Violet-light suppression of thermogenesis by opsin 5 hypothalamic neurons.

The opsin family of G-protein-coupled receptors are used as light detectors in animals. Opsin 5 (also known as neuropsin or OPN5) is a highly conserved opsin that is sensitive to visible violet light1,2. In mice, OPN5 is a known photoreceptor in the retina3 and skin4 but is also expressed in the hypothalamic preoptic area (POA)5. Here we describe a light-sensing pathway in which POA neurons that express Opn5 regulate thermogenesis in brown adipose tissue (BAT). We show that Opn5 is expressed in glutamatergic warm-sensing POA neurons that receive synaptic input from several thermoregulatory nuclei. We further show that Opn5 POA neurons project to BAT and decrease its activity under chemogenetic stimulation. Opn5-null mice show overactive BAT, increased body temperature, and exaggerated thermogenesis when cold-challenged. Moreover, violet photostimulation during cold exposure acutely suppresses BAT temperature in wild-type mice but not in Opn5-null mice. Direct measurements of intracellular cAMP ex vivo show that Opn5 POA neurons increase cAMP when stimulated with violet light. This analysis thus identifies a violet light-sensitive deep brain photoreceptor that normally suppresses BAT thermogenesis.

Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice.

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus-induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

Retinal ganglion cell interactions shape the developing mammalian visual system.

Retinal ganglion cells (RGCs) serve as a crucial communication channel from the retina to the brain. In the adult, these cells receive input from defined sets of presynaptic partners and communicate with postsynaptic brain regions to convey features of the visual scene. However, in the developing visual system, RGC interactions extend beyond their synaptic partners such that they guide development before the onset of vision. In this Review, we summarize our current understanding of how interactions between RGCs and their environment influence cellular targeting, migration and circuit maturation during visual system development. We describe the roles of RGC subclasses in shaping unique developmental responses within the retina and at central targets. Finally, we highlight the utility of RNA sequencing and genetic tools in uncovering RGC type-specific roles during the development of the visual system.

Encephalopsin (OPN3) is required for normal refractive development and the GO/GROW response to induced myopia.

Purpose: Myopia, or nearsightedness, is the most common form of refractive error and is increasing in prevalence. While significant efforts have been made to identify genetic variants that predispose individuals to myopia, these variants are believed to account for only a small portion of the myopia prevalence, leading to a feedback theory of emmetropization, which depends on the active perception of environmental visual cues. Consequently, there has been renewed interest in studying myopia in the context of light perception, beginning with the opsin family of G-protein coupled receptors (GPCRs). Refractive phenotypes have been characterized in every opsin signaling pathway studied, leaving only Opsin 3 (OPN3), the most widely expressed and blue-light sensing noncanonical opsin, to be investigated for function in the eye and refraction. Methods: Opn3 expression was assessed in various ocular tissues using an Opn3eGFP reporter. Weekly refractive development in Opn3 retinal and germline mutants from 3 to 9 weeks of age was measured using an infrared photorefractor and spectral domain optical coherence tomography (SD-OCT). Susceptibility to lens-induced myopia was then assessed using skull-mounted goggles with a -30 diopter experimental and a 0 diopter control lens. Mouse eye biometry was similarly tracked from 3 to 6 weeks. A myopia gene expression signature was assessed 24 h after lens induction for germline mutants to further assess myopia-induced changes. Results: Opn3 was found to be expressed in a subset of retinal ganglion cells and a limited number of choroidal cells. Based on an assessment of Opn3 mutants, the OPN3 germline, but not retina conditional Opn3 knockout, exhibits a refractive myopia phenotype, which manifests in decreased lens thickness, shallower aqueous compartment depth, and shorter axial length, atypical of traditional axial myopias. Despite the short axial length, Opn3 null eyes demonstrate normal axial elongation in response to myopia induction and mild changes in choroidal thinning and myopic shift, suggesting that susceptibility to lens-induced myopia is largely unchanged. Additionally, the Opn3 null retinal gene expression signature in response to induced myopia after 24 h is distinct, with opposing Ctgf, Cx43, and Egr1 polarity compared to controls. Conclusions: The data suggest that an OPN3 expression domain outside the retina can control lens shape and thus the refractive performance of the eye. Prior to this study, the role of Opn3 in the eye had not been investigated. This work adds OPN3 to the list of opsin family GPCRs that are implicated in emmetropization and myopia. Further, the work to exclude retinal OPN3 as the contributing domain in this refractive phenotype is unique and suggests a distinct mechanism when compared to other opsins.

Modulation of Both Intrinsic and Extrinsic Factors Additively Promotes Rewiring of Corticospinal Circuits after Spinal Cord Injury.

Axon regeneration after spinal cord injury (SCI) is limited by both a decreased intrinsic ability of neurons to grow axons and the growth-hindering effects of extrinsic inhibitory molecules expressed around the lesion. Deletion of phosphatase and tensin homolog (Pten) augments mechanistic target of rapamycin (mTOR) signaling and enhances the intrinsic regenerative response of injured corticospinal neurons after SCI. Because of the variety of growth-restrictive extrinsic molecules, it remains unclear how inhibition of conserved inhibitory signaling elements would affect axon regeneration and rewiring after SCI. Moreover, it remains unknown how a combinatorial approach to modulate both extrinsic and intrinsic mechanisms can enhance regeneration and rewiring after SCI. In the present study, we deleted RhoA and RhoC, which encode small GTPases that mediate growth inhibition signals of a variety of extrinsic molecules, to remove global extrinsic pathways. RhoA/RhoC double deletion in mice suppressed retraction or dieback of corticospinal axons after SCI. In contrast, Pten deletion increased regrowth of corticospinal axons into the lesion core. Although deletion of both RhoA and Pten did not promote axon regrowth across the lesion or motor recovery, it additively promoted rewiring of corticospinal circuits connecting the cerebral cortex, spinal cord, and hindlimb muscles. Our genetic findings, therefore, reveal that a combinatorial approach to modulate both intrinsic and extrinsic factors can additively promote neural circuit rewiring after SCI.SIGNIFICANCE STATEMENT SCI often causes severe motor deficits because of damage to the corticospinal tract (CST), the major neural pathway for voluntary movements. Regeneration of CST axons is required to reconstruct motor circuits and restore functions; however, a lower intrinsic ability to grow axons and extrinsic inhibitory molecules severely limit axon regeneration in the CNS. Here, we investigated whether suppression of extrinsic inhibitory cues by genetic deletion of Rho as well as enhancement of the intrinsic pathway by deletion of Pten could enable axon regrowth and rewiring of the CST after SCI. We show that simultaneous elimination of extrinsic and intrinsic signaling pathways can additively promote axon sprouting and rewiring of the corticospinal circuits. Our data demonstrate a potential molecular approach to reconstruct motor pathways after SCI.

Proceedings of the 2022 National Toxicology Program Satellite Symposium.

The 2022 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri," was held in Austin, Texas at the Society of Toxicologic Pathology's 40th annual meeting during a half-day session on Sunday, June 19. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers' talks along with select images that were used by the audience for voting and discussion. Various lesions and topics covered during the symposium included induced and spontaneous neoplastic and nonneoplastic lesions in the mouse lung, spontaneous lesions in the reproductive tract of a female cynomolgus macaque, induced vascular lesions in a mouse asthma model and interesting case studies in a rhesus macaque, dog and genetically engineered mouse model.

Opsin 3-Gαs Promotes Airway Smooth Muscle Relaxation Modulated by G Protein Receptor Kinase 2.

Recently, we characterized blue light-mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, we characterized the cellular signaling mechanisms of photorelaxation. We confirmed the functional role of OPN3 in blue light photorelaxation using trachea from OPN3 null mice (maximal relaxation 52 ± 13% compared with wild-type mice 90 ± 4.3%, P < 0.05). We then demonstrated colocalization of OPN3 and Gαs using co-IP and proximity ligation assays in primary human ASM cells, which was further supported by an increase in cAMP in mouse trachea treated with blue light compared with dark controls (23 ± 3.6 vs. 14 ± 2.6 pmol cAMP/ring, P < 0.05). Downstream PKA (protein kinase A) involvement was shown by inhibiting photorelaxation using Rp-cAMPS (P < 0.0001). Moreover, we observed converging mechanisms of desensitization by chronic ß2-agonist exposure in mouse trachea and correlated this finding with colocalization of OPN3 and GRK2 (G protein receptor kinase) in primary human ASM cells. Finally, an overexpression model of OPN1LW (a red light photoreceptor in the same opsin family) in human ASM cells showed an increase in intracellular cAMP levels following red light exposure compared with nontransfected cells (48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg protein, P < 0.01), suggesting a conserved photorelaxation mechanism for wavelengths of light that are more tissue penetrant. Together, these results demonstrate that blue light photorelaxation in ASM is mediated by the OPN3 receptor interacting with Gαs, which increases cAMP levels, activating PKA and modulated by GRK2.

Mechanisms underlying spontaneous phasic contractions and sympathetic control of smooth muscle in the rat caudal epididymis.

Here we investigate mechanisms underlying spontaneous phasic contractions (SPCs) and sympathetic control of contractility in the rat epididymis, a long tubular duct involved in transportation and maturation of sperm. Longitudinal contractions of short segments (~ 1.5 mm) of rat proximal and distal caudal epididymal duct were measured + / - nerve stimulation. The extent of sympathetic innervation of these duct regions was determined by immunohistochemistry. Proximal caudal duct segments (150-300 µm dia.) exhibited SPCs, while distal segments (350-500 µm) were quiescent in ~ 80% of preparations. SPC amplitude and frequency were reduced by the L-type voltage-dependent Ca2+ channel (LVDCC) blocker nifedipine (1 µM), with the T-type voltage-dependent Ca2+ channel (TVDCC) blocker ML218 (1 µM) specifically decreasing SPC frequency. SPCs were inhibited upon blockade of the SR/ER Ca2+-ATPase (CPA 10 µM). SPCs were also inhibited by caffeine (1 µM), 2-APB (100 µM), niflumic acid (100 µM), or by lowering extracellular [Cl-] from 134.4 to 12.4 mM but not by ryanodine (25 µM) or tetracaine (100 µM). Electrical field stimulation (EFS) at 2 Hz for 60 s caused a sustained α1-adrenoceptor-sensitive contraction in distal segments and enhanced and/or induced α2-adrenoceptor-sensitive oscillatory phasic contractions in proximal and distal segments, the latter mimicked by application of the α2-adrenoceptor agonist clonidine. We hypothesise that SPCs in the proximal cauda are triggered by pacemaker mechanisms involving rhythmic IP3 receptor-operated SR/ER store Ca2+ release and resultant activation of CaCC with TVDCCs and possibly LVDCCs subserving in this process. Sympathetic nerve-released noradrenaline induces α2-adrenoceptor-mediated phasic contractions in the proximal and distal cauda. These findings provide new pharmacological targets for male infertility and contraception.

Developmental vascular regression is regulated by a Wnt/ß-catenin, MYC and CDKN1A pathway that controls cell proliferation and cell death.

Normal development requires tight regulation of cell proliferation and cell death. Here, we have investigated these control mechanisms in the hyaloid vessels, a temporary vascular network in the mammalian eye that requires a Wnt/ß-catenin response for scheduled regression. We investigated whether the hyaloid Wnt response was linked to the oncogene Myc, and the cyclin-dependent kinase inhibitor CDKN1A (P21), both established regulators of cell cycle progression and cell death. Our analysis showed that the Wnt pathway co-receptors LRP5 and LRP6 have overlapping activities that mediate the Wnt/ß-catenin signaling in hyaloid vascular endothelial cells (VECs). We also showed that both Myc and Cdkn1a are downstream of the Wnt response and are required for hyaloid regression but for different reasons. Conditional deletion of Myc in VECs suppressed both proliferation and cell death. By contrast, conditional deletion of Cdkn1a resulted in VEC overproliferation that countered the effects of cell death on regression. When combined with analysis of MYC and CDKN1A protein levels, this analysis suggests that a Wnt/ß-catenin and MYC-CDKN1A pathway regulates scheduled hyaloid vessel regression.

Functional heterogeneity of PDGFRα (+) cells in spontaneously active urogenital tissues.

AIMS: As PDGFRα (+) cells appear not to suppress the excitability of detrusor smooth muscle by generating SK3-dependent hyperpolarising as proposed in the gastrointestinal tract, we further explored the functional roles of PDGFRα (+) cells in regulating the spontaneous activity of urogenital tissues. METHODS: Using PDGFRα-eGFP mice, intracellular Ca2+ signaling in PDGFRα (+) cells of the bladder lamina propria, renal pelvis, and seminal vesicle were visualized using Cal-590 fluorescence. The distribution and SK3 expression of PDGFRα (+) cells were also examined by immunohistochemistry. RESULTS: In the bladder lamina propria, SK3 (-) PDGFRα (+) cells exhibited spontaneous Ca2+ transients and responded to stimulation of P2Y1 purinoceptors with MRS2365 (100 nM) or adenosine diphosphate (ADP) (100 µM) by developing Ca2+ transients. In the proximal renal pelvis, PDGFRα (+) cells were distributed in the mucosal, muscular and serosal layers but did not express SK3 immunoreactivity. PDGFRα (+) cells in the musculature resembling atypical smooth muscle cells generated spontaneous Ca2+ transients that were partially suppressed upon P2Y1-stimulation, while vigorously responding to human angiotensin II (100 nM). In the seminal vesicle, PDGFRα (+) cells in the musculature but not mucosa expressed SK3 immunoreactivity. In the mucosa, the P2Y1 stimulation evoked Ca2+ transients in both PDGFRα (+) cells and PDGFRα (-) cells. CONCLUSION: PDGFRα (+) cells in spontaneously active urogenital tissues display heterogeneity in terms of their SK3 expression and P2Y1-induced Ca2+ responses. Muscular PDGFRα (+) cells in the renal pelvis and mucosal PDGFRα (+) cells in the seminal vesicle may generate depolarizing signals to drive smooth muscle cells.

YAP/TAZ-CDC42 signaling regulates vascular tip cell migration.

Angiogenesis and vascular remodeling are essential for the establishment of vascular networks during organogenesis. Here we show that the Hippo signaling pathway effectors YAP and TAZ are required, in a gene dosage-dependent manner, for the proliferation and migration of vascular endothelial cells (ECs) during retinal angiogenesis. Intriguingly, nuclear translocation of YAP and TAZ induced by Lats1/2-deletion blocked endothelial migration and phenocopied Yap/Taz-deficient mutants. Furthermore, overexpression of a cytoplasmic form of YAP (YAPS127D) partially rescued the migration defects caused by loss of YAP and TAZ function. Finally, we found that cytoplasmic YAP positively regulated the activity of the small GTPase CDC42, deletion of which caused severe defects in endothelial migration. These findings uncover a previously unrecognized role of cytoplasmic YAP/TAZ in promoting cell migration by activating CDC42 and provide insight into how Hippo signaling in ECs regulates angiogenesis.

Crim1 regulates integrin signaling in murine lens development.

The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1(glcr11), which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1(null) and Crim1(cko), we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active ß1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development.

A direct and melanopsin-dependent fetal light response regulates mouse eye development.

Vascular patterning is critical for organ function. In the eye, there is simultaneous regression of embryonic hyaloid vasculature (important to clear the optical path) and formation of the retinal vasculature (important for the high metabolic demands of retinal neurons). These events occur postnatally in the mouse. Here we have identified a light-response pathway that regulates both processes. We show that when mice are mutated in the gene (Opn4) for the atypical opsin melanopsin, or are dark-reared from late gestation, the hyaloid vessels are persistent at 8 days post-partum and the retinal vasculature overgrows. We provide evidence that these vascular anomalies are explained by a light-response pathway that suppresses retinal neuron number, limits hypoxia and, as a consequence, holds local expression of vascular endothelial growth factor (VEGFA) in check. We also show that the light response for this pathway occurs in late gestation at about embryonic day 16 and requires the photopigment in the fetus and not the mother. Measurements show that visceral cavity photon flux is probably sufficient to activate melanopsin-expressing retinal ganglion cells in the mouse fetus. These data thus show that light--the stimulus for function of the mature eye--is also critical in preparing the eye for vision by regulating retinal neuron number and initiating a series of events that ultimately pattern the ocular blood vessels.

Pacemaker Mechanisms Driving Pyeloureteric Peristalsis: Modulatory Role of Interstitial Cells.

The peristaltic pressure waves in the renal pelvis that propel urine expressed by the kidney into the ureter towards the bladder have long been considered to be 'myogenic', being little affected by blockers of nerve conduction or autonomic neurotransmission, but sustained by the intrinsic release of prostaglandins and sensory neurotransmitters. In uni-papilla mammals, the funnel-shaped renal pelvis consists of a lumen-forming urothelium and a stromal layer enveloped by a plexus of 'typical' smooth muscle cells (TSMCs), in multi-papillae kidneys a number of minor and major calyces fuse into a large renal pelvis. Electron microscopic, electrophysiological and Ca2+ imaging studies have established that the pacemaker cells driving pyeloureteric peristalsis are likely to be morphologically distinct 'atypical' smooth muscle cells (ASMCs) that fire Ca2+ transients and spontaneous transient depolarizations (STDs) which trigger propagating nifedipine-sensitive action potentials and Ca2+ waves in the TSMC layer. In uni-calyceal kidneys, ASMCs predominately locate on the serosal surface of the proximal renal pelvis while in multi-papillae kidneys they locate within the sub-urothelial space. 'Fibroblast-like' interstitial cells (ICs) located in the sub-urothelial space or adventitia are a mixed population of cells, having regional and species-dependent expression of various Cl-, K+, Ca2+ and cationic channels. ICs display asynchronous Ca2+ transients that periodically synchronize into bursts that accelerate ASMC Ca2+ transient firing. This review presents current knowledge of the architecture of the proximal renal pelvis, the role Ca2+ plays in renal pelvis peristalsis and the mechanisms by which ICs may sustain/accelerate ASMC pacemaking.

Excitation-Contraction Coupling in Ureteric Smooth Muscle: Mechanisms Driving Ureteric Peristalsis.

The ureter acts as a functional syncytium and is controlled by a propagating plateau-type action potential (AP) which gives rise to a wave of contraction (ureteral peristalsis) via a process called excitation-contraction (E-C)coupling. The second messenger Ca2+ activates Ca2+/calmodulin-dependent myosin light chain kinase-dependent phosphorylation of 20-kDa regulatory light chains of myosin which leads to ureteric contraction. Ca2+ entry from the extracellular space via voltage-gated L-type Ca2+ channels (VGCCs) provides the major source of activator Ca2+, responsible for generation of both the AP and a Ca2+ transient that appears as an intercellular Ca2+ wave. The AP, inward Ca2+ current, Ca2+ transient and twitch contraction are all fully blocked by the selective L-type Ca2+ channel blocker nifedipine. Ca2+ entry via VGCCs, coupled to activation of Ca2+-sensitive K+ (KCa) or Cl- (ClCa) channels, acts as a negative or positive feedback mechanism, respectively, to control excitability and the amplitude and duration of the plateau component of the AP, Ca2+ transient and twitch contraction. The ureter, isolated from the pelvis, is not spontaneously active. However, spontaneous activity can be initiated in the proximal and distal ureter by a variety of biological effectors such as neurotransmitters, paracrine, endocrine and inflammatory factors. Applied agonists depolarise ureteric smooth muscles cells to threshold of AP activation, initiating propagating intercellular AP-mediated Ca2+ waves to produce antegrade and/or retrograde ureteric peristalsis. Several mechanisms have been proposed to describe agonist-induced depolarization of ureteric smooth muscle, which include suppression of K+ channels, stimulation of ClCa current and activation of non-selective cation receptor/store operated channels.

Wnt ligands from the embryonic surface ectoderm regulate 'bimetallic strip' optic cup morphogenesis in mouse.

The Wnt/ß-catenin response pathway is central to many developmental processes. Here, we assessed the role of Wnt signaling in early eye development using the mouse as a model system. We showed that the surface ectoderm region that includes the lens placode expressed 12 out of 19 possible Wnt ligands. When these activities were suppressed by conditional deletion of wntless (Le-cre; Wls(fl/fl)) there were dramatic consequences that included a saucer-shaped optic cup, ventral coloboma, and a deficiency of periocular mesenchyme. This phenotype shared features with that produced when the Wnt/ß-catenin pathway co-receptor Lrp6 is mutated or when retinoic acid (RA) signaling in the eye is compromised. Consistent with this, microarray and cell fate marker analysis identified a series of expression changes in genes known to be regulated by RA or by the Wnt/ß-catenin pathway. Using pathway reporters, we showed that Wnt ligands from the surface ectoderm directly or indirectly elicit a Wnt/ß-catenin response in retinal pigment epithelium (RPE) progenitors near the optic cup rim. In Le-cre; Wls(fl/fl) mice, the numbers of RPE cells are reduced and this can explain, using the principle of the bimetallic strip, the curvature of the optic cup. These data thus establish a novel hypothesis to explain how differential cell numbers in a bilayered epithelium can lead to shape change.

Neuropsin (OPN5)-mediated photoentrainment of local circadian oscillators in mammalian retina and cornea.

The molecular circadian clocks in the mammalian retina are locally synchronized by environmental light cycles independent of the suprachiasmatic nuclei (SCN) in the brain. Unexpectedly, this entrainment does not require rods, cones, or melanopsin (OPN4), possibly suggesting the involvement of another retinal photopigment. Here, we show that the ex vivo mouse retinal rhythm is most sensitive to short-wavelength light but that this photoentrainment requires neither the short-wavelength-sensitive cone pigment [S-pigment or cone opsin (OPN1SW)] nor encephalopsin (OPN3). However, retinas lacking neuropsin (OPN5) fail to photoentrain, even though other visual functions appear largely normal. Initial evidence suggests that OPN5 is expressed in select retinal ganglion cells. Remarkably, the mouse corneal circadian rhythm is also photoentrainable ex vivo, and this photoentrainment likewise requires OPN5. Our findings reveal a light-sensing function for mammalian OPN5, until now an orphan opsin.

Interstitial cell modulation of pyeloureteric peristalsis in the mouse renal pelvis examined using FIBSEM tomography and calcium indicators.

Typical and atypical smooth muscle cells (TSMCs and ASMCs, respectively) and interstitial cells (ICs) within the pacemaker region of the mouse renal pelvis were examined using focused ion beam scanning electron (FIB SEM) tomography, immunohistochemistry and Ca2+ imaging. Individual cells within 500-900 electron micrograph stacks were volume rendered and associations with their neighbours established. 'Ribbon-shaped', Ano1 Cl- channel immuno-reactive ICs were present in the adventitia and the sub-urothelial space adjacent to the TSMC layer. ICs in the proximal renal pelvis were immuno-reactive to antibodies for CaV3.1 and hyperpolarization-activated cation nucleotide-gated isoform 3 (HCN3) channel sub-units, while basal-epithelial cells (BECs) were intensely immuno-reactive to Kv7.5 channel antibodies. Adventitial to the TSMC layer, ASMCs formed close appositions with TSMCs and ICs. The T-type Ca2+channel blocker, Ni2+ (10-200 µM), reduced the frequency while the L-type Ca2+ channel blocker (1 µM nifedipine) reduced the amplitude of propagating Ca2+ waves and contractions in the TSMC layer. Upon complete suppression of Ca2+ entry through TSMC Ca2+ channels, ASMCs displayed high-frequency (6 min-1) Ca2+ transients, and ICs distributed into two populations of cells firing at 1 and 3 min-1, respectively. IC Ca2+ transients periodically (every 3-5 min-1) summed into bursts which doubled the frequency of ASMC Ca2+ transient firing. Synchronized IC bursting and the acceleration of ASMC firing were inhibited upon blockade of HCN channels with ZD7288 or cell-to-cell coupling with carbenoxolone. While ASMCs appear to be the primary pacemaker driving pyeloureteric peristalsis, it was concluded that sub-urothelial HCN3(+), CaV3.1(+) ICs can accelerate ASMC Ca2+ signalling.

CRIM1 haploinsufficiency causes defects in eye development in human and mouse.

Colobomatous macrophthalmia with microcornea syndrome (MACOM, Online Mendelian Inheritance in Man (OMIM) 602499) is an autosomal dominantly inherited malformation of the eye, which is characterized by microcornea with increased axial length, coloboma of the iris and of the optic disc, and severe myopia. We performed whole-exome sequencing (WES) in two affected individuals from the 2p23-p16-linked MACOM family, which includes 13 affected individuals in 3 generations. As no shared novel variation was found on the linked haplotype, we performed copy number variation (CNV) analysis by comparing the coverage of all exons in the WES data sets of the 2 patients with the coverage of 26 control exomes. We identified a heterozygous deletion predicted to span 22 kb including exons 14-17 of CRIM1 (cysteine-rich transmembrane bone morphogenetic protein (BMP) regulator 1). Quantitative PCR (qPCR) analysis confirmed the deletion, which was present in 11 affected individuals. Split-read analysis of WES data followed by breakpoint PCR and Sanger sequencing determined both breakpoints flanked by a 4-bp microhomology (CTTG). In the mouse, Crim1 is a growth-factor-binding protein with pleiotropic roles in the development of multiple organs, including the eye. To investigate the role of Crim1 during eye development in mice, we crossed a Crim1(flox) mouse line with the Ap2α-cre mouse line, which expresses Cre in the head surface ectoderm. Strikingly, we observed alterations of eye development in homozygous mice leading to severe anatomical and morphological changes overlapping with the anomalies observed in MACOM patients. Taken together, these findings identify CRIM1 as the causative gene for MACOM syndrome and emphasize the importance of CRIM1 in eye development.

p120-catenin-dependent junctional recruitment of Shroom3 is required for apical constriction during lens pit morphogenesis.

Apical constriction (AC) is a widely utilized mechanism of cell shape change whereby epithelial cells transform from a cylindrical to conical shape, which can facilitate morphogenetic movements during embryonic development. Invertebrate epithelial cells undergoing AC depend on the contraction of apical cortex-spanning actomyosin filaments that generate force on the apical junctions and pull them toward the middle of the cell, effectively reducing the apical circumference. A current challenge is to determine whether these mechanisms are conserved in vertebrates and to identify the molecules responsible for linking apical junctions with the AC machinery. Utilizing the developing mouse eye as a model, we have uncovered evidence that lens placode AC may be partially dependent on apically positioned myosin-containing filaments associated with the zonula adherens. In addition we found that, among several junctional components, p120-catenin genetically interacts with Shroom3, a protein required for AC during embryonic morphogenesis. Further analysis revealed that, similar to Shroom3, p120-catenin is required for AC of lens cells. Finally, we determined that p120-catenin functions by recruiting Shroom3 to adherens junctions. Together, these data identify a novel role for p120-catenin during AC and further define the mechanisms required for vertebrate AC.