a skeletal-cutaneous phenotype, suggesting that augmented hemichannel activity could play a role in skin and skeletal disorders caused by human Cx43 mutations.

Lens Connexin Channels Show Differential Permeability to Signaling Molecules.

Gap junction channels mediate the direct intercellular passage of small ions as well as larger solutes such as second messengers. A family of proteins called connexins make up the subunits of gap junction channels in chordate animals. Each individual connexin forms channels that exhibit distinct permeability to molecules that influence cellular signaling, such as calcium ions, cyclic nucleotides, or inositol phosphates. In this review, we examine the permeability of connexin channels containing Cx43, Cx46, and Cx50 to signaling molecules and attempt to relate the observed differences in permeability to possible in vivo consequences that were revealed by studies of transgenic animals where these connexin genes have been manipulated. Taken together, these data suggest that differences in the permeability of individual connexin channels to larger solutes like 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) could play a role in regulating epithelial cell division, differentiation, and homeostasis in organs like the ocular lens.

A novel autosomal recessive GJB2-associated disorder: Ichthyosis follicularis, bilateral severe sensorineural hearing loss, and punctate palmoplantar keratoderma.

Ichthyosis follicularis, a distinct cutaneous entity reported in combination with atrichia, and photophobia has been associated with mutations in MBTPS2. We sought the genetic cause of a novel syndrome of ichthyosis follicularis, bilateral severe sensorineural hearing loss and punctate palmoplantar keratoderma in two families. We performed whole exome sequencing on three patients from two families. The pathogenicity and consequences of mutations were studied in the Xenopus oocyte expression system and by molecular modeling analysis. Compound heterozygous mutations in the GJB2 gene were discovered: a pathogenic c.526A>G; p.Asn176Asp, and a common frameshift mutation, c.35delG; p.Gly12Valfs*2. The p.Asn176Asp missense mutation was demonstrated to significantly reduce the cell-cell gap junction channel activity and increase the nonjunctional hemichannel activity in the Xenopus oocyte expression system. Molecular modeling analyses of the mutant Cx26 protein revealed significant changes in the structural characteristics and electrostatic potential of the Cx26, either in hemichannel or gap junction conformation. Thus, association of a new syndrome of an autosomal recessive disorder of ichthyosis follicularis, bilateral severe sensorineural hearing loss and punctate palmoplantar keratoderma with mutations in GJB2, expands the phenotypic spectrum of the GJB2-associated disorders. The findings attest to the complexity of the clinical consequences of different mutations in GJB2.

Connexin 43 Mutations Lead to Increased Hemichannel Functionality in Skin Disease.

Gap junctional channels are specialized components of the cellular membrane that allow the intercellular passage of small metabolites, ions, and second messengers to maintain homeostasis. They are comprised of members of the connexin gene family that encode a wide array of proteins that are expressed in nearly every tissue type. Cx43 is perceived to be the most broadly expressed connexin in humans, with several genetic skin diseases being linked to Cx43 mutations specifically. These mutations, in large, produce a gain of functional hemichannels that contribute to the phenotypes of Erythrokeratoderma Variabilis et Progressiva (EKVP), Palmoplantar Keratodemra Congenital Alopecia-1 (PPKCA1), and others that produce large conductance and increased permselectivity in otherwise quiescent structures. Gaining functional hemichannels can have adverse effects in the skin, inducing apoptosis via Ca2+ overload or increased ATP permeability. Here, we review the link between Cx43 and skin disease. We aim to provide insight into the mechanisms regulating the normal and pathophysiological gating of these essential proteins, as well as address current therapeutic strategies. We also demonstrate that transient transfection of neuro-2a (N2a) cells with mutant Cx43 cDNA resulted in increased hemichannel activity compared to wild-type Cx43 and untransfected cells, which is consistent with other studies in the current literature.

Connexin channels in congenital skin disorders.

Gap junctions and hemichannels comprised of connexins influence epidermal proliferation and differentiation. Significant advances in our understanding of the functional role of connexins in the skin have been made by studying the diseases caused by connexin mutations. Eleven clinically defined cutaneous disorders with an overlapping spectrum of phenotypes are caused by mutations in five different connexin genes, highlighting that disease presentation must be deciphered with an understanding of how connexin functions are affected. Increasing evidence suggests that the skin diseases produced by connexin mutations result from dominant gains of function. In palmoplantar keratoderma with deafness, the connexin 26 mutations transdominantly alter the function of wild-type connexin 43 and create leaky heteromeric hemichannels. In keratitis-ichthyosis-deafness syndrome, different connexin 26 mutations can either form dominant hemichannels with altered calcium regulation or increased calcium permeability, leading to clinical subtypes of this syndrome. It is only with detailed understanding of these subtle functional differences that we can hope to create successful pathophysiology driven therapies for the connexin skin disorders.

Human diseases associated with connexin mutations.

Gap junctions and hemichannels comprised of connexins impact many cellular processes. Significant advances in our understanding of the functional role of these channels have been made by the identification of a host of genetic diseases caused by connexin mutations. Prominent features of connexin disorders are the inability of other connexins expressed in the same cell type to compensate for the mutated one, and the ability of connexin mutants to dominantly influence the activity of other wild-type connexins. Functional studies have begun to identify some of the underlying mechanisms whereby connexin channel mutation contributes to the disease state. Detailed mechanistic understanding of these functional differences will help to facilitate new pathophysiology driven therapies for the diverse array of connexin genetic disorders. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Indications for surgical stabilization of rib fractures in patients without flail chest: surveyed opinions of members of the Chest Wall Injury Society.

PURPOSE: There are currently no evidence-based indications for surgical stabilization of rib fractures (SSRF) in patients without flail chest. The purpose of this survey was to identify patients for whom there is relative equipoise (operative vs. non-operative) in order to assist in designing a randomized clinical trial. METHODS: Members of the Chest Wall Injury Society were sent an online survey, in which 18 patient scenarios were presented. The baseline patient had ≥ three displaced, contiguous fractures and had no other contraindications for surgery. This default scenario was then varied based upon patient age, degree of traumatic brain injury (TBI), fracture series location, and number of abnormal pulmonary physiologic variables (oxygen requirement, respiratory rate, incentive spirometry ability, cough, and numeric pain score). RESULTS: Thirty respondents provided a total of 540 answers. Overall, the majority of responses were in favor of SSRF (n = 413, 84.1%). Furthermore, the vast majority of responses indicated that some degree of pulmonary compromise was necessary to recommend SSRF (n = 44, 90.4%), with ≥ two abnormal parameters being the most common threshold (n = 156, 31.8%). Decision to recommend SSRF varied significantly by number of abnormal clinical variables, age, and degree of TBI, but not by fracture series location. Patients aged 85 years old and those with moderate TBI were the least likely to be recommended for SSRF, regardless of abnormal pulmonary physiologic variables. The most appropriate cutoff for equipoise appeared to be a patient aged 21-79 years old, with no or mild TBI, ≥ two abnormal pulmonary parameters, and regardless of fracture location (44.8% consensus for SSRF). CONCLUSIONS: SSRF was recommended for most patients with non-flail, displaced rib fractures. However, this recommendation was contingent upon patient age, degree of TBI, and pulmonary clinical status. Results of this survey may be used to inform inclusion criteria for a future randomized, clinical trial.

Lens gap junctions in growth, differentiation, and homeostasis.

The cells of most mammalian organs are connected by groups of cell-to-cell channels called gap junctions. Gap junction channels are made from the connexin (Cx) family of proteins. There are at least 20 isoforms of connexins, and most tissues express more than 1 isoform. The lens is no exception, as it expresses three isoforms: Cx43, Cx46, and Cx50. A common role for all gap junctions, regardless of their Cx composition, is to provide a conduit for ion flow between cells, thus creating a syncytial tissue with regard to intracellular voltage and ion concentrations. Given this rather simple role of gap junctions, a persistent question has been: Why are there so many Cx isoforms and why do tissues express more than one isoform? Recent studies of lens Cx knockout (KO) and knock in (KI) lenses have begun to answer these questions. To understand these roles, one must first understand the physiological requirements of the lens. We therefore first review the development and structure of the lens, its numerous transport systems, how these systems are integrated to generate the lens circulation, the roles of the circulation in lens homeostasis, and finally the roles of lens connexins in growth, development, and the lens circulation.

Feedback Regulation of Intracellular Hydrostatic Pressure in Surface Cells of the Lens.

In wild-type lenses from various species, an intracellular hydrostatic pressure gradient goes from ∼340 mmHg in central fiber cells to 0 mmHg in surface cells. This gradient drives a center-to-surface flow of intracellular fluid. In lenses in which gap-junction coupling is increased, the central pressure is lower, whereas if gap-junction coupling is reduced, the central pressure is higher but surface pressure is always zero. Recently, we found that surface cell pressure was elevated in PTEN null lenses. This suggested disruption of a feedback control system that normally maintained zero surface cell pressure. Our purpose in this study was to investigate and characterize this feedback control system. We measured intracellular hydrostatic pressures in mouse lenses using a microelectrode/manometer-based system. We found that all feedback went through transport by the Na/K ATPase, which adjusted surface cell osmolarity such that pressure was maintained at zero. We traced the regulation of Na/K ATPase activity back to either TRPV4, which sensed positive pressure and stimulated activity, or TRPV1, which sensed negative pressure and inhibited activity. The inhibitory effect of TRPV1 on Na/K pumps was shown to signal through activation of the PI3K/AKT axis. The stimulatory effect of TRPV4 was shown in previous studies to go through a different signal transduction path. Thus, there is a local two-legged feedback control system for pressure in lens surface cells. The surface pressure provides a pedestal on which the pressure gradient sits, so surface pressure determines the absolute value of pressure at each radial location. We speculate that the absolute value of intracellular pressure may set the radial gradient in the refractive index, which is essential for visual acuity.

Connexin 46 (cx46) gap junctions provide a pathway for the delivery of glutathione to the lens nucleus.

Maintenance of adequate levels of glutathione (GSH) in the lens nucleus is critical for protection of lens proteins from the effects of oxidative stress and for lens transparency. How GSH is transported to the nucleus is unknown. We show that GSH diffuses to the nucleus from the outer cortex, where a high concentration of the anti-oxidant is established by synthesis or uptake, via the network of gap junctions. Using electrophysiological measurements, we found that channels formed by Cx46 and Cx50, the two connexin isoforms expressed in the lens, were moderately cation-selective (P(Na)/P(Cl) ∼5 for Cx46 and ∼3 for Cx50). Single channel permeation of the larger GSH anion was low but detectable (P(Na)/P(GSH) ∼12 for Cx46 and ∼8 for Cx50), whereas permeation of divalent anion glutathione disulfide (GSSG) was undetectable. Measurement of GSH levels in the lenses from connexin knock-out (KO) mice indicated Cx46, and not Cx50, is necessary for transport of GSH to the core. Levels of GSH in the nucleus were markedly reduced in Cx46 KO, whereas they were unaffected by Cx50 KO. We also show that GSH delivery to the nucleus is not dependent on the lens microcirculation, which is believed to be responsible for extracellular transport of other nutrients to membrane transporters in the core. These results indicate that glutathione diffuses from cortical fiber cells to the nucleus via gap junction channels formed by Cx46. We present a model of GSH diffusion from outer cells to inner fiber cells through gap junctions.

Connexin hemichannels influence genetically determined inflammatory and hyperproliferative skin diseases.

Connexin mutations underlie numerous human genetic diseases. Several connexin genes have been linked to skin diseases, and mechanistic studies have indicated that a gain of abnormal channel function may be responsible for pathology. The topical accessibility of the epidermal connexins, the existence of several mouse models of human skin disease, and the ongoing identification of pharmacological inhibitors targeting connexins provide an opportunity to test new therapeutic approaches.

Functional effects of Cx50 mutations associated with congenital cataracts.

Mutations in connexin50 (Cx50) cause dominant cataracts in both humans and mice. The exact mechanisms by which mutations cause these variable phenotypes are poorly understood. We have examined the functional properties of gap junctions made by three Cx50 mutations, V44E, D47N, and V79L, expressed in mammalian cell lines. V44E trafficked to the plasma membrane properly and formed gap junctional plaques. However, the mutant did not form functional gap junctions when expressed alone, or with wild-type (WT) Cx46 and Cx50, indicating that V44E is a dominant negative inhibitor of WT connexin function. In contrast, D47N subunits did not localize to junctional plaques or form functional homotypic gap junctions; however, mixed expression of D47N and WT subunits of either Cx50 or Cx46 resulted in functional intercellular channels, with high levels of coupling. Single-channel studies indicated that D47N formed heteromeric channels with WT Cx46 with unique properties. Unlike either V44E or D47N, V79L formed functional homotypic intercellular channels. However, the mutation caused an alteration in voltage gating and a dramatic reduction in the single-channel open probability, resulting in much lower levels of conductance in cells expressing V79L alone, or together with WT connexin subunits. Thus, each mutation produced distinct changes in the properties of junctional coupling. V44E failed to form intercellular channels in any configuration, D47N formed only heteromeric channels with WT connexins, and V79L formed homotypic and heteromeric channels with altered properties. These results suggest that unique interactions between mutant and wild-type lens connexins might underlie the development of various cataract phenotypes in humans.

[Surgical reconstruction of chest wall instability : Indications, contraindications and timing of surgery]. / Operative Rekonstruktion der Thoraxwandinstabilität : Indikationen, Kontraindikationen und Operationszeitpunkt.

The impact of energy on the thorax can lead to serial rib fractures, sternal fractures, the combination of both and to injury of intrathoracic organs depending on the type, localization and intensity. Sometimes this results in chest wall instability with severe impairment of the respiratory mechanics. In the last decade the importance of surgical chest wall reconstruction in cases of chest wall instability has greatly increased. The evidence for a surgical approach has in the meantime been supported by prospective randomized multicenter studies, multiple retrospective data analyses and meta-analyses based on these studies, including a Cochrane review. The assessment of form and severity of the trauma and the degree of impairment of the respiratory mechanism are the basis for a structured decision on an extended conservative or surgical reconstructive strategy as well as the timing, type and extent of the operation. The morbidity (rate of pneumonia, duration of intensive care unit stay and mechanical ventilation) and fatality can be reduced by a timely surgery within 72 h after trauma. In this article the already established and evidence-based algorithms for surgical chest wall reconstruction are discussed in the context of the current evidence.

Alterations in Lens Free Water Distribution Are Associated with Shape Deformation in Accommodation.

Objective: To investigate whether a redistribution of water within the crystalline lens is associated with the shape deformation that occurs during accommodation. Design: Observational, cross sectional study. Subjects: Eleven young adults without presbyopia (aged 18-39 years) and 9 middle-aged adults with presbyopia (aged 40-55 years). Methods: Magnetic resonance imaging (MRI) scans of the lens were acquired on a 3 Tesla clinical MRI scanner, without and with the presentation of a 3 Diopter accommodative stimulus. The MRIs were postprocessed using established methods to extract the geometric dimensions and spatial maps of water distribution of the lens. Main Outcome Measures: Accommodative changes in the full 3-dimensional description of lens shape, the lens total-water distribution profile, and the lens free-water distribution profile. Results: Viewing of an accommodative stimulus by young subjects elicited an elastic shape deformation of the lens consistent with accommodation that was associated with an elevated, smoother free-water distribution, primarily in the anterior region of the lens. In contrast, viewing of an accommodative stimulus by presbyopic subjects produced an atypical shape deformation of the lens that was instead associated with a lowered free-water distribution, primarily in the anterior region of the lens. No discernible changes to the lens total-water distribution were observed in response to the accommodative stimulus in either subject cohort. Conclusions: The present study suggests that protein-mediated alterations in the free-water distribution of the anterior region of the lens influence the shape deformation in accommodation, presenting pharmacological modulation of free-water distribution as an attractive novel approach for treating presbyopia. Financial Disclosures: The authors have no proprietary or commercial interest in any materials discussed in this article.

An initiative to assess and improve the resources and patient care processes used among Chest Wall Injury Society collaborative centers study (CWIS-CC2).

BACKGROUND: Over the last two decades, the acute management of rib fractures has changed significantly. In 2021, the Chest Wall injury Society (CWIS) began recognizing centers that epitomize their mission as CWIS Collaborative Centers. The primary aim of this study was to determine the resources, surgical expertise, access to care, and institutional support that are present among centers. METHODS: A survey was performed including all CWIS Collaborative Centers evaluating the resources available at their hospital for the treatment of patients with chest wall injury. Data about each chest wall injury center care process, availability of resources, institutional support, research support, and educational offerings were recorded. RESULTS: Data were collected from 20 trauma centers resulting in an 80% response rate. These trauma centers were made up of 5 international and 15 US-based trauma centers. Eighty percent (16 of 20) have dedicated care team members for the evaluation and management of rib fractures. Twenty-five percent (5 of 20) have a dedicated rib fracture service with a separate call schedule. Staffing for chest wall injury clinics consists of a multidisciplinary team: with attending surgeons in all clinics, 80% (8 of 10) with advanced practice providers and 70% (7 of 10) with care coordinators. Forty percent (8 of 20) of centers have dedicated rib fracture research support, and 35% (7 of 20) have surgical stabilization of rib fracture (SSRF)-related grants. Forty percent (8 of 20) of centers have marketing support, and 30% (8 of 20) have a web page support to bring awareness to their center. At these trauma centers, a median of 4 (1-9) surgeons perform SSRFs. In the majority of trauma centers, the trauma surgeons perform SSRF. CONCLUSION: Considerable similarities and differences exist within these CWIS collaborative centers. These differences in resources are hypothesis generating in determining the optimal chest wall injury center. These findings may generate several patient care and team process questions to optimize patient care, patient experience, provider satisfaction, research productivity, education, and outreach. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level V.

The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity.

Mutations in the human gene encoding connexin 26 (Cx26 or GJB2) cause either nonsyndromic deafness or syndromic deafness associated with skin diseases. That distinct clinical disorders can be caused by different mutations within the same gene suggests that different channel activities influence the ear and skin. Here we use three different expression systems to examine the functional characteristics of two Cx26 mutations causing either mild (Cx26-D50A) or lethal (Cx26-A88V) keratitis-ichthyosis-deafness (KID) syndrome. In either cRNA-injected Xenopus oocytes, transfected HeLa cells, or transfected primary human keratinocytes, we show that both Cx26-D50A and Cx26-A88V form active hemichannels that significantly increase membrane current flow compared with wild-type Cx26. This increased membrane current accelerated cell death in low extracellular calcium solutions and was not due to increased mutant protein expression. Elevated mutant hemichannel currents could be blocked by increased extracellular calcium concentration. These results show that these two mutations exhibit a shared gain of functional activity and support the hypothesis that increased hemichannel activity is a common feature of human Cx26 mutations responsible for KID syndrome.

Pathological hemichannels associated with human Cx26 mutations causing Keratitis-Ichthyosis-Deafness syndrome.

Connexin (Cx) proteins form intercellular gap junction channels by first assembling into single membrane hemichannels that then dock to connect the cytoplasm of two adjacent cells. Gap junctions are highly specialized structures that allow the direct passage of small molecules between cells to maintain tissue homeostasis. Functional activity of nonjunctional hemichannels has now been shown in several experimental systems. Hemichannels may constitute an important diffusional exchange pathway with the extracellular space, but the extent of their normal physiological role is currently unknown. Aberrant hemichannel activity has been linked to mutations of connexin proteins involved in genetic diseases. Here, we review a proposed role for hemichannels in the pathogenesis of Keratitis-Ichthyosis-Deafness (KID) syndrome associated with connexin26 (Cx26) mutations. Continued functional evaluation of mutated hemichannels linked to human hereditary disorders may provide additional insights into the mechanisms governing their regulation in normal physiology and dysregulation in disease. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Single-Cell RNA Sequencing Analysis of the Early Postnatal Mouse Lens Epithelium.

Purpose: The lens epithelium maintains the overall health of the organ. We used single-cell RNA sequencing (scRNA-seq) technology to assess transcriptional heterogeneity between cells in the postnatal day 2 (P2) epithelium and identify distinct epithelial cell subtypes. Analysis of these data was used to better understand lens growth, differentiation, and homeostasis on P2. Methods: scRNA-seq on P2 mouse lenses was performed using the 10x Genomics Chromium Single Cell 3' Kit (v3.1) and short-read Illumina sequencing. Sequence alignment and preprocessing of data were conducted using 10x Genomics Cell Ranger software. Seurat was employed for preprocessing, quality control, dimensionality reduction, and cell clustering, and Monocle was utilized for trajectory analysis to understand the developmental progression of the lens cells. CellChat and GO analyses were used to explore cell-cell communication networks and signaling interactions. Results: Lens epithelial cells (LECs) were divided into seven subclusters, classified by specific gene markers. The expression of crystallin, cell-cycle, and metabolic genes was not uniform, indicating distinct functional roles of LECs. Trajectory analysis predicted a bifurcation of differentiating and cycling cells from an Igfbp5+ progenitor pool. We also identified heterogeneity in signaling molecules and pathways, suggesting that cycling and progenitor subclusters have prominent roles in coordinating crosstalk. Conclusions: scRNA-seq corroborated many known markers of epithelial differentiation and proliferation while providing further insight into the pathways and genes directing these processes. Interestingly, we demonstrated that the developing epithelium can be divided into distinct subpopulations. These clusters reflect the transcriptionally diverse roles of the epithelium in proliferation, signaling, and maintenance.