Quantification of Metamorphopsia in Resolved Idiopathic Central Serous Chorioretinopathy: An Analysis Using M-CHARTS, Amsler Grid, and Optical Coherence Tomography.

Purpose: To quantify metamorphopsia in patients with resolved idiopathic Central Serous Chorioretinopathy (CSCR) using M-CHARTS and compare the results with the traditional Amsler grid and optical coherence tomography (OCT). Patients and Methods: For the purpose of this study, all consecutive cases of patients with resolved CSCR were evaluated for metamorphopsia (using the standard Amsler grid and M-CHARTS) and spectral domain OCT. The OCT images were analyzed for the following five parameters: central macular thickness, pigment epithelial detachment, retinal pigment epithelial bumps, discontinuation in the inner segment/outer segment junction or the external limiting membrane, fibrinous exudates in the subretinal space, and hyperreflective dots in the intraretinal and/or subretinal layer. Binary logistic regression was used to find the association between metamorphopsia and foveal morphology. Cohen's Kappa was used to determine the agreement between the M-CHARTS and Amsler grid for diagnosing metamorphopsia. The sensitivity, specificity, and accuracy in the diagnosis of metamorphopsia were calculated against the Amsler grid. Results: Of 41 eyes, Amsler Grid detected metamorphopsia in 39.02%, and M-CHARTS detected metamorphopsia in 53.66%. The agreement rate of detection between the two tests was moderate (Kappa=0.52). M-CHARTS had a sensitivity of 87.50%, a specificity of 68.00%, a positive predictive value of 63.64%; and a negative predictive value of 89.47% for the diagnosis of metamorphopsia compared to the Amsler grid. The presence of PED in OCT was significantly associated with metamorphopsia. Conclusion: M-CHARTS can be a useful ancillary test to detect and quantify metamorphopsia even after fluid resolution in CSCR. Structural changes in macular morphology as observed with OCT can predict the likelihood of metamorphopsia.

Impact of active vision therapy compared to conventional patching therapy on visual acuity and stereoacuity in children with amblyopia.

PURPOSE: To compare improvements in visual acuity (VA) and stereoacuity between active vision therapy (AVT) and conventional patching therapy in children with amblyopia. METHODS: This study included 65 children aged 5 to 16 years (mean age±SD, 11.00±3.29 years) with unilateral amblyopia. Among them, 31 children underwent active vision therapy (AVT group), and 34 children underwent conventional patching therapy (patching group). AVT group underwent three sequential phases of AVT: Monocular phase (pursuit, saccades, fixation, visuomotor, eye-hand coordination, and central peripheral activities), biocular phase (diplopia awareness, antisuppression, monocular fixation in a binocular field, accommodative activities, bilateral integration, and fine motor activities) and binocular phase (fusion and stereopsis). Patching group patched their fellow eyes as per guidelines by Pediatric Eye Disease Investigator Group. Best-corrected monocular VA and stereoacuity were measured at baseline and after three months of therapy in both groups. RESULTS: There were significant improvements in the mean acuities in amblyopic eye (AE) in both AVT (0.32±0.11 logMAR, p <0.001) and patching groups (0.27±0.19 logMAR, p Ë‚ 0.001). However, there was no significant difference in mean acuity gains in AE between AVT and patching groups (p = 0.059). Mean gains in stereoacuities (log seconds of arc) were statistically significant in both AVT (0.81±0.34, p < 0.001) and patching groups (0.32±0.34, p < 0.001). The stereoacuity gain in the AVT group was significantly higher compared to patching group (p < 0.001). CONCLUSION: Active vision therapy had a better impact than conventional patching therapy in terms of improvement of stereoacuity but not in terms of VA when used for treating children with amblyopia.

Kaleidos-based binocular vision analyzer for the estimation of refractive errors and horizontal ocular deviation in children.

CLINICAL RELEVANCE: Knowing the agreement and interobserver variability of the 2WIN-S photorefractometer with cycloplegic retinoscopy and prism cover test is important for judging the clinical usefulness of the device. BACKGROUND: This study aims to determine an agreement and interobserver reliability between 2WIN-S photorefractometer and cycloplegic retinoscopy for estimating refractive errors and between 2WIN-S and prism cover test for measuring horizontal ocular deviation in children. METHODS: Two groups of 129 and 64 participants (range, 5 to 16 years) were recruited for the agreement and interobserver reliability studies, respectively. Non-cycloplegic 2WIN-S refraction was compared with cycloplegic retinoscopy for low and moderate myopia and hyperopia. Similarly, corneal reflex-function (CR-function) of 2WIN-S was compared with prism cover test for horizontal ocular deviation. Bland-Altman plots were used to depict agreement, and the intraclass correlation coefficient (ICC) was used for determining interobserver reliability of repeated measures. RESULTS: The mean differences in spherical equivalent, J0 (Cartesian astigmatism) and J45 (oblique Jackson cross-cylinder) between cycloplegic retinoscopy and 2WIN-S were 1.10 (95% CI = 0.97 to 1.21, p < 0.001), -0.06 (95% CI = -0.09 to -0.03, p < 0.001) and 0.009 (95% CI = -0.01 to 0.03, p = 0.35), respectively. The overall median difference in the horizontal ocular deviation between prism cover test and 2WIN-S was -4.5 (Interquartile range = -12.0 to 4.0). For constant strabismus, this difference was insignificant (median= -2.0, interquartile range = -11.0 to 6.7, p = 0.19) between prism cover test and 2WIN-S. The ICC was high for the spherical equivalent (0.997; p < 0.001), J0 (0.932; p < 0.001) and J45 (0.901; p < 0.001), whereas it was low-to-moderate for horizontal ocular deviation (0.50; p < 0.001). CONCLUSION: 2WIN-S refraction was in agreement with cycloplegic retinoscopy for low and moderate myopia and hyperopia, and requires an adjustment for the consistent differences between cycloplegic retinoscopy and 2WIN-S refraction. The measurement of constant strabismus with 2WIN-S is comparable to that of prism cover test.

Bilateral Iridochorioretinal Coloboma Managed with Low Vision Rehabilitation: A Case Report.

Ocular coloboma is a rare congenital disability. If involving the macula, it affects the patient's vision and subsequently affects childhood development and quality of life in the future. Appropriate low vision aid and timely rehabilitation can provide the best possible quality of life for visually impaired children. We report a 9-year-old boy who presented with a diminution of vision in both eyes, and who was just enrolled in pre-school. He was diagnosed with bilateral iridochorioretinal coloboma associated with nystagmus and unilateral cataract. After all the necessary evaluation, a telescope was prescribed for distance and a dome magnifier for near. Furthermore, a peaked cap and photo grey lens were given for outdoor activities. This case highlights the importance of low vision intervention in a visually impaired child. Appropriate low vision aid and rehabilitation can improve patients' lifestyle and academic performance who are diagnosed with iridochorioretinal coloboma. Keywords: case reports; coloboma; ocular; rehabilitation; training.

Burden and Risk Factors of Diabetic Retinopathy Among Diabetic Patients Attending a Multispecialty Tertiary Eye Hospital in Nepal.

INTRODUCTION: As the number of people with diabetes mellitus is increasing because of urbanization and change in dietary habits and sedentary lifestyle, the number of diabetic retinopathy is also expected to increase in future. [1] [sa2] We aimed to find out the prevalence of diabetic retinopathy and associated risk factors among diabetic patients in the tertiary eye hospital. MATERIALS AND METHODS: This is the observational cross-sectional study enrolling 420 diabetic patients visiting the multispecialty tertiary eye hospital between March 2020 and February 2021. Anthropometry measurement, laboratory risk profiles and blood pressure were recorded Results: The prevalence of any diabetic retinopathy, proliferative diabetic retinopathy, and diabetic macular edema were 30.96 %, 6.19 %, and 5.95 % respectively. The duration of DM (p=0.001), hypertension (p=0.04), high SBP (p=0.023), abdominal obesity (p=0.015), high LDL(p=0.011) cholesterol, low HDL cholesterol(p=0.012), and creatinine (p=0.001) were associated with DR in our study. CONCLUSION: A holistic approach should target to control the modifiable risk factors like blood sugar, blood pressure, lipid profile, kidney function, and obesity to prevent DR. Anthropometric assessment of waist to height and waist circumference should be included in the holistic health promotion strategy in Nepal as BMI may not be risk factors for DR in Nepalese people.

Clinico-epidemiological Study of Patients with Glaucoma in a Tertiary Eye Center, Nepal.

INTRODUCTION: Major cause of irreversible blindness is glaucoma which is increasing as a significant global health problem. Knowledge of glaucoma burden and its clinical characteristics in this region is essential. MATERIALS AND METHODS: This was a cross-sectional hospital -based study conducted at Glaucoma unit in CHEERS Hospital, Bhaktapur from August 2015 to July 2019. Patients' demographic profile, along with comprehensive eye examination findings were reviewed from the records. Data analysis was done with IBM SPSS version 24 and was presented in mean (±SD), frequency and proportions and a chi- square test was applied. P value < 0.05 was considered statistically significant. RESULTS: Total of 310 glaucoma cases were included in this study. The mean age presentation was 61.89±14.4 years, and the majority of patients (52.9%) were in the age group 60-79 years. Most patients had primary open angle glaucoma (171, 55.2%) followed by primary angle closure glaucoma (92, 29.7%). CONCLUSION: Primary open angle glaucoma is the most common form of glaucoma. The proportion of glaucoma increased with increasing age (> 40 years). Provision of screening programs targeting persons with risk factors and opportunistic eye examinations would be beneficial in detection of glaucoma.

Clinical variations and therapeutic challenges in the management of symptomatic retinal artery macroaneurysm: a tertiary center experience.

PURPOSE: To find out clinical characteristics, therapeutic options, and visual outcome in symptomatic retinal artery macroaneurysm (RAMA) patients. METHOD: Newly diagnosed cases of symptomatic RAMA from January 2015 to December 2019 were included. Fifteen eyes of 15 patients with mean age 62.46 years ± 14.89 (SD) fulfilled the inclusion criteria. RESULT: Hypertension was present in 66.6% of the patients, and the commonest site was superotemporal (12 eyes, 80%). The most commonly employed treatment was Nd: Yag laser hyaloidotomy in 4 eyes (26.6%). Other treatments were intravitreal bevacizumab in 3 eyes (20%), focal laser with intravitreal bevacizumab in 3 eyes (20%), PPV with focal laser (13.3%) in 2, focal laser only in one (6.6%), PPV with focal and intravitreal bevacizumab in one (6.6%). The mean best-corrected visual acuity (BCVA) at baseline was 1.35 ± 0.84 LogMAR, which improved to 0.39 ± 0.53 LogMAR at the last follow-up. Presenting VA 0.77 (±0.40) improved to 0.20 (±0.17) p value (0.180) in intravitreal bevacizumab only group, 1.29 (±0.35) to 0.75 (±0.15) p value 0.66 in Nd: Yag laser group, 2.67 (±0.58) to 0.46 (±0.28) p value 0.019 in PPV group and did not improve in combined anti-VEGF and focal laser group due to dense hard exudates at the fovea in one and persistent cystoid macular edema in another case. CONCLUSION: With regard to its presentation, which can vary tremendously, there are no approved guidelines for its treatment. The present study reinforces the need for a treatment guideline development. Customization of treatment should be considered depending on the clinical presentation of each case.

Treatment Strategies in Acute Post-operative Endophthalmitis after Cataract Surgery at a Tertiary Eye Hospital in Nepal: A 5-year Retrospective Review.

INTRODUCTION: Despite best possible preventive measures, acute postoperative endophthalmitis (POE) is still the most devastating, sight-threatening complication after intraocular surgery and the most feared complication by treating surgeons. MATERIALS AND METHODS: It is a retrospective study of 22 eyes diagnosed as acute POE following cataract surgery in the last 5 years (2015-2019), aimed to evaluate the treatment strategies used in its management. Main outcome measures evaluated were rates of repeat intravitreal injection, adjunctive therapeutic regimens, pars plana vitrectomy (PPV) and visual outcome. RESULTS: Twenty one eyes (95.45%) received repeated intravitreal injection. Adjuvant intravitreal steroid was used in 12 eyes (54.54%), oral steroid in 16 eyes (72.72%) and oral antibiotic in 8 eyes (36.36%). PPV was done in 8 eyes (34.78%) and all 8 eyes that underwent PPV had a vision of Hand Movement (HM) close to face. 7 eyes (87.5%) had early PPV within 1 week of diagnosis. The median best corrected visual acuity (BCVA) improved from 1.00 logMar to 0.8 logMar following treatment at 3 months follow up (p= 0.117). CONCLUSION: Repeat intravitreal injections were commonly employed. Early PPV was performed more commonly regardless of the visual acuity at the time of diagnosis of acute POE.

Use of Intravitreal Bevacizumab Injection among Patients Undergoing Surgical Retinal Interventions at Tertiary Eye Hospital: A Descriptive Cross-sectional Study.

INTRODUCTION: Intravitreal Bevacizumab injection has now become a routine procedure for retina specialists throughout the world. Easy availability of this monoclonal antibody molecule even in Nepal has brought a revolution in the management of various retinal diseases. This study aims to find out the prevalence of the use of intravitreal Bevacizumab for retinal diseases at the tertiary eye hospital. METHODS: This descriptive cross-sectional study was carried out in the retina department at a tertiary care hospital from January 2017 to December 2019 after obtaining ethical clearance from Nepal Health Research Council (Ref: 125/2020P). The sample size was calculated and the study enrolled all patients who received intravitreal Bevacizumab for retinal diseases using convenience sampling technique. Data were analyzed using Statistical Package for Social Science Version 21. Point estimate at 95% Confidence Interval was calculated, along with frequency and percentage for binary data. RESULTS: Out of 959 total surgical retinal interventions done 296 (30.86%) at 95% Confidence Interval (27.93-33.78) patients received intravitreal Bevacizumab. Out of total intravitreal Bevacizumab injections, 143 (36.7%) injections were given to retinal vein occlusions patients, 127 (32.6%) injections were given to diabetic retinopathy patients and 66 (17%) injections was given to age-related macular degeneration patients. Males 176 (59.5%) outnumbered the females 120 (40.5%) in receiving intravitreal Bevacizumab. Mean baseline Logarithm of the Minimal Angle of Resolution visual acuity, 1.1, improved to, 0.75, after 3 months of intravitreal Bevacizumab. CONCLUSIONS: Intravitreal Bevacizumab was one of the commonest retinal interventions used. Retinal vein occlusion, diabetic retinopathy, and age-related macular degeneration were the commonest retinal diseases needing intravitreal Bevacizumab.

Baseline Predictive Factors of Visual Outcome and Persistence of Subretinal Fluid Based on Morphologic Changes in Spectral Domain Optical Coherence Tomography in Patients with Idiopathic Central Serous Chorioretinopathy.

BACKGROUND: To determine the influence of spectral domain optical coherence tomography (OCT) changes on visual outcome and persistence of subretinal fluid (SRF) in patients with idiopathic central serous chorioretinopathy (CSCR). MATERIALS AND METHODS: In a retrospective study done in 48 eyes of 45 patients diagnosed as CSCR, all eyes were subjected to fundus photography, spectral domain OCT, and fluorescein angiography (FA) in selected cases. RESULTS: Retinal pigment epithelium detachment was present in 22.91% of the cases at presentation. The logMar best corrected visual acuity improved from 0.46±0.29 at presentation to 0.18±0.22 at 3 months (P-value<0.01). The mean foveal thickness was 486.81±146.06 µm at presentation and 259±94.5 µm at 3 months (P-value<0.01) (paired T-test). OCT factors that were associated with poor visual outcome (BCVA>0.3 logMar) were disruption in the inner segment (IS)/outer segment (OS) junction or external limiting membrane (ELM) line and hyper-reflective dots in the intra/subretinal layer (P-value<0.05) (Fischer's Exact test). Out of the total 48 eyes, 26 had persistent SRF at 3 months. The presence of discontinuation in IS/OS junction and hyper-reflective dots in the intra/subretinal layer were the only two OCT factors that were associated with the persistence of SRF (P-value<0.01) (Pearson's Chi-square test). CONCLUSION: Visual outcome and persistence of subretinal fluid at 3 months can be predicted on the basis of early morphologic changes in OCT. This will aid in counseling patients regarding its course and may guide us in its management.

Nd:YAG laser hyaloidotomy in the management of Premacular Subhyaloid Hemorrhage.

BACKGROUND: Premacular subhyaloid hemorrhage results in a sudden profound loss of vision. Among the modalities for its treatment, Nd:YAG laser hyaloidotomy is a non invasive method enabling rapid drainage of the obstructed macular area and improved vision within days. This study was aimed to evaluate the efficacy, visual outcome and complications following Nd:YAG laser hyaloidotomy for premacular subhyaloid hemorrhage. METHODS: Patients with premacular subhyaloid hemorrhage of more than 3 disc diameters (DD) of various etiologies, attending Tilganga Institute of Ophthalmology, Nepal from August, 2014 to February, 2015, were included. A comprehensive ocular evaluation was conducted and fundus photographs were taken to measure the size of the subhyaloid hemorrhage. Optical coherence tomography (OCT) were performed before and after treatment and on subsequent follow up visits. Fundus fluorescence angiography was done whenever necessary. Q switched Nd:YAG laser was applied to create an opening in the posterior hyaloids membrane for draining subhyaloid hemorrhage. The main outcome measures were success rate in performing hyaloidotomy, drainage of subhyaloid blood into vitreous cavity and its resorption, improvement in visual acuity, need for further intervention and postoperative complications. RESULTS: There were 21 eyes of 19 patients, 17(89.48%) male and 2(10.52%) female. In 3, premacular subhyaloid hemorrhage was bilateral. Mean age was 41.68 ± 17.08 years and a mean duration of symptoms 15.04 days. Mean pretreatment hemorrhage was 6.27DD. Nd:YAG laser hyaloidotomy was successful in 19 eyes(86.4%). In 2 patients, one each with Eales' disease and retinal vein occlusion the procedure was unsuccessful, necessitating pars plana vitrectomy, while in a case with proliferative diabetic retinopathy (PDR), vitrectomy was resorted for non clearing vitreous hemorrhage. Vision improved from a median of 3/60 pre-operatively to 6/6, at 6 months follow up. At 3 months, 2 patients with Eales' disease, one developed tractional detachment at macula while the other, an epiretinal membrane. No other complications were noted at 6 months. CONCLUSION: Nd:YAG laser hyaloidotomy is an inexpensive, effective and a safe outpatient procedure for premacular subhyaloid hemorrhage, producing rapid drainage with restoration of visual function avoiding more invasive procedures and enabling early assessment of the underlying retina. The final visual prognosis however, rests on the underlying cause of the subhyaloid hemorrhage and any accompanying retinal changes.

Ocular manifestations of childhood acute leukemia in a tertiiary level eye centre of Kathmandu, Nepal.

INTRODUCTION: In some instances, the understanding of the ocular manifestations in childhood leukemia is not only important to establish the diagnosis but also reflects the disease state and prognosis. OBJECTIVE: To study the ocular manifestations of childhood acute leukemia among the children attending a tertiary-level hospital in Nepal. MATERIALS AND METHODS: A cross-sectional, descriptive study was undertaken at the B.P. Koirala Lions Centre for Ophthalmic Studies (BPKLCOS) and Kanti Children Hospital (KCH), Kathmandu, over a period of one-and-a-half years. Children diagnosed with acute childhood leukemia referred to the BPKLCOS from the Oncology Unit of the KCH and the Emergency Department of the Tribhuvan University Teaching Hospital (TUTH) were included in the study, using a non-probability sampling method. RESULTS: Of the 71 cases with childhood acute leukemia, 55 (77.5%; 95% CI = 66% - 85%) had acute lymphoblastic leukemia(ALL)whereas the other 16 (23%) had acute myeloblastic leukemia (AML). Ocular involvement were seen in 33 cases (46%) and were more frequent in cases of AML as compared to those with ALL (p=0.001, OR 5.0, 95% CI= 1.4 - 17.5). Direct ocular involvement and secondary ocular involvement were observed in 12 (16.9%) and 29 (40.8%) subjects, respectively. Ocular symptoms were present in only 11 cases (15.49%). Cerebro-spinal fluid (CSF) and bone marrow examination in cases with direct ocular involvement showed 10 cases (83.3%) positive for blast cells in the CSF and 6 cases (50%) positive for blast cells in bone marrow. The most common secondary manifestation was retinal haemorrhage, seen in 23 cases (32.4%). CONCLUSION: In view of the high asymptomatic ocular involvement and the significant visual morbidity, a routine ophthalmic examination is recommended as an integral part of the medical examination in all cases of childhood acute leukemia.

Is laser photocoagulation still effective in diabetic macular edema? Assessment with optical coherence tomography in Nepal.

AIM: To find out the outcome of laser photocoagulation in clinically significant macular edema (CSME) by optical coherence tomography (OCT) METHODS: It was a prospective, non-controlled, case series study enrolling 81 eyes of 64 patients with CSME between August 2008 and January 2010. All patients received modified grid photocoagulation with frequency doubled Nd: YAG laser. Each patient was evaluated in terms of best-corrected visual acuity (BCVA) and regression or progression of maculopathy after laser therapy at 1, 3 and 6 months. Spearman's correlation test was used to show the correlation between BCVA and total macular volume (TMV). Analysis of variance (ANOVA) was used to compare among groups and independent t-test was used to compare in each group. RESULTS: There is high correlation between BCVA and TMV (P≤0.001). BCVA improved in 50.6 %, remained static in 39.5% and deteriorated in 9.9% patients after 6 month of treatment. The Baseline TMV (mean and SD) were 9.26±1.83, 10.4±2.38), 11.5±3.05), 8.89±0.75 and 9.47±1.98mm(3) for different OCT patterns, ST (sponge like thickening), CMO (cystoid macular edema), SFD (subfoveal detachment), VMIA (Vitreo macular interface abnormality) and average TMV respectively (P=0.04). After 6 months of laser treatment, the mean TMV decreased from 9.47±1.98mm(3) to 8.77±1.31mm(3) (P=0.01). In ST there was significant decrease in TMV, P=0.01, Further within these groups at 6 months, they were significantly different, P=0.01. CONCLUSION: OCT showed the different morphological variant of CSME while the response of treatment is different. TMV decreased the most and hence showed the improvement in vision after 6 months of laser treatment. In the era of Anti vascular endothelial growth factors (VEGFs), efficacy of laser seems to be in shadow but it is still first line of treatment in developing nation like Nepal where antiVEGFs may not be easily available and affordable.

Inferior conjunctival autografting for pterygium surgery: an alternative way of preserving the glaucoma filtration site in far western Nepal.

BACKGROUND: The purpose of this study was to evaluate the outcome of pterygium excision with inferior conjunctival autografting for primary pterygium. METHODS: This was a prospective noncomparative interventional case series study enrolling 50 eyes of 50 patients with primary pterygium between November 1, 2010 and October 30, 2011. All patients underwent the standard surgical technique for pterygium excision with inferior conjunctival autografting. The sampling method was purposive. The study variables were complications of surgery and recurrence rates during a follow-up period of 6 months. RESULTS: The mean age of the patients was 43 ± 7.97 (range 26-64) years. Grade 1 pterygium comprised 64% while grade 2 pterygium comprised 36% of cases. The mean size of pterygium was 3.2 ± 0.60 mm. Minor complications did occur, but only 4% required resuturing. Recurrence occurred in two eyes (4%) which we detected 3 months after surgery in both cases. We observed conjunctival scarring at the donor site in four eyes (8%); however, there was no symblepharon formation or restriction of upgaze. CONCLUSION: Inferior conjunctival autografting is an effective technique with a low recurrence rate. This is a useful technique when it is not possible or desirable to use the superior conjunctiva as a donor source. It is an especially good option for preserving the glaucoma filtration site for the future.

MIM regulates vertebrate neural tube closure.

Neural tube closure is a critical morphogenetic event that is regulated by dynamic changes in cell shape and behavior. Although previous studies have uncovered a central role for the non-canonical Wnt signaling pathway in neural tube closure, the underlying mechanism remains poorly resolved. Here, we show that the missing in metastasis (MIM; Mtss1) protein, previously identified as a Hedgehog response gene and actin and membrane remodeling protein, specifically binds to Daam1 and couples non-canonical Wnt signaling to neural tube closure. MIM binds to a conserved domain within Daam1, and this interaction is positively regulated by Wnt stimulation. Spatial expression of MIM is enriched in the anterior neural plate and neural folds, and depletion of MIM specifically inhibits anterior neural fold closure without affecting convergent extension movements or mesoderm cell fate specification. Particularly, we find that MIM is required for neural fold elevation and apical constriction along with cell polarization and elongation in both the superficial and deep layers of the anterior neural plate. The function of MIM during neural tube closure requires both its membrane-remodeling domain and its actin-binding domain. Finally, we show that the effect of MIM on neural tube closure is not due to modulation of Hedgehog signaling in the Xenopus embryo. Together, our studies define a morphogenetic pathway involving Daam1 and MIM that transduces non-canonical Wnt signaling for the cytoskeletal changes and membrane dynamics required for vertebrate neural tube closure.

TRPM7 regulates gastrulation during vertebrate embryogenesis.

During gastrulation, cells in the dorsal marginal zone polarize, elongate, align and intercalate to establish the physical body axis of the developing embryo. Here we demonstrate that the bifunctional channel-kinase TRPM7 is specifically required for vertebrate gastrulation. TRPM7 is temporally expressed maternally and throughout development, and is spatially enriched in tissues undergoing convergent extension during gastrulation. Functional studies reveal that TRPM7's ion channel, but not its kinase domain, specifically affects cell polarity and convergent extension movements during gastrulation, independent of mesodermal specification. During gastrulation, the non-canonical Wnt pathway via Dishevelled (Dvl) orchestrates the activities of the GTPases Rho and Rac to control convergent extension movements. We find that TRPM7 functions synergistically with non-canonical Wnt signaling to regulate Rac activity. The phenotype caused by depletion of the Ca(2+)- and Mg(2+)-permeant TRPM7 is suppressed by expression of a dominant negative form of Rac, as well as by Mg(2+) supplementation or by expression of the Mg(2+) transporter SLC41A2. Together, these studies demonstrate an essential role for the ion channel TRPM7 and Mg(2+) in Rac-dependent polarized cell movements during vertebrate gastrulation.

Non-redundant roles for Profilin2 and Profilin1 during vertebrate gastrulation.

Gastrulation is a critical morphogenetic event during vertebrate embryogenesis, and it is comprised of directional cell movement resulting from the polarization and reorganization of the actin cytoskeleton. The non-canonical Wnt signaling pathway has emerged as a key regulator of gastrulation. However, the molecular mechanisms by which the Wnt pathway mediates changes to the cellular actin cytoskeleton remains poorly defined. We had previously identified the Formin protein Daam1 and an effector molecule XProfilin1 as links for Wnt-mediated cytoskeletal changes during gastrulation. We report here the identification of XProfilin2 as a non-redundant and distinct effector of Daam1 for gastrulation. XProfilin2 interacts with FH1 domain of Daam1 and temporally interacts with Daam1 during gastrulation. In the Xenopus embryo, XProfilin2 is temporally expressed throughout embryogenesis and it is spatially expressed in cells undergoing morphogenetic movement during gastrulation. While we have previously shown XProfilin1 regulates blastopore closure, overexpression or depletion of XProfilin2 specifically affects convergent extension movement independent of mesodermal specification. Specifically, we show that XProfilin2 modulates cell polarization and axial alignment of mesodermal cells undergoing gastrulation independent of XProfilin1. Together, our studies demonstrate that XProfilin2 and XProfilin1 are non-redundant effectors for Daam1 for non-canonical Wnt signaling and that they regulate distinct functions during vertebrate gastrulation.

Mechanism of activation of the Formin protein Daam1.

The Formin proteins are central players in mediating cytoskeletal reorganization and are epistatically positioned in a pathway downstream of Rho activation. These proteins exist in the cytoplasm in an autoinhibited state, which is mediated by intramolecular interactions between the amino-terminal GTPase binding domain (GBD) that encompasses the diaphanous inhibitory domain (DID) and the carboxyl-terminal diaphanous autoregulatory domain (DAD). It has been proposed that the binding of Rho within the GBD releases this molecule from autoinhibition by disrupting the DID/DAD interactions. Here we report that Daam1 is not significantly activated by Rho binding but rather by its interaction with Dishevelled (Dvl). Removal of the DAD domain disrupts interactions between Dvl and Daam1, and the binding of Dvl to Daam1 disrupts the interaction between the GBD and DAD that mediates Daam1 autoinhibition. Mutations within or removal of the DAD converts Daam1 into an active protein that can induce Rho activation. We further demonstrate that Dvl synergizes with Daam1 to regulate gastrulation during Xenopus embryogenesis and that expression of activated Daam1 can rescue impaired convergent extension movements resulting from deregulated noncanonical Wnt signaling. Our studies together define the importance of a carboxyl-terminal binding partner, Dvl, that leads to the activation of Daam1.

Profilin is an effector for Daam1 in non-canonical Wnt signaling and is required for vertebrate gastrulation.

Non-canonical Wnt signaling plays important roles during vertebrate embryogenesis and is required for cell motility during gastrulation. However, the molecular mechanisms of how Wnt signaling regulates modification of the actin cytoskeleton remain incompletely understood. We had previously identified the Formin homology protein Daam1 as an important link between Dishevelled and the Rho GTPase for cytoskeletal modulation. Here, we report that Profilin1 is an effector downstream of Daam1 required for cytoskeletal changes. Profilin1 interacted with the FH1 domain of Daam1 and was localized with Daam1 to actin stress fibers in response to Wnt signaling in mammalian cells. In addition, depletion of Profilin1 inhibited stress fiber formation induced by non-canonical Wnt signaling. Inhibition or depletion of Profilin1 in vivo specifically inhibited blastopore closure in Xenopus but did not affect convergent extension movements, tissue separation or neural fold closure. Our studies define a molecular pathway downstream of Daam1 that controls Wnt-mediated cytoskeletal reorganization for a specific morphogenetic process during vertebrate gastrulation.