Age-related neurodegenerative diseases are a growing burden to society, and many are sporadic, meaning that the environment, diet and lifestyle play significant roles. Cerebrospinal fluid (CSF)-mediated clearing of brain waste products via perivascular pathways, named the glymphatic system, is receiving increasing interest, as it offers unexplored perspectives on understanding neurodegenerative diseases. The glymphatic system is involved in clearance of metabolic by-products such as amyloid-β from the brain, and its function is believed to lower the risk of developing some of the most common neurodegenerative diseases. Here, we present magnetic resonance imaging (MRI) data on the heart cycle’s control of CSF flow in humans which corroborates findings from animal studies. We also review the importance of sleep, diet, vascular health for glymphatic clearance and find that these factors are also known players in brain longevity. This raised the question of the significance of CSF for clearing toxic metabolites from the brain, and in 2012, the glial-lymphatic (“glymphatic”) system, that describes a mechanism for brain clearance via a perivascular  CSF flow pathway was characterised.4 Indeed, the glymphatic system plays a role in clearance of amyloid-β,47 tau,9 and α-synuclein.10As life expectancy has increased in many countries, aging-related neurodegenerative disorders such as dementia or movement disorders are growing in prevalence. Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common neurodegenerative diseases. Currently, no cure is available although epidemiological studies suggest that the risk of developing neurodegenerative diseases can be modulated by lifestyle-related factors, suggesting that some cases could be prevented. The toxic accumulation, misfolding or mis-localisation of proteins leading to neuronal loss, i.e. proteinopathies, are key pathological features of age-related neurodegenerative diseases.11 The system was named the glia-lymphatic or “glymphatic” system upon its discovery in 2012 as astrocyte end feet are a main structural component of the fluid exchange pathway.4 CSF is predominantly produced in the choroid plexus in the 3rd and lateral ventricles, and it is circulated from the ventricles to the subarachnoid space surrounding the brain primarily by arterial pulsations.12 The subarachnoid space is continuous with the periarterial spaces of the pial vessels, from which the CSF enters the brain parenchyma, where it facilitates the clearance of solutes, although the efflux routes are less described (13 The interchange of CSF and ISF is dependent on aquaporin 4 (AQP4) water channels on astrocyte endfeet that enwrap the cerebral vasculature.6 Changes in AQP4 expression or polarisation – referring to the differential distribution of AQP4 in the endfeet versus rest of the cell – are associated with disturbances in glymphatic function.15 In line with the observation that the glymphatic system can clear amyloid-β, decreased glymphatic function caused by deletion of the Aqp4 gene in an animal model of Alzheimer’s disease leads to increased accumulation of amyloid-β2 and tau.16 Abnormalities in AQP4 polarisation are also seen in Alzheimer’s patients, which provides some evidence that glymphatic function might also play a role in Alzheimer’s disease in humans.17The glymphatic brain clearance mechanism relies on interchange of CSF and interstitial fluid (ISF) that allows waste to be transferred to the CSF and transported out of the brain.escribed .4,13 TheResearch in animals has shown that glymphatic activity can be modulated by a number of dietary, behavioural, and physiological changes which can be affected by lifestyle in humans. Here we carried out a 7 Tesla magnetic resonance (7 T MR) visualisation of the fact that the CSF flow in the aqueduct is correlated with the heart cycle in humans, and we discuss these results in the context of diet, vascular function and the glymphatic system. Additionally, we review the interplay between lifestyle and brain health.18 The CSF flow measurement was acquired in the cerebral aqueduct, with an image resolution of 0.3x0.3 mm2 and a 5 mm thick imaging slice. A high resolution is crucial to avoid underestimation of flow in the narrow aqueduct. The slice was positioned perpendicularly to the aqueduct after identifying it on a 3 D T1 weighted MR image. The 2 D PC MR acquisition was synchronised with the heart beat using ECG detection. In one subject, we additionally acquired 4 D PC flow data covering the whole aqueduct. The technique can be used to assess changes is the flow in longitudinal studies of disease or after physiological challenges.We used ultra-high field 7 T MR  with a dual-channel transmit/32-channel receive head coil  to perform 2 D PC MR velocity and flow quantification in 9 healthy volunteers, as previously described.All volunteers gave informed consent and the experiment had ethical approval and followed the ethical guidelines from the Swedish Regional Ethical Review Board (Etikprövningsmyndigheten No. 2012-428).http://segment.heiberg.se) by manually delineating the cerebral aqueduct in the cardiac phase with maximal flow and copying that delineation to all time frames. Statistical analysis was performed with Prism v8.2.1 . To study the CSF flow patterns over the heart cycle, the RR-interval, as recorded by 7 T, was normalised to 60 bpm in all subjects.CSF flow data were analysed using Segment v2.2 R7052 .25 As CSF is primarily produced in the choroid plexus of the lateral, third and fourth ventricles, the net CSF flow in the aqueduct between the third and fourth ventricle can be used as a proxy for the amount of CSF added or re-circulated to the downstream compartments (27 By using ultra-high field MRI (7 T) we obtained a higher image resolution which is particularly beneficial for accuracy when measuring flow in small regions such as the aqueduct.18 We synchronised the 2 D PC MRI with the heartbeat using ECG detection to obtain the flow curve over the cardiac cycle. CSF flow measurements were acquired at 7 T with a resolution of 0.3x0.3 mm2 and a 5 mm thick imaging slice positioned perpendicular to the aqueduct. The data show that the CSF pulsatility in the aqueduct is highly correlated with discrete phases of the cardiac cycle. Consistently for all healthy subjects, the CSF efflux to the fourth ventricle peaks during systole while reflux back into the third ventricle peaks during the late diastolic phase  is a driver of glymphatic function,31 and there is a growing body of evidence suggesting that dysregulation of CBF plays a role in both vascular and neurodegenerative dementias.39 Restriction of CBF reduces arterial wall pulsatility, thus reducing CSF-ISF exchange in the brain, suggesting a functional connection between cerebral haemodynamics and the glymphatic system.31 Furthermore, the potential for enhancement of perivascular flow by improving vascular compliance would be in line with the data showing that hypertension reduces both vascular compliance and glymphatic function.40 The pulse wave velocity (PWV) is increased in peripheral arteries as vascular compliance is reduced.41 Increased PWV can be seen in patients with mild cognitive impairment (MCI) or dementia,41 which suggests a connection between compliance and cognitive function. The link between exercise, vascular compliance and the glymphatic system is interesting, as it might clarify the link between vascular diseases and neurodegeneration. This points to a dual role for the circulatory system in terms of maintaining CNS health, as it drives perivascular fluid movement in addition to blood supply itself, in line with blood flow being a marker for a healthy brain. Taken together, the evidence presented above converge on the suggestion that modulation of CBF or cerebral vasomotion can enhance perivascular clearance and improve CSF-ISF exchange, under similar conditions that are known to enhance cognition in humans. Cerebrovascular insufficiencies as encountered during aging or vascular diseases of the brain could perhaps result in an inadequate waste clearance due to impaired vasomotion, leading or contributing to neurodegenerative diseases such as AD.Additionally, exercise exerts beneficial effects on vascular health and increases vascular compliance.42 Glymphatic function emerged as a potential link between poor sleep and neurodegenerative diseases, as glymphatic function is almost exclusively active in the sleep stage,5 assuming that reduced interstitial clearing of e.g. amyloid-β plays a role in developing PD and AD. Owing to the apparent importance of sleep on brain clearance, we here summarise what is known on the relation between sleep and brain health.Disruptions of circadian rhythm and fragmented sleep are believed to be connected to the development of PD and AD in a bidirectional manner, suggesting sleep disturbances are both a symptom and a potential contributor to these diseases.43 and some cancers.44 Sleep deprivation has been connected to cognitive dysfunction.46 Interestingly, sleep disturbances and circadian dysfunction are common in AD and PD patients,48 and even observed in mouse models of AD.49 Huntington’s disease and Lewy body dementia are strongly associated with changing sleep quality, while frontotemporal dementia is associated with sleeping problems.50Sleep is necessary for a wealth of biological functions, and short or disrupted sleep have both acute and chronic adverse effects on health. Chronic sleep deprivation, sleep disruption and even shift work are connected to an increased prevalence of pathologies, including the metabolic syndrome, cardiovascular disease51 which is one of the main regulators of the sleep/wake cycle.52 Some studies report increased hypocretin levels in AD and MCI patients53 which is linked to night time wakefulness.54 Interestingly, the hypocretin levels also correlated with increased levels of amyloid-β.54 A small clinical study suggested that autoimmunity to AQP4 may cause hypothalamic lesions and lead to disruption of hypocretin signalling in Multiple Sclerosis patients.55 Building upon the hypothesis that AQP4 expression can influence sleep, it was suggested that genetic variation in AQP4 leads to hypothalamic dysfunction, thus providing a possible connection between sleep problems and increased amyloid burden.56 Another sleep-regulating neuropeptide, melatonin, is also reduced in AD patients at night and increased during the day due to damage to the suprachiasmatic nucleus.57 In PD, it is believed that the damage to the dopaminergic neurons causes disruptions in sleep, and in later stages of PD α-synuclein aggregates in sleep-associated areas thus exacerbating sleeping disorders.58 Furthermore, neurodegeneration seems to lead to disruption of pathways needed for glymphatic function and regulation of sleep. AD patients show a higher degree of perivascular enlargement compared to healthy controls.59 Interestingly, enlargement of perivascular spaces in itself is connected to poor sleep quality.61 A population study found that enlarged perivascular spaces and sleep disruptions are linked, and it was therefore hypothesised that sleep deficiency may lead to structural changes in the perivascular spaces.61 Another study found a connection between the quantity of enlarged perivascular spaces and decreased sleep time.62 Taken together, these data suggest that disturbed sleep and circadian dysfunctions as a consequence of AD and PD are at least partly due to direct cell damage to the brain regions governing sleep and wakefulness, and sleep disturbances may further aggravate disease progression via accumulation of metabolic by-products.Post mortem tissue from AD patients with daytime sleepiness showed loss of hypocretin (orexin),63 Sleep disturbances start already in the preclinical stages, i.e. cognitively normal individuals with poor sleep quality have significantly higher amyloid levels, suggesting that sleep disturbances may be a risk factor for AD.63 In line with the hypothesis that disturbed sleep patterns lead to reduced brain clearance and proteinopathies, there is a strong inverse correlation observed between hours slept and amyloid-β levels in the cortex of humans.64 Both animal and clinical studies suggest that deep sleep in particular is vital for brain clearance, as slow (delta) waves that occur during stage three of NREM sleep are associated with high levels of glymphatic influx65 and amyloid-β clearance.66 The connection between delta waves and increased glymphatic function can also be seen in anaesthetised mice, where anaesthetics that promote delta waves more have the highest influx in the entire brain.68In fact, increased daytime napping – symptomatic of circadian dysregulation – correlates with amyloid deposits.69 In rodents, the levels of amyloid-β6971 and tau70 are relatively high during wakefulness and low during sleep. In a natural sleep cycle in humans, levels of amyloid-β40 in CSF peak during the evening and is lowest in the morning.69 The circadian fluctuations in both the glymphatic activity and levels of waste products point to the importance of the circadian rhythm as a regulator of brain clearance.72 However, since the sleep-induced fluctuations can be manipulated with sleep deprivation70 or anaesthetics,5 it appears that the state dependent changes are a more powerful regulator of glymphatic function than the circadian timepoint alone.72 This conclusion is supported by studies that use anaesthetics to activate the glymphatic system. This would suggest that not the circadian time point, but presumably the sleep or a sleep-like brain state is a main regulator of glymphatic activity.In addition to the state-dependent changes in awake versus sleep state, diurnal fluctuations in neurodegenerative-associated protein levels in the ISF have been observed already before the glymphatic system was conceptualized.74 A small clinical study in humans has shown that neurodegenerative disease patients tend to spend more time sleeping in supine position (on the back) than healthy controls, who instead prefer lateral positions (on the side).74 In rats, CSF flow is optimal when positioned in lateral and supine positions, while correspondingly reduced in the prone position.7 Head position also has an effect on amyloid-β clearance rate.7 Thus, the beneficial effect of sleep is affected by something as simple as the position of the head, further indicating a role for fluid dynamics as a player in brain clearance.Sleeping posture is a proposed risk factor for neurodegeneration.64 amyloid-β42,75 ISF tau70 and CSF tau and α-synuclein.70 Similar to the effects seen after acute sleep deprivation, chronic insomnia patients had higher CSF levels of amyloid-β42 than normally sleeping individuals.76 There is also a correlation between amyloid-β42 levels and sleep quality as well as time since onset of insomnia76 suggesting detrimental effects of insomnia display dose-dependent patterns.In healthy humans, studies have shown that one night of sleep deprivation leads to increases in amyloid-β plaques in the hippocampus,69 In AD mice, chronic sleep deprivation also resulted in an increase in insoluble amyloid-β42, both acutely but interestingly even after sleep discovery.45 Likewise, tau pathology in the parenchyma is exacerbated in a mouse model of AD following chronic sleep deprivation.78 In terms of glymphatic function, sleep deprivation induces reactive astrocytes and reduces perivascular flow and waste clearance.79 Sleep appears to influence amyloid-β accumulation via several mechanisms; amyloidogenesis appears to be increased via increased amyloid precursor protein (APP) cleavage as a result of sleep deprivation,80 while clearing via glymphatic pathways is simultaneously reduced.These findings can be recapitulated in animal models. Increase in ISF amyloid-β levels can be seen as an effect of a single night of sleep deprivation and when chronic, sleep deprivation leads to increased amyloid-β plaque burden leads in an AD mouse model.79 In humans, some AQP4 single nucleotide polymorphisms (SNPs) correlated with worsened self-reported sleep quality and an increased amyloid-β burden.56 This suggests genetic variation in AQP4 can not only impact amyloid- β accumulation, but also sleep quality.Sleep deprivation leads to changes in AQP4 expression.8183 Stress response also goes hand in hand with inflammation,81 decreased AQP4 expression and polarisation.84 Furthermore, chronic stress reduced CSF influx and increased Aβ42 build up in mice.85 Reduced brain clearance appears to be mediated via the glucocorticoid receptor (GR), as GR agonists result in reduction of glymphatic function, AQP4 expression and increased amyloid build-up, while anti-glucocorticoids rescue the phenotype and prevent glymphatic dysfunction.84 Disruption of glymphatic pathways might exacerbate the negative effect chronic stress has on the brain.85Stress caused by sleep deprivation might exacerbate the negative effects of disturbed sleep, as stress itself has been indicated as a risk factor for AD, PD and Huntington’s disease.Therefore, the glymphatic system could be of interest for future studies addressing the connection between various neurological problems that are observed as a result of sleep deprivation. The role that sleep plays that brain clearance further highlights its importance for combatting proteinopathies.87 while high-glycaemic-index and high-fat diets are associated with increased amyloid accumulation and thus higher risk of developing AD.88 The effects that dietary choices have on neurodegeneration and brain health are complex, connected to nutrition-related changes in the function of multiple organ systems, and it often takes decades for effects to manifest.Eating habits have a profound effect on health and can significantly increase or reduce the risk of developing a number of diseases. For instance, the ‘Mediterranean diet’ has been associated with reduced risk for developing AD,89 Obesity and T2D are risk factors for hypertension and other cardiovascular morbidities.91 In humans, hypertension increases risk for dementia.9294 Although the cause of hypertension varies between patients, the contributions of excess dietary sodium intake are widely recognised.95 Both T2D 96 and hypertension have been linked to decreased glymphatic function,40 thus offering one potential explanation for the relationship between T2D, hypertension and neurodegeneration. A study found that spontaneously hypertensive rats had higher CSF volume but decreased CSF influx, suggesting that perivascular CSF flow is compromised.40 The presence of tracer in the subarachnoid space surrounding the basal brain regions was the same in both hypertensive and normotensive groups, indicating comparable subarachnoid circulation, which suggests that the disruption of the CSF-ISF interchange is likely due to altered perivascular flow and clearance.40 An MRI study of patients with cerebral small vessel disease (SVD) found that decreases in cerebrovascular compliance and adaptivity lead to higher sheer forces generated by the pulse wave, while the total blood flow might actually be reduced due to the decreased compliance.97 Increases in pulsatility (difference in blood flow between systolic and diastolic phase) were also observed in SVD patients following intracerebral haemorrhage.98 This increase in vascular pulsatility led to decreased CSF pulsatility in the foramen magnum.97 Interestingly, many of these patients were also hypertensive, which may further contribute to decreased vascular compliance.97 Although not fully understood, this suggests that deviations from normal cerebrovascular function due to decreased compliance and subsequent high vascular pulsatility affect CSF circulation in both the ventricular system and the perivascular compartments. Conditions affecting vascular compliance, such as T2D or hypertension will thus likely exacerbate disturbances in CSF flow, which might lead to dysfunctional CSF-mediated brain clearance and in part explain the increased dementia risk associated with these conditions.Diet is the main cause of obesity, metabolic syndrome and type 2 diabetes mellitus (T2D), and these metabolic changes increase the risk for developing AD.99 High intake of docosahexaenoic acid (DHA), a type of omega 3 fatty acid, has been associated with lower risk of AD and PD, and interestingly post-mortem studies indicate deficiency of DHA in AD patients.100 PUFAs have recently been suggested to support glymphatic function as mice on a high PUFA diet showed increased CSF tracer influx and clearance, supporting the role of PUFAs in maintaining normal glymphatic function.102 Furthermore, transgenic mice expressing increased levels of omega 3 fatty acids in the brain showed protection against neuronal death from increased levels of amyloid beta.101 Interestingly, this protective effect was not seen in AQP4 knockout mice where the glymphatic system was inhibited.101Polyunsaturated fatty acids (PUFAs), and particularly n-3 PUFAs (omega 3 fatty acids), are essential fatty acids that are important for maintaining vascular health. Central to this function appear to be their anti-hypertensive and anti-inflammatory properties, and their role in regulating platelet aggregation and blood lipid metabolism.103 However, at least some studies suggest that low consumption of alcohol leads to a decrease of all-cause mortality compared to zero consumption.105 This positive impact of low to moderate alcohol consumption on health may seem paradoxical, especially considering the detrimental effects of heavy drinking. Even one episode of binge drinking can result in adverse changes in the brain.106 Nevertheless, some studies suggest that light drinkers have reduced incidence of PD107 and dementia or AD109 when compared to non-drinkers, although not all studies show the same association. The effects of alcohol on the glymphatic system also show a J-shaped dose-response curve, where low levels seem to induce, and high levels inhibit the activity of the glymphatic system. When alcohol use is chronic, the glymphatic impairment is not rapidly reversed.110 Alcohol is a modulator of glymphatic function, resulting in an increased and decreased glymphatic function at low and high levels, respectively.111 The enhanced CSF-mediated clearance was attributed to nitric oxide (NO)-mediated dilation of blood vessels as an effect of low doses of alcohol.113 A role for vascular smooth muscle activity has also been implied as a mediator of the effect of alcohol on solute clearance.112 On the other hand, moderate levels of alcohol treatment in mice increased AQP4 levels but decreased polarisation, while low levels showed no difference compared to the control. Interestingly, based on the levels of glial fibrillary acidic protein (GFAP), reactive gliosis increased with binge-drinking levels of alcohol.111 Increased GFAP levels have also previously been connected to glymphatic dysfunction in mice35 and reported in AD patients.114 Furthermore, an EEG based sleep study on the effects of binge drinking in college students found that alcohol changed the EEG patterns to patterns associated with disrupted sleep.115 Thus, besides the cellular changes, alcohol may modulate glymphatic function by disrupting sleeping patterns.115 Thus, high levels of alcohol consumption have disruptive effects on glymphatic function mediated by both acute and chronic effects on various players in the glymphatic pathway, including AQP4 polarisation and disruption of sleep patterns. Low levels of alcohol on the other hand might have a beneficial effect on glymphatic function.Alcohol use is a multifaceted public health issue; it increases the risk of various adverse health outcomes including several types of cancer and cardiovascular disease.116119 Similar to glymphatic animal studies, it is possible to study the distribution of contrast agent injected into the CSF in humans.120 Assessment of perivascular contrast agent enhancement makes it possible to study CSF distribution in the key anatomical compartments of glymphatic function, and support the persistence of the glymphatic system in humans.119 MRI measurements suggest that CSF contrast agent enters the brain and is subsequently cleared, and the clearance process is delayed in patients with dementia.116 Measurement of apparent diffusion coefficients using diffusion tensor imaging (DTI) can be used to study mechanisms of fluid transport in the brain, and help to establish whether fluid movement can be explained by diffusion or if bulk flow is likely to form a part of the flow.118 As clearance rates appear reduced in patients with dementia,116 measuring clearance might have predictive value in clinical settings.The glymphatic system has been indicated as a potential player that links CSF-mediated clearance to prevention of aggregated protein accumulation and thus decreased risk of neurodegenerative diseases. Many of the life style components that affect the glymphatic system also modulate the risk of neurodegenerative diseases . Dissect117 However, the invasive nature of such methods involving gadolinium-based contrast agents for experimentally assessing glymphatic function makes it largely unsuitable to directly replicate rodent studies in healthy volunteers. To avoid the use of contrast agents, macroscopic CSF flow can be measured non-invasively in the ventricular system, for example in the cerebral aqueduct27 or the foramen magnum.97 One study found CSF pulsatility to be increased in MCI and even more so in normal pressure hydrocephalus.19 However, in AD patients the pulsatility had returned to normal range. This suggests that CSF pulsatility might be useful for differential diagnosis of AD or normal pressure hydrocephalus. Furthermore, longitudinal studies may offer predictive value of MCI progression to AD.Use of contrast agents injected into the CSF is required to enhance visibility of perivascular or intraparenchymal CSF flow to measure it using MRI. Lumbar injections of gadolinium-based contrast agents to assess CSF leakage in patients is already in use.Development of new imaging techniques for studying CSF flow and homeostasis is vital for better understanding the CSF-mediated brain clearance in humans. As a step towards such an approach, we showed how ultra-high field 7 T MRI can be used to quantify flow of CSF in the cerebral aqueduct and its correlation with the heart cycle in humans, as an attempt to bridge the translational gap between animal studies and clinical observations. Further studies, particularly in higher mammals and humans, are needed to assess whether glymphatic dysfunction significantly contributes to the progression of neurodegenerative disease. Another important question is whether it is possible to prevent or alleviate neurodegenerative diseases by further enhancing brain clearance via therapies targeting glymphatic pathways.


Laparoscopic surgery has been described as a minimally invasive surgery. The purpose of this study is to clarify its minimal invasive features using a patient questionnaire on the postoperative quality of life (QOL) over various time periods following either laparoscopic hysterectomy (LH) or abdominal hysterectomy (AH) and to compare the results.This study enrolled 28 patients who underwent total hysterectomy for uterine fibroids in 2012 (14 AH cases and 24 LH cases) were enrolled in this study. The 36-Item Short Form Survey (SF-36) questionnaire was completed on postsurgical day 3; weeks 1, 2, and 4; and month 6. The results were compared between the two groups.Patients who underwent LH scored significantly higher on physical functioning on postoperative day 3 and week 2; physical role and bodily pain on day 3 and week 1; general health on postoperative day 3, weeks 1, 2, and 4, and month 6; social functioning on day 3; and emotional role on day 3 and week 1. No significant differences were found between vitality and mental health at any time point or in the categories above at any other time point.Postoperative QOL in LH cases was improved on day 3 and week 1; however, no significant differences between the LH and AH groups were found in most categories at week 4 and month 6. LH leads to superior short-term QOL early in the postoperative period relative to AH. Uterine fibroids are a typical benign gynecological disease in sexually mature women, and total hysterectomy is the treatment of choice for symptomatic patients. Although the size of uterine fibroids influences the surgical choice, laparoscopic hysterectomy (LH) is less invasive than abdominal hysterectomy (AH) and is becoming increasingly popular despite limitations in its application dependent on lesion size , 2.Laparoscopic surgery is reported to be less susceptible to surgical blood loss or wound infection and facilitates a shorter hospital stay and faster return to society compared with abdominal surgery , 2. HoweWe considered that although laparoscopy unequivocally yields smaller surface wounds, it may be more accurately classified as a minimal access—rather than minimally invasive—procedure given that the degree of intra-abdominal maneuvering is similar to that required for open surgery. Likewise, a question regarding the nature of invasiveness is raised, that is, whether it is minimally invasive in the long term as well as in the short term. Few surveys have been published to date on quality of life (QOL) and satisfaction with laparoscopic and open surgeries.To our knowledge, no studies to date have investigated the QOL of patients with benign gynecological diseases over short or long postoperative periods using a measure such as the Short Form 36-Item Health Survey (SF-36). In the present study, we compared self-reported health-related postoperative QOL between patients who underwent LH or AH for uterine fibroids. The SF-36 was used to gather self-reported data on patients’ postoperative QOL in the short and long terms to investigate whether laparoscopic surgery is truly a minimally invasive procedure that meaningfully improves QOL.This retrospective study was conducted at Kindai University Hospital and Kindai University Nara Hospital, with the approval of the Ethics Committee. Written informed consent was obtained from all participating respondents . Eligibility criteria comprised patients diagnosed with uterine fibroids and adenomyosis on preoperative magnetic resonance imaging (MRI) who underwent total hysterectomy. Exclusion criteria comprised those who had underwent LH or AH when preoperative MRI suspected malignancy or written consent could not be obtained. The type of hysterectomy was categorized as either open surgery  or LH. Kindai University Nara Hospital does not perform LH, opting for AH in all cases. On the other hand, Kindai University Hospital treats approximately all total hysterectomies for uterine fibroids and adenomyosis via LH. Therefore, all LH cases included in our study were performed at Kindai University Hospital, and all AH cases were performed at Kindai University Nara Hospital. Thirty-eight patients who underwent total hysterectomy for uterine fibroids in 2012 (14 AH and 24 LH cases) were included. Of the 14 AH patients, 12 had uterine fibroids and two had adenomyosis. Of the 24 LH patients, 19 had uterine fibroids and six had adenomyosis.Patient background  and surgical outcomes  were examined for both AH and LH.The SF-36  was administered on postoperative day 3; weeks 1, 2, and 4; and months 6 [t-test to compare the mean values between the two groups and the χ2 test to compare proportions. A P-value < 0.05 was considered statistically significant.Statistical analysis was performed using Student’s AH and LH cases are shown in Table Comparisons of the QOL between patients receiving AH or LH are shown in Fig. This is the first report to investigate postoperative QOL using the SF-36 to compare LH and AH for uterine fibroids and adenomyosis. In this context, Andersen et al.  conducteIn the field of obstetrics and gynecology, Janda et al.  reportedIn addition, the postoperative QOL may vary depending on the number of complications and surgeon experience. Agarwal et al.  suggesteOur study revealed that both LH and AH reduce QOL in the immediate postoperative period. Even in this period, however, the QOL was significantly greater in the LH group compared with the AH group. Subsequently, both groups’ scores recovered over time to the national average baseline of 50 points for most variables, and no difference was observed in the long-term QOL between the LH and AH groups in most variables at week 4 and thereafter. Here, the general health scores were significantly higher for LH through 6 months, whereas the mental health and vitality scores showed no significant differences at any time-point. These findings suggest that laparoscopic surgery may be advantageous patients for in the short term regarding physical function and pain but without particular benefits for mental health. Finally, the general health scores were significantly greater for LH at all time points, potentially suggesting that the patients perceive LH as a healthier option.One of our study’s limitations is the small sample size given the relatively brief study period  and the small proportion of patients who met the eligibility criteria and provided informed consent during this period.Additionally, the study was conducted in two different facilities with different teams which may have created a bias due to the difference in the study environment of the two groups. The retrospective nature of our study is another limitation; future large-scale prospective studies are encouraged. In addition, the SF-36 used in our study did not include items related to the duration of return to a normal sex life, a metric of importance from the perspective of QOL after hysterectomy.The results demonstrate that QOL is increased with laparoscopy in the short postoperative term, improves thereafter in both procedures, and shows no inter-group differences in the long term.


We show that, in virtual absence of proliferation, RPE cells stretch and flatten, thereby matching the retinal curvature and promoting OV folding. Localized interference with the RPE cytoskeleton disrupts tissue stretching and OV folding. Thus, extreme RPE flattening and accelerated differentiation are efficient solutions adopted by fast-developing species to enable timely optic cup formation. This mechanism differs in amniotes, in which proliferation drives RPE expansion with a much-reduced need of cell flattening.The vertebrate eye primordium consists of a pseudostratified neuroepithelium, the optic vesicle (OV), in which cells acquire neural retina or retinal pigment epithelium (RPE) fates. As these fates arise, the OV assumes a cup shape, influenced by mechanical forces generated within the neural retina. Whether the RPE passively adapts to retinal changes or actively contributes to OV morphogenesis remains unexplored. We generated a zebrafish Tg, and a thicker inner layer called the neural retina. If this process fails, the animal may be born blind or visually impaired.How this flat two-layered structure becomes round is still being investigated. In fish, studies have shown that the inner cell layer – the neural retina – generates mechanical forces that cause the developing tissue to curve inwards to form a cup-like shape. But it was unclear whether the outer layer of cells (the RPE) also contributed to this process.Moreno-Marmol et al. were able to investigate this question by genetically modifying zebrafish to make all new RPE cells fluoresce. Following the early development of the zebrafish eye under a microscope revealed that RPE cells flattened themselves into long thin structures that stretched to cover the entire neural retina. This change was made possible by the cell’s internal skeleton reorganizing. In fact, preventing this reorganization stopped the RPE cells from flattening, and precluded the optic cup from acquiring its curved shape. The results thus confirmed a direct role for the RPE in generating curvature.The entire process did not require the RPE to produce new cells, allowing the curved shape to emerge in just a few hours. This is a major advantage for fast-developing species such as zebrafish. In species whose embryos develop more slowly, such as mice and humans, the RPE instead grows by producing additional cells – a process that takes many days. The development of the eye thus shows how various species use different evolutionary approaches to achieve a common goal. The retinal pigment epithelium (RPE) is an essential component of the vertebrate eye, composed of a monolayer of pigment-enriched epithelial cells abutting the neural retina (NR) with a primary role in photoreception . Despiteojoplano medaka fish mutant – affecting a transmembrane protein localized at the basal end feet of NR cells , enables RPE progenitors to acquire an NR fate  zebrafish transgenic line with which we followed the beginning of RPE morphogenesis under both normal and interfered conditions. We show that, whereas in amniotes, including humans, the developing RPE undergo proliferation to increase its surface with a less evident cell flattening, zebrafish RPE cells rapidly cease proliferation and expand their surface by reducing their length along the apico-basal axis and extending in the medio-lateral direction with a tissue autonomous process that depends on cytoskeletal reorganization. Localized interference with either the retinal or the RPE actomyosin and microtubule cytoskeleton shows that RPE flattening generates a mechanical force that actively contributes to OV folding, complementing the force generated by the basal constriction of the NR. This mechanism represents an efficient solution to match the increased apical surface of the NR layer in a fast-developing vertebrate species such as zebrafish.Here, we report the generation of a Tg(E1-bhlhe40) gene, a basic helix-loop-helix family member, encodes a light and hypoxia-induced transcription factor  involved in cell proliferation and differentiation as well as in the control of circadian rhythms  used in this study.We used predictive enhancer and promoter epigenetic marks at different zebrafish developmental stages  to scan rs E1–4;  appearedrs E1–4; . These ers E1–4;  as potenbhlhe40:GFP) progeny confirmed that the transgenic line faithfully recapitulated the bhlhe40 mRNA expression profile detected with ISH (bhlhe40 mRNA distribution (bhlhe40:GFP) line over other presently available transgenic lines that allow visualizing the RPE  line for the identification of the very first RPE cells is supported by the onset of the reporter expression in the dorso-medial OV region, coinciding with previous fate map predictions  embryos were thereafter used to dissect the extensive changes in cell shape that are associated with the acquisition of RPE identity (2 vs. PN‾a: 43.7 ± 7.8 μm2). In contrast, the abutting apical surface of NR cells slightly shrank as compared to that of PN cells (2 vs. PN‾a: 43.7 ± 7.8 μm2) while maintaining a constant apico-basal length. The latter observation agrees with previous reports showing that the cone-like morphology of NR progenitors represents only a slight modification of the progenitor columnar shape  reporter was discretized into seven different segments that were individually analysed along the recording time  line or from the Tg(E1-bhlhe40:GFP) line injected with the pCS2:H2B-RFP mRNA (4 μm3/hr from 17 to 22 hpf and 3.6 × 104 μm3/hr from 24 to 37 hpf) as compared to that of the RPE  – despite the rather drastic morphological changes of its cells – whereas the whole OV expands at a pace ~25 times faster  embryos were bathed either in blebbistatin or its diluent (DMSO) at 17 hpf  17 hpf embryos were bathed in Ableb or in DMSO and irradiated in a small region of either the dorsal outer layer (RPE) or the inner retinal layer (retina) of the OV . Embryos were then let develop until 24 hpf. During this period, the irradiated RPE cells underwent anterior and medio-lateral spreading – likely coinciding with the reported pinwheel ‘movement’ (bhlhe40:GFP) transgene in any experimental condition, indicating that cellular tension and morphology did not affect RPE specification.Tg embryos injected with the mRNA of EB3:GFP, a protein that binds to the plus end of growing microtubules (bhlhe40:GFP) embryos in nocodazole, a drug that interferes with microtubule polymerization, or its vehicle (DMSO) at either 16 or 17 hpf (otx1 and mitf) and apico-basal polarity (zo-1 and laminin) markers , a key regulator of microtubule depolymerization (STMN1) driving the simultaneous production of GFP and STMN1 under the same regulatory sequences (rx3:GAL4) embryos. We reasoned that, although rx3 drives transgene expression in both NR and RPE progenitors, the random and sparse expression that occurs in F0 would be sufficient to separate the effect in the two tissues. RPE cells expressing STMN1 – and notably also those nearby – retained a cuboidal-like shape with an abnormally increased apico-basal axis as compared to GFP-positive cells in control UAS:GFP-injected embryos. Even though cells in the inner OV layer appeared to still undergo basal constriction  embryos from early OV (17 hpf) to late OC stages (48 hpf) showed a marked reduction of cell proliferation in the OV outer layer  = 32,023; p < 0.001). During this period, the apico-basal axis of individual RPE cells flattened reaching a length of 3 μm at 22–23 hpf. Thus, acquisition of RPE identity, cell shape changes, and OV folding are associated with a progressive reduction of cell proliferation in the OV outer layer.5-Bromo-2′-deoxyuridine (BrdU) incorporation in Tg13 . A fullyCollectively, these data indicate that, in the absence of sufficient time for cell proliferation, flattening is an efficient solution adopted by zebrafish RPE cells to enlarge the whole tissue to the extent needed for OV folding. In other vertebrates, in which slower development allows for more rounds of cell division, the RPE grows in a conventional proliferation-based mode that correlates with a less evident flattening of RPE cells .bhlhe40:GFP), in which GFP expression appears in the domain fated to originate the RPE. Following the cells arising from this domain, we show that RPE surface expansion is an active and tissue autonomous process required for OV folding. This expansion largely occurs by extreme cell flattening with little contribution of cell proliferation, a mechanism that sets zebrafish RPE morphogenesis apart from that of other analysed vertebrate species, in which proliferation accounts for RPE growth.The cup shape of the vertebrate eye is thought to optimize vision . This shbhlhe40 expression coincides spatially and temporally with that of zebrafish RPE specification. Thus, the Tg(E1-bhlhe40:GFP) line serves as an early tissue-specific marker that even precedes the appearance of previously accepted Otx or Mitf tissue specifiers, as confirmed in a parallel transcriptomic analysis . One possible reason for the absence of an evident RPE phenotype is functional redundancy with other untested members of the large family of the BHLH transcription factors or that the gene has only later functions as reported  and regulators of epidermal specification  were maintained under standard conditions at 28°C on 14/10 hr light/dark cycles. AB/Tübingen strain was used to generate the transgenic lines and as control wild type. Embryos and larvae were kept in E3 medium  supplemented with Methylene Blue (Sigma) at 28°C and staged according to somite number and morphology (bhlhe40:GFP) and Tg  line. Wild-type medaka fish (Oryzias latipes) of the cab strain were maintained at 28°C on a 14/10 hr light/dark cycle. Embryos were staged as described (Adult zebrafish (rphology . The Tg(UAS:RFP)  lines weescribed . Fertilihttp://hdbr.org). Sections corresponded to samples CS13, -14, -15, and -16. CS staging allowed to determine the age of embryo as days post ovulation based on morphological landmarks (Paraffin sections of human embryonic eye primordia were provided by the Joint MRC/Wellcome Trust (grant# MR/R006237/1) Human Developmental Biology Resource  with aP-97 Flaming/Brown Micropipette Puller (Sutter Instrument Company). A total of 30 pg for DNA and between 50 and 100 pg for mRNA in 1 nl volume were injected in the embryos in the cell or the yolk, respectively. Drug treatments were performed on manually dechorionated embryos at the desired developmental stage in E3 medium. The following compounds were used: blebbistatin ; paranitroblebbistatin , Ableb , and nocodazole .The pCS2:Kaede, pCS2:EB3-GFP, and pCS2:H2B-RFP constructs were linearized and transcribed using the mMessage mMachine SP6 transcription kit (Invitrogen), following manufacturer’s instructions. After transcription mRNAs were purified using the NucleoSpin RNA Clean-up kit (Machery Nagel).otx1 (previously known as otx1b) and mitfa probes were gifts from Prof. Steve Wilson . The bhlhe40 probe was generated by PCR from 24 hpf cDNA with specific primers g for 30 min, washed with ethanol 70%, and re-suspended in 15 µl of RNAse-free water and 15 µl Ultra-Pure Formamide (Panreac). ISH were performed as described (In situ hybridization (ISH) escribed .bhlhe40:GFP) zebra- and wild-type medaka fish groups of 15 embryos of stages comprised between 16 ss and 48 hpf were dechorionated and placed in BrdU solution (5 mg/ml in E3 medium) for 30 min on ice and then washed with fresh E3 medium. Embryos were let recover at 28°C for 10 min before fixation in paraformaldehyde (PFA) 4% overnight at 4°C. For analysis in chick, BrdU (50 mg/egg) was added to each embryo 30 min before fixation. For analysis in mouse, pregnant dams were injected intraperitoneally with BrdU (50 μg/g), sacrificed 1 hr later and fixed. Chick and mouse embryos were immersion fixed in 4% PFA in 0.1 M phosphate buffer, pH 7 at 4°C for 4 hr and then washed in PBS and cryoprotected in 15% and 30% saccharose in 0.1 M phosphate buffer . All embryos were cryo-sectioned and the sections hydrated with PBS 1X during 5 min and incubated in HCl during 40 min at 37°C. After HCl treatment, sections were rinsed with PBS 1X 10 times, and then processed for immunofluorescence as described below. The percentage of RPE proliferating progenitors was determined as the proportion of BrdU-positive cells over the total number of GFP (for E1-bhlhe40:GFP) or Otx2/Hoechst  positive cells in the RPE layer in each section. A minimum of three embryos and sections per embryo were counted (both eyes).BrdU (Roche) was re-suspended in DMSO (Sigma) to generate stocks of 50 mg/ml that were kept at –20°C. For Tg(E1-Zebrafish embryos at the corresponding stage for each experiment were fixed with 4% (wt/vol) PFA (Merck) in 0.1 M phosphate buffer overnight at 4°C. Whole-mount immunofluorescence was performed as described . AlternaWild-type embryos were injected with Kaede mRNA. Embryos at 15 hpf with homogeneous green fluorescence were selected, mounted, and visualized under the Nikon AR1+ Confocal Microscope using a 20×/0.75 Plan-Apochromat objective. A region of interest (ROI) was drawn in the outer layer, corresponding to the putative position of the RPE progenitors, at a specific z-position and irradiated with the 405 nm laser at 21% of power for 10 loops to switch Kaede emission from green to red fluorescence. Due to confocality, photoconversion occasionally extended further than the selected plane, so that the tissues present above or below (i.e. ectoderm) also underwent photoconversion. After photoconversion embryos were let develop up to approximately 30 hpf stage, fixed and analysed by confocal microscopy for red fluorescence distribution.2 inside the ROI).Ableb  was photR + High Definition Resonant Scanning Confocal Microscope connected to an Inverted Eclipse Ti-E Microscope  or with a Zeiss LSM710 Confocal Laser Scanning Microscope connected to a Vertical AxioImager M2 Microscope .Embryos were mounted with the appropriate orientation in 1.5% low melting point agarose (Conda) diluted in E3 medium (for in vivo recording) or PBS (for fixed samples). Images were acquired either with a Nikon A13D videos i.e.  were genbhlhe40:GFP; rx3:GAL4;UAS:RFP) or Tg(E1-bhlhe40:GFP) and H2B-RFP-injected embryos (3) were calculated as the number of voxels with a value higher than 0. RPE surface (μm2) was calculated applying a second-order linear adjustment on the plane YZ corresponding to the plane of the OV/OC hinges with the fit function available in Matlab . RPE thickness (μm) was determined as the result of volume (μm3)/surface (μm2). Unfortunately, semi-manual RPE image extraction was not perfect, when GFP signal associated to CMZ development arises. To account for this problem, the GFP signal for each frame was divided into seven equivalent blocks using the x, y coordinates from the z-projection of each frame. In this case, RPE volume and surface were calculated independently in each one of the regions up to 20 hpf, when the most anterior block (now corresponding to the arising CMZ) was discarded from the analysis. For the subsequent frames the two anterior most blocks were discarded (3) was determined using the red fluorescence from the Tg embryos at 17–22hpf. H2B expression was used to determine the volume of the OC  using the XYZ coordinates of the processed images or Fiji . This an embryos , from wh embryos . For alliscarded . The totAll statistical analysis was performed with IBM SPSS Statistics version 20.0. The method used is indicated in each case together with the sample size. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The authors describe a novel tool [Tg(E1-bhlhe40:GFP)] that can be used to monitor the earliest phases of zebrafish retinal pigment epithelium (RPE) development. The results demonstrate the RPE cell flattening in zebrafish occurs independent of the basal constriction / apical expansion of neural retina progenitor cells and that this process is affected by perturbation of actomyosin activity or microtubule organization. This study provides a deeper understanding of RPE and optic cup morphogenesis.Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by Marianne Bronner as the Senior Editor and Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Stretching of the retinal pigment epithelium contributes to zebrafish optic cup morphogenesis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.Summary:Reviewer #1:Moreno-Marmol and colleagues describe the general morphogenic rearrangements of zebrafish RPE cells during optic cup formation, providing novel insights to the autonomous nature of RPE cell flattening from the changes occurring in the neural retinal progenitors. They postulate that in fast developing species, RPE progenitor cells stretch to cover the optic vesicle and aid in its development whereas in slower developing species, RPE progenitors undergo more extensive and prolonged proliferation to provide the cell coverage to envelop the optic cup. Both reviewers see considerable merit in the work and find the data interesting and thought-provoking. However, they also suggest some important and feasible modifications. These are described in more detail in the full reviews which are appended below.In their study, Moreno-Marmol and colleagues describe the general morphogenic rearrangements of zebrafish RPE cells during optic cup formation. Novel insights to the autonomous nature of RPE cell flattening from the changes occurring in the neural retinal progenitors is presented. Through comparative analysis of RPE progenitor cell proliferation, the Authors put forward and provide data that supports the hypothesis that in fast developing species, RPE progenitor cells stretch to cover the optic vesicle and aid in its development. In slower developing species, RPE progenitors undergo more extensive and prolonged proliferation to provide the cell coverage needed to envelop the optic cup.Overall the paper is well written and addresses questions that will garner the attention of those interested in ocular development. The authors describe a novel tool [Tg(E1-bhlhe40:GFP)] that can be used to monitor the earliest phases of zebrafish RPE development. There is lost opportunity however, in that neither Gal4 nor Cre / CreERT2 lines were not also developed. This would have been useful in better parsing the interactions between RPE and NR progenitors. Still, experiments described in their paper convincingly demonstrate the RPE cell flattening in zebrafish occurs independent of the basal constriction / apical expansion of the NR progenitors cells. They also convincingly show that this process is affected when either the actomyosin activity or microtubule organization is perturbed. These are outstanding starting observations to provide a deeper understanding of RPE and optic cup morphogenesis, but given the limited scope of the analysis, this work is better suited for a more specialized journal. Providing more in depth analysis of E1-bhlhe40 activation and conservation across species, or more detailed mechanisms into the cytoskeletal regulation and reorganization required for RPE cell flattening is warranted .Substantive Concerns:1. In Figure 3B, it was unclear (to me) the utility of the H2B construct, and how the H2B volume was calculated 2. For the experiment described in Figure 4, inactivation of myosinII (ABleb irradiation) appears to have a stronger effect on RPE in the ventral part of the eye. Given the spot irradiation occurred at the region of the eye-bud specified to give rise to RPE , could 'pinwheeling' migration of the RPE progenitors contribute the phenotype? Inactiviation of actomyosin function at several stages might be informative.Reviewer #2:3. With regard to use of the bidirectional UAS constructs that facilitated expression of STMN1 or CCND1, how faithful is the concordance of GFP and STMN1 or CCND1 protein? Similarly, is it possible that low expressing cells (faint or undetectable GFP), still produce enough STMN1 or CCND1 protein to affect microtubule network / proliferation, even if stoichiometrically produced with GFP? This is particularly important given the use of a the rx3 promoter which expresses in both RPE and NR progenitors. Finally, given the migration of RPE cells, those that are isolated from non-expressing NR may have shortly before been quite proximal to NR that did express STMN1. A more elegant approach to distinguish the autonomous from non-autonomous effects would be to use promoters more specific to each cell type (such as the E1-bhlhe40 element for RPE).In the present work, Moreno-Mármol and colleagues investigate the morphological changes of RPE cells during optic cup formation in zebrafish. Using a novel transgenic line that allows them to follow the process in 4D and from earlier stages than previously available lines, they show that RPE cells in zebrafish stretch during optic vesicle folding. Following a number of elegant approaches, they show an autonomous process happening in the RPE that accompanies the NR folding, and speculate that RPE flattening is an evolutionary adaptation due to the fast dynamics of the process. Finally they show comparative data using other vertebrates that develop slower – in what is perhaps the highest point of the manuscript – revealing a different strategy that involves a sustained proliferation of RPE cells; the additional number of cells, therefore, allows for the RPE covering the folding NR without the need of such a massive stretching.I have some points that I would like the authors to address.#1 It is clear from their results that flattening can occur in RPE cells regardless of OC folding . But is it a driving force for the OC folding? I was left with the impression that the authors are ambiguous about it. Their interpretation from figure 5F is that retina can still fold in the absence of RPE flattening . In 5J, however, they conclude that flattening is indeed affecting NR invagination. A unifying quantification of the NR folding  would help assessing the role of RPE flattening as a driving force for OC folding in zebrafish. In general, I would like to see a clearer statement on the co-occurence of both processes in the RPE and NR. Are both of them tissue-autonomous? Are they independent of each other? Or is there some degree of inter-dependance, and at what level does it happen?#2 Section "RPE flattening is a cell autonomous process required for proper OV folding". The authors show that RPE cell flattening is a tissue autonomous process, not a cell autonomous process. The experiments illustrated in Figure 4 report an all-or-non response in the RPE. Form the experiments shown on lines 275-276 (UAS:stmn1), it seems that the flattening effect is non cell autonomous. In fact, the authors use during the discussion "tissue autonomous", which is more accurate. The authors should either demonstrate cell autonomy by generating a mosaic RPE in the experiments of figure 4, or change their statement to tissue autonomy. If they go for tissue autonomy (as their experiments demonstrate), please discuss about the non-cell autonomy of the flattening response.#3 The experiments using UAS:stmn1 shown that, although the expression is clonal and targets only a few RPE cells, the non-flattening effects involved pretty much the entire RPE . Is the RPE responding as one unit? Along the manuscript there is no case in which the RPE responds in a mosaic manner. Why is that? The authors leave this point unexplored. If RPE cells are coordinated in their response, how does it happen? Any hint in this regard will help framing the results in a better context.#4 The data obtained by using UAS:ccnd1 contrasts with the quality of the other results shown along the manuscript. It is not clear to me whether the forced expression of ccnd1 is indeed promoting proliferation in this system , nor whether the reported differences (52.5% vs 47.5%) are significant or not. They seem too small differences to be responsible of the reported phenotype. Given the FO mosaic approach implemented, the authors could rapidly assess more candidates to drive sustained proliferation in RPE cells. Being the last part of the manuscript about other species that resolve the same problem in a different manner – i.e., adding more cells rather than stretching the existing ones – I find this to be a critical point that needs to be shown clearly.eLife. Your revised article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by Marianne Bronner as the Senior Editor and Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for resubmitting your article "Stretching of the retinal pigment epithelium contributes to zebrafish optic cup morphogenesis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.While the reviewers are largely satisfied, one remaining revision is (please see complete review below).Essential revisions:1. Reviewer 3 raises and important point concerning over-expression of ccnd1 in RPE cells – the point that sets the basis for the "proliferation-OR-stretching". This was weak in the previous version and has not improved during the revision and would require the generation of a new transgenic line which may already be in place.Reviewer #1:2. Lines 320 to 330 are problematic, since they show only a weak trend .Overall the paper is well written and addresses questions that will garner the attention of those interested in ocular development and epithelial morphogensis. The authors describe a novel tool [Tg(E1-bhlhe40:GFP)] that can be used to monitor the earliest phases of zebrafish RPE development. Ideally, some experiments would have been better performed using promoters that drive gene expression specifically in RPE and NR progenitors, as opposed to the rx3 promoter which expresses in both cell types. Still, experiments described in their paper convincingly demonstrate the RPE cell flattening in zebrafish occurs independent of the basal constriction / apical expansion of the NR progenitors cells. They also convincingly show that this process is affected when either the actomyosin activity or microtubule organization is perturbed. These are outstanding starting observations to provide a deeper understanding of RPE and optic cup morphogenesis. This study sets the stage for more in depth analysis of E1-bhlhe40 activation and conservation across species, as well as a more detailed mechanisms into the cytoskeletal regulation and reorganization required for RPE cell flattening.Reviewer #3:The Authors have adequately addressed my concerns and the paper reads very nicely. Ideally, the cell type specific expression experiments would be included in this version, but experimental challenges and time to generate the new lines are appreciated. I highly encourage the Authors to follow through with updates on the planned experiments and the results as a "Research Advance" to be associated with this paper.In the revised version of their manuscript, Moreno-Mármol and colleagues have addressed most of the main concerns I had raised. The authors have improved their previous versions by emphasising the main concept of the work and providing a better explanation for their hypothesis. Overall, the work now describes the inter-relation between the NR and the RPE during retinogenesis, highlighting that morphological changes occurring in the zebrafish RPE are critical for the retina morphogenesis. The authors report that species in which the RPE stretching does nor occur show an increase RP proliferation, suggesting two alternative ways to coordinate the grow of RPE and NR. The observation stays descriptive, since the attempts from the authors to switch stretching-to-proliferation modes in zebrafish were not successful – see recommendation for authors below.One of my main points was to provide more accuracy in the "cell-autonomous" effect of RPE cells. The authors have now changed their statement to a "tissue-autonomous" effects, which in my view fits better the data they have acquired. The idea of the RPE behaving in a syncytial-like manner is very interesting indeed, and the discussion has been enriched by this addition.The one point I do not find satisfactory addressed is the one concerning over-expression of ccnd1 in RPE cells – the point that sets the basis for the "proliferation-OR-stretching". This is a point that was raised as well by reviewer 1; it was weak in the previous version and has not improved during the revision. I consider the experiment to be critical, in light of the exciting results the authors provide for different species. The generation of a new transgenic line is an enterprise that can take longer that the time allowed for a revision, but I understand that the authors already have the tools to go for it? ("… a new construct we have developed"). Furthermore, the mosaic approach followed by the authors in their initial submission allows the analysis in the injected generation and therefore within days, so I would have expected this point to be tackled experimentally. In their current form, the lines 320 to 330 are the most problematic of the entire manuscript, since they show only a weak trend . Presenting additional data in a subsequent "Research Advance" seems adequate for other experiments required by reviewer 1 and myself, but I feel this point is central to the story and I can not picture major problems in performing transient experiments to strengthen the proposed statement – keeping the proliferative state impairs RPE flattening. Is it realistic that the authors perform a transient over-expression of ccnd1 using an RPE specific promoter? – a construct they already have. If the authors are experiencing restrictions to the lab due to the pandemic and it is not possible to perform additional experiments, then I would suggest removing the lines 320 to 330. The manuscript would miss an interesting, important aspect but will gain in accuracy.I would strongly encourage the authors to tackle this point in the present version of the manuscript, though. If so, I would ask the authors to consider reporting this dataset at the end of the manuscript – after the report on the difference species in Figure 7. In this manner, they could show: (a) first, the inverse correlation between flattening and proliferation across vertebrates, (b) then prove that in one model species the switch can be induced experimentally. Reviewer #1:In their study, Moreno-Marmol and colleagues describe the general morphogenic rearrangements of zebrafish RPE cells during optic cup formation. Novel insights to the autonomous nature of RPE cell flattening from the changes occurring in the neural retinal progenitors is presented. Through comparative analysis of RPE progenitor cell proliferation, the Authors put forward and provide data that supports the hypothesis that in fast developing species, RPE progenitor cells stretch to cover the optic vesicle and aid in its development. In slower developing species, RPE progenitors undergo more extensive and prolonged proliferation to provide the cell coverage needed to envelop the optic cup.Overall the paper is well written and addresses questions that will garner the attention of those interested in ocular development. The authors describe a novel tool [Tg(E1-bhlhe40:GFP)] that can be used to monitor the earliest phases of zebrafish RPE development. There is lost opportunity however, in that neither Gal4 nor Cre / CreERT2 lines were not also developed. This would have been useful in better parsing the interactions between RPE and NR progenitors. Still, experiments described in their paper convincingly demonstrate the RPE cell flattening in zebrafish occurs independent of the basal constriction / apical expansion of the NR progenitors cells. They also convincingly show that this process is affected when either the actomyosin activity or microtubule organization is perturbed. These are outstanding starting observations to provide a deeper understanding of RPE and optic cup morphogenesis, but given the limited scope of the analysis, this work is better suited for a more specialized journal. Providing more in depth analysis of E1-bhlhe40 activation and conservation across species, or more detailed mechanisms into the cytoskeletal regulation and reorganization required for RPE cell flattening is warranted .bhlhe40 enhancer would have been a great addition to our toolkit and in fact we did attempt to generate such a line. Unfortunately, the result was not satisfactory, as the line did not show the expected specificity, in retrospect, likely due to a problem of insulators. This is why we resorted to the use of the rx3:Gal4-line. To overcome such a problem, we have now taken a different approach and modified the original plasmid that we used to generate the Tg(E1-bhlhe40:GFP) line so that now we can use it to overexpress any gene of interest in the RPE. We have already verified that the new construct faithfully reproduces the pattern of the Tg(E1-bhlhe40:GFP), and we have further used it to generate new transgenic lines. We will exploit this construct to force STMN1 and ccnd1 expression in the RPE as detailed below. However, these experiments will require several months and therefore, following the indication of the Editor, we will report them as a “Research Advances” later on, linking the obtained results to this manuscript.We agree with the reviewer that generating a Gal4/Cre line based on the Substantive Concerns:1. In Figure 3B, it was unclear (to me) the utility of the H2B construct, and how the H2B volume was calculated rx3 line used at early stages is less visible later on. We therefore used H2B to counterstain the optic primordium. Although a cytoplasmic marker would have avoided the process of artificially “filling the gaps”, H2B provided a bright and reliable signal. The methods used to fill the gaps and infer the volume of the whole eye primordium are explained in the “morphometric analysis” section of the methods section together with the procedures used to quantify the entire eye volume. The approach that we took has been carefully designed and does not affect the rigor of the analysis. We have now re-written and clarified this description (page 21-22).The RFP reporter expression of the 2. For the experiment described in Figure 4, inactivation of myosinII (ABleb irradiation) appears to have a stronger effect on RPE in the ventral part of the eye. Given the spot irradiation occurred at the region of the eye-bud specified to give rise to RPE , could 'pinwheeling' migration of the RPE progenitors contribute the phenotype? Inactiviation of actomyosin function at several stages might be informative.Indeed, in the ABleb experiment the most severe alterations were observed in the RPE cells closer to the ventral region. As we now indicate in the text (page 8) this is likely because cells flatten and spread in the medio-lateral direction, exactly following the “pinwheel movement” described by Kwan et al., (2012 Development) -to which we believe the reviewer refers to. We cannot exclude that cell movements do exist and contribute to the localization of the cells ventrally. However, RPE cells spreading may give the impression of “migration”.bhlhe40:GFP to identify RPE cells, whereas at later stages the optic vesicle is already folded and the RPE already flattened, making it very challenging to irradiate exclusively RPE cells and in sufficient number. Besides, the question we addressed in our manuscript focuses on the contribution of RPE morphogenesis to OV folding. We expect that once the optic cup is formed, interference with RPE cytoskeleton will have only a minor impact on the overall shape of the eye.The reviewer also suggests interfering with actomyosin at several stages. There is a technical and a conceptual reason why we have not done so. We cannot assess the effect at earlier stages as we rely on the onset of expression of E1-3. With regard to use of the bidirectional UAS constructs that facilitated expression of STMN1 or CCND1, how faithful is the concordance of GFP and STMN1 or CCND1 protein?STMN1 or ccnd1 expression. To clarify this point we now mention how the vector works in the corresponding section in methods (page 18) and in results (page 8). We also added the reference of Paquet et al., not included in our previous version.The bidirectional UAS construct we have used was originally described in Paquet et al., 2009, J Clin Inv. 119, 1382–1395 and then further used in Distel et al., 2010, J Cell Biol. 191, 875–90 and Distel et al., 2011, Commun Integr Biol. 4, 336-9. In the original publication, Paquet et al., show that the bidirectional UAS efficiently express both of the tested proteins at similar levels, although they did not perform an explicit stoichiometric analysis. This vector has been successfully used in several additional publications (see https://zfin.org/ZDB-TGCONSTRCT-111028-1 for a list). We are thus confident that there is concordance between GFP and either Similarly, is it possible that low expressing cells (faint or undetectable GFP), still produce enough STMN1 or CCND1 protein to affect microtubule network / proliferation, even if stoichiometrically produced with GFP? This is particularly important given the use of a the rx3 promoter which expresses in both RPE and NR progenitors. Finally, given the migration of RPE cells, those that are isolated from non-expressing NR may have shortly before been quite proximal to NR that did express STMN1. A more elegant approach to distinguish the autonomous from non-autonomous effects would be to use promoters more specific to each cell type (such as the E1-bhlhe40 element for RPE).bhlhe40 plasmid. These experiments however will require some time and we have therefore decided to follow the editor’s recommendation and incorporate the new data to this study in the form of an associated Research Advance report.We fully agree. As mentioned in answer to point 1, this was our original, although so far failed, plan. As also mentioned, we will perform cell specific studies using a modified ZED-E1-Reviewer #2:In the present work, Moreno-Mármol and colleagues investigate the morphological changes of RPE cells during optic cup formation in zebrafish. Using a novel transgenic line that allows them to follow the process in 4D and from earlier stages than previously available lines, they show that RPE cells in zebrafish stretch during optic vesicle folding. Following a number of elegant approaches, they show an autonomous process happening in the RPE that accompanies the NR folding, and speculate that RPE flattening is an evolutionary adaptation due to the fast dynamics of the process. Finally they show comparative data using other vertebrates that develop slower – in what is perhaps the highest point of the manuscript – revealing a different strategy that involves a sustained proliferation of RPE cells; the additional number of cells, therefore, allows for the RPE covering the folding NR without the need of such a massive stretching.I have some points that I would like the authors to address.#1 It is clear from their results that flattening can occur in RPE cells regardless of OC folding . But is it a driving force for the OC folding? I was left with the impression that the authors are ambiguous about it. Their interpretation from figure 5F is that retina can still fold in the absence of RPE flattening . In 5J, however, they conclude that flattening is indeed affecting NR invagination. A unifying quantification of the NR folding  would help assessing the role of RPE flattening as a driving force for OC folding in zebrafish. In general, I would like to see a clearer statement on the co-occurence of both processes in the RPE and NR. Are both of them tissue-autonomous? Are they independent of each other? Or is there some degree of inter-dependance, and at what level does it happen?Indeed, we propose that the RPE is a driving force for OV folding. As represented in the model illustrated in Figure 8, the mechanical force generated by RPE stretching synergizes with those generated by rim involution and neural retina basal constriction to the overall OV folding. We apologize if this message was not explicit enough, although we believe that the heading in page 7 indeed stated this message that now reads “RPE flattening is a tissue autonomous process required for proper OV folding” (we changed cell for tissue). We have also edited a few additional sentences in the abstract, end of the introduction, results and discussion to make this message more explicit than what it was before.#2 Section "RPE flattening is a cell autonomous process required for proper OV folding". The authors show that RPE cell flattening is a tissue autonomous process, not a cell autonomous process. The experiments illustrated in Figure 4 report an all-or-non response in the RPE. Form the experiments shown on lines 275-276 (UAS:stmn1), it seems that the flattening effect is non cell autonomous. In fact, the authors use during the discussion "tissue autonomous", which is more accurate. The authors should either demonstrate cell autonomy by generating a mosaic RPE in the experiments of figure 4, or change their statement to tissue autonomy. If they go for tissue autonomy (as their experiments demonstrate), please discuss about the non-cell autonomy of the flattening response.STMN1 or ccnd1 in RPE cells. We apologize if this was not clearly explained in the text. As addressed in the point below, there is evidence in the literature indicating that RPE cells are interconnected and behave as a community. Following the reviewer’s advice, we have now changed the terminology throughout the text from cell-autonomous to tissue-autonomous.We appreciate the reviewer remark. We believe that overall our studies call for “tissue autonomy” rather than individual cell autonomy. The activation of ABleb occurs in a limited number of cells but a larger number of cells seem to remain cuboidal in shape. A similar “community effect” is observed upon mosaic Gal4-mediated expression of #3 The experiments using UAS:stmn1 shown that, although the expression is clonal and targets only a few RPE cells, the non-flattening effects involved pretty much the entire RPE . Is the RPE responding as one unit? Along the manuscript there is no case in which the RPE responds in a mosaic manner. Why is that? The authors leave this point unexplored. If RPE cells are coordinated in their response, how does it happen? Any hint in this regard will help framing the results in a better context.++ waves in the brain . In unpublished results, we have observed that Gap-43 is one of the earliest markers of RPE cells in different species. This expression is maintained at adult stages when gap junctions connecting RPE cells have been described . Likely these connections are responsible of the described chemically coupling observed in RPE cells and the propagation of Ca++ waves, as observed in astrocytes . In addition, in a parallel study , we have shown that st18 RPE cells already express many desmosomal proteins, indicating that the cells establish tight connections very soon. These connections promote cohesion that in other contexts, as for example zebrafish epiboly , are known to promote coordinated cell spreading. Such a coordinated behavior seems to occur also in the RPE. Finally, it might be worth mentioning that the existence of “a community effect” during embryonic development has been described decades ago for different tissues, including the eye . We have included some of these ideas in the discussion to provide an answer to the reviewer questions (page 14).The reviewer points to a very interesting aspect of RPE behavior that has caught our attention and indeed needs further investigation. However, we have not done so in this manuscript as we felt and still feel that asking whether the RPE behaves in “syncytial-like” manner was somewhat distracting from the main question of the present manuscript: does RPE stretching contribute to OV folding? Although at present we can just offer a speculation, we believe that the RPE indeed acts in a “syncytial-like” manner. This kind of behavior is for example observed among astrocytes, which, through a Gap-43 mediated connections, can propagate Ca#4 The data obtained by using UAS:ccnd1 contrasts with the quality of the other results shown along the manuscript. It is not clear to me whether the forced expression of ccnd1 is indeed promoting proliferation in this system , nor whether the reported differences (52.5% vs 47.5%) are significant or not. They seem too small differences to be responsible of the reported phenotype. Given the FO mosaic approach implemented, the authors could rapidly assess more candidates to drive sustained proliferation in RPE cells. Being the last part of the manuscript about other species that resolve the same problem in a different manner – i.e., adding more cells rather than stretching the existing ones – I find this to be a critical point that needs to be shown clearly.ccnd1 with a new construct we have developed. With this approach we will also use different genes that may force RPE proliferation. However, as we mentioned the results of these experiments will be reported later as a Research advance. Nevertheless, the use of the UAS:ccnd1 construct shows that its forced expression prevents cell flattening and interferes bhlhe40 expression, although the data related to forced proliferation are weak with no statistical significance (now indicated in the text), likely due to the limited number of embryos we could analyze.We agree with the reviewer. As mentioned in our answer to reviewer 1 (point 1) we will repeat this experiment using RPE specific overexpression of Essential revisions:1. Reviewer 3 raises and important point concerning over-expression of ccnd1 in RPE cells – the point that sets the basis for the "proliferation-OR-stretching". This was weak in the previous version and has not improved during the revision and would require the generation of a new transgenic line which may already be in place.We do agree that this point is weak. As we fully explain below in our answer to reviewer 3, our attempt to generate a new transgenic line has failed. The reasons are unclear and we can only put forward a few hypotheses as we explain below.2. Lines 320 to 330 are problematic, since they show only a weak trend .UAS:ccdn1 construct.We agree with this appreciation and given that we have been unable to force proliferation in the prospective RPE (see answer to reviewer 3 for more information), we have removed these lines from the text, together with the corresponding panels and legends in Figure 6. We have also deleted the methods related to the generation of the In addition to the changes mentioned above we have revised the text at our best to catch all typos, as indicated by reviewer 3, and improve the readability of a few sentences. We have also expanded our description of the work reported in ref 22 as it supports well the notion that proliferation has a minimal contribution to RPE expansion in zebrafish . We apologize for not having includedthis information in the first version, as the data escaped our attention.Reviewer #1:Overall the paper is well written and addresses questions that will garner the attention of those interested in ocular development and epithelial morphogensis. The authors describe a novel tool [Tg(E1-bhlhe40:GFP)] that can be used to monitor the earliest phases of zebrafish RPE development. Ideally, some experiments would have been better performed using promoters that drive gene expression specifically in RPE and NR progenitors, as opposed to the rx3 promoter which expresses in both cell types. Still, experiments described in their paper convincingly demonstrate the RPE cell flattening in zebrafish occurs independent of the basal constriction / apical expansion of the NR progenitors cells. They also convincingly show that this process is affected when either the actomyosin activity or microtubule organization is perturbed. These are outstanding starting observations to provide a deeper understanding of RPE and optic cup morphogenesis. This study sets the stage for more in depth analysis of E1-bhlhe40 activation and conservation across species, as well as a more detailed mechanisms into the cytoskeletal regulation and reorganization required for RPE cell flattening.The Authors have adequately addressed my concerns and the paper reads very nicely. Ideally, the cell type specific expression experiments would be included in this version, but experimental challenges and time to generate the new lines are appreciated. I highly encourage the Authors to follow through with updates on the planned experiments and the results as a "Research Advance" to be associated with this paper.We thank the reviewer for the comments and her/his appreciation of the experimental challenges associated with the generation of new zebrafish line. As we explain below in our answer to reviewer 3, so far, we have been unable to generate new lines limiting the expression of specific genes to the RPE. In particular, we failed to force proliferation in the RPE, likely because this tissue differentiates “en bloc”, as we suggest in our manuscript .Reviewer #3:In the revised version of their manuscript, Moreno-Mármol and colleagues have addressed most of the main concerns I had raised. The authors have improved their previous versions by emphasising the main concept of the work and providing a better explanation for their hypothesis. Overall, the work now describes the inter-relation between the NR and the RPE during retinogenesis, highlighting that morphological changes occurring in the zebrafish RPE are critical for the retina morphogenesis. The authors report that species in which the RPE stretching does nor occur show an increase RP proliferation, suggesting two alternative ways to coordinate the grow of RPE and NR. The observation stays descriptive, since the attempts from the authors to switch stretching-to-proliferation modes in zebrafish were not successful – see recommendation for authors below.One of my main points was to provide more accuracy in the "cell-autonomous" effect of RPE cells. The authors have now changed their statement to a "tissue-autonomous" effects, which in my view fits better the data they have acquired. The idea of the RPE behaving in a syncytial-like manner is very interesting indeed, and the discussion has been enriched by this addition.The one point I do not find satisfactory addressed is the one concerning over-expression of ccnd1 in RPE cells – the point that sets the basis for the "proliferation-OR-stretching". This is a point that was raised as well by reviewer 1; it was weak in the previous version and has not improved during the revision. I consider the experiment to be critical, in light of the exciting results the authors provide for different species. The generation of a new transgenic line is an enterprise that can take longer that the time allowed for a revision, but I understand that the authors already have the tools to go for it? ("… a new construct we have developed"). Furthermore, the mosaic approach followed by the authors in their initial submission allows the analysis in the injected generation and therefore within days, so I would have expected this point to be tackled experimentally. In their current form, the lines 320 to 330 are the most problematic of the entire manuscript, since they show only a weak trend . Presenting additional data in a subsequent "Research Advance" seems adequate for other experiments required by reviewer 1 and myself, but I feel this point is central to the story and I can not picture major problems in performing transient experiments to strengthen the proposed statement – keeping the proliferative state impairs RPE flattening. Is it realistic that the authors perform a transient over-expression of ccnd1 using an RPE specific promoter? – a construct they already have. If the authors are experiencing restrictions to the lab due to the pandemic and it is not possible to perform additional experiments, then I would suggest removing the lines 320 to 330. The manuscript would miss an interesting, important aspect but will gain in accuracy.I would strongly encourage the authors to tackle this point in the present version of the manuscript, though. If so, I would ask the authors to consider reporting this dataset at the end of the manuscript – after the report on the difference species in Figure 7. In this manner, they could show: (a) first, the inverse correlation between flattening and proliferation across vertebrates, (b) then prove that in one model species the switch can be induced experimentally.E1-bhlhe40:GFP construct contained in the ZED vector, replacing the GFP sequence for a multicloning site that allows for insertion of any gene of interest. To test that this modification did not interfere with tissue specificity, we re-inserted GFP and generated F0 embryos, in which the fluorescent signal was detected only in RPE cells. We then generated and injected an E1-bhlhe40:ccnd1-HA construct and determined the expression of cyclin D1 with antibodies against the HA tag. When the construct was injected in the embryos, we could detect only very few and sparse HA-positive cells in the RPE of the folding OV. These cells incorporated also EdU, although this was not always the case. More importantly, the large majority of HA+/EdU+ cells we detected were isolated and only very rarely we have observed small clusters of HA positive cells  line, which needed several months. We have crossed already a large number of grown up fishes with no success.We appreciate the reviewer’s concern and agree with her/him that the results we reported of forced cell proliferation in the zebrafish RPE were rather weak. We also felt that forcing proliferation in the zebrafish RPE would have allowed to “switch the zebrafish RPE” to “an amniote RPE”. We also could “not picture major problems in performing transient experiments to strengthen the proposed statement”, as the reviewer indicates. We proved ourselves wrong and perhaps for reasons that, eventually, should have been evident. To manipulate gene expression specifically in the forming RPE, we modified the original pression , suggestccdn1) is insufficient to force cell proliferation in the RPE. The best explanation is likely because the RPE differentiates “en bloc”, explaining why, at difference of the amniote RPE, the zebrafish one has not been reported to acquire a neural retina fate, as we discuss in our manuscript. Further explanations may relate to the lack of the enhancer activity as we have explained above, or because forced proliferation induces rapid cell death preventing the expansion of cell proliferation. Some apoptotic cells were for example observed when we used the UAS:ccd1 in the Rx3Gal4 line, perhaps explaining, in retrospective, the weak results.Although we cannot exclude that a different experimental design may provide the expected results, we have concluded that expression of a single proliferative gene  were seeking for. We have thus taken the reviewer’ suggestion of removing lines 320 to 330 from the manuscript (the description of forced expression of cyclin D1). Although frustrating, this seems the easiest option. To reinforce the message that proliferation has a limited contribution to RPE enlargement, we have emphasized an experiment performed by the lab of S. McFarlane (ref 22) in which they show that blocking cell proliferation has no effect of the enlargement of the RPE.