Transition Food, Healthy Food and Traditional Food. The course of development in dietary patterns from 9–36 mo indicated tracking for a relatively large group of participants in the three patterns. Transition Food and Healthy Food were associated with some of the investigated outcomes. Children with lower adherence to the Transition Food pattern than average at 18 and 36 mo irrespectively of intake at 9 mo had higher BMI z-scores at 36 mo. Similar trend was identified for higher fat mass indices. Children with lower adherence to the Healthy Food pattern than average at all three ages compared to children with higher adherence to the Healthy Food pattern at the first two registrations, 9 and 18 mo had higher total cholesterol and LDL. Hence, this could represent undesirable development of dietary patterns in toddlers. In conclusion, development of dietary patterns can be exploratory characterised by PCA and related to potential cardiovascular risk markers in toddlers even within a relatively homogeneous population with a high socioeconomic status. The tracking of dietary patterns from 9 mo of age indicates a need for early and sustained promotion of healthy diets.Little is known about the development of dietary patterns during toddlerhood and the relation to growth and health. The study objective was to characterise the development of dietary patterns from 9–36 mo of age and investigate the association to body size, body composition and metabolic risk markers at 36 mo. Food records were filled out at 9, 18 and 36 mo of age (n = 229). Dietary patterns were identified by principal component analysis (PCA). Three dietary patterns were identified:  This is the most extensive dietary change made throughout life, and furthermore this is the period of the most rapid growth. The first years of life are a window of opportunity because early nutrition and growth seem to have long-lasting consequences for the risk of obesity and cardiovascular diseases The consequences of breastfeeding versus formula-feeding is widely investigated Firm characterisations of the development of dietary patterns during the first years of life as well as knowledge about the impact on key anthropometric and metabolic outcomes are important to support health guidance. The aim of this study was to characterise the development in dietary patterns from 9 to 36 mo of age and to explore the association between the development in dietary patterns and body size, body composition and metabolic risk markers at 36 mo.2.2.1.Data were from a Danish longitudinal observational cohort study  which monitored children at 9 (+/-14 days), 18 (+/-30days) and 36 (+/-90 days) mo of age from year 2007 to 2010 and was previously described by Madsen and colleagues 2.2.www.Foodcomp.dk, visited May 2011) as described previously The diet of each child was recorded by parents after an oral and written introduction for seven consecutive days at 9, 18 and 36 mo of age using a seven day food record method. This method has been shown to be valuable to estimate dietary intake in this age groups when comparing with double-labelled water Possible over- and under-reporters of energy intake were identified based on Goldberg cut-off The food group variables for the PCA model were the same for all three ages selected based on nutritional knowledge trying to cover most aspects of the official recommendations, nutrition evidence and typical toddler diet in Denmark. In total the PCA was based on 25 food groups which are named with a short, compressed description, such as ‘FatsAnimal’ and ‘SugaryDrink’. The mean intake (g/day) of all food groups, except for breast milk, was divided by total body weight  for each participant at each age to make the contribution of each food to the whole diet comparable across different ages and requirements. Intake of breast milk was estimated as number of breastfeeding sessions per day according to interview with parents  and recoded as the mean of each interval. These categories (feedings/day) were used in the PCA while an estimate of 99 g/feeding 2.3.2) was calculated from weight and height. Child height and BMI were converted to z-scores using the WHO growth standard as reference and the software program WHO Anthro 2005 Weight was measured without clothes to the nearest 0.1 kg using a digital scale . Height at 36 mo was measured by a stationary digital height measurer , which made readings to the nearest 0.01 cm. Height was performed in triplicates, and the average was used in analysis. BMI , high density lipoprotein (HDL), insulin-like growth factor 1 (IGF-I) and insulin-like growth factor binding protein 3 (IGFBP3) were measured in a blood sample  taken at the 36 mo visit after a two hour fast. Mean fasting time (±SD) was 174 ± 36 minutes with the exception of 29 children, who fasted from the night before the examination. Composition of the last meal before fasting was recorded and analysed using the software programme Dankost . Insulin, IGF-I, and IGFBP3 were determined on an Immulite 1000 analyser ; glucose was determined in EDTA on HemoCue ; and total cholesterol, LDL, HDL and triacylglycerol were determined on a Pentra 400 analyser . To estimate the IGF-I/IGFBP3 molar ratio, which is an expression of the bioactive fraction of IGF-I, the following conversion equivalents were used: 1 ng/mL IGF-I = 0.133 nM IGF-I and 1 ng/mL IGFBP3 = 0.033 nM IGFBP3. More details about blood sampling have been published elsewhere 2.5.2 (kg/m2) and fat mass index (FMI) as fat mass/height2 (kg/m2).Predictive equations for fat free mass and fat mass have previously been generated using bioelectrical impedance , and height and weight from this cohort at 36 mo 2.6.2). If weight and height measures from the 36 mo visit were not available, then self-reported values from questionnaires were used.A background questionnaire at 9 mo included questions about parental age at child's birth, total household income, educational level of parents (updated at 36 mo) and parity. Total household income was divided into < 800,000 DKK  or unknown and > 800,000 DKK per year. The mean household income for a Danish family with two parents and two children is 684,000 DKK per year  2.7.2 test. Continuous variables are represented as mean ± SD (n) and compared between the groups with or without three complete diet registrations by the unpaired t-test, together for boys and girls, while the descriptive presentation is divided by gender.Categorical variables are represented as % (n) and groups with or without three complete diet registrations were compared by the chi9mo*p18mo*p36mo*n for the category Above-Above-Above (AAA) and as (1-p9mo)*(1-p18mo)*(1-p36mo)*n for the category Below-Below-Below (BBB), where p = proportion of children above mean, and n = total sample size. If the observed number is higher than the theoretically calculated number the individual adherence to a dietary pattern at different time points are not independent and can be interpreted as tracking.For the identification of dietary patterns, a PCA was carried out including food groups at 9, 18 and 36 mo in long format meaning that the data is structured so that each observation (person at specific time point) is considered as a row in the data matrix, Hence the same person appears in three rows in the matrix with the 25 food groups as variables (columns). This way, a PCA model of the data will provide an estimate of the underlying food patterns that are common over different time points. The score for each observation, on the other hand, will then indicate how much the person adhere to a particular dietary pattern at a particular time point. Thus, by assuming that a general set of latent dietary patterns exist over time, the dynamics of these dietary patterns can be monitored in the scores as a function of time. To normalise data for the PCA they were centred and scaled to unit variance  www.r-project.org). All p-values were evaluated at a 5% significance level.The relation between different categories of development in dietary patterns and body size , body composition  and metabolic risk markers  were analysed by ANCOVA adjusted for potential confounders. All analyses were adjusted for sex, parity, parental BMI, parental age, parental education level and household income. Metabolic risk markers were further adjusted for fasting time and energy content in last meal. In ANCOVA analysis, all variables of categories of development in dietary patterns  were included in the initial model, and backward stepwise elimination was used to identify which of the dietary patterns were significant. Post hoc pairwise comparisons of the eight categories within a dietary pattern were adjusted for multiple testing using the single step method 3.3.1.In the SKOT I cohort, 330 children were enrolled, including one child who was excluded before analysis because of late manifestation of a severe chronic disorder. More information about dropouts has been published previously 3.2.The mean energy intake for children with all three dietary records was 397 kJ/kg BW/day (including estimate of energy from breast milk) at 9 mo, 408 kJ/kg BW/day at 18 mo and 353 kJ/kg BW/day at 36 mo. Of these children 1%, 4% and 15% were possible under-reporters and 16%, 6% and 1% were possible over-reporters at 9, 18 and 36 mo respectively. All these children were included in the analysis, as no major changes were observed in dietary patterns by exclusion. Large variations were seen in the percentage of children eating different foods in the record periods  to the versatile intake of different more solid foods which can be eaten by the whole family at later stages. Another 8% of the variation in food groups was described in the second component, named “Healthy Food”. This dietary pattern describes a gradient of food intake ranging from potentially unhealthy foods, like SweetsCake, SugaryDrink and Chips, with lowest loadings to healthy food groups, like Fruit, Vegetable and Fish with the highest loadings. The third dietary pattern, explaining 5% of variation, was named “Traditional Food” because this pattern differentiated children with highest intake of FatsAnimal, Potato, Meat and WheatBreadNoWholegrain from children with highest intake of foods such as FruitNutSnack, RyeBread, BreakfastCerealsNoAddSugar and Fish.The general trend from 3.3.Transition Food pattern than average at all ages (category BBB) was the most common development for this pattern. It was also common to stay either below or above the average of the cohort at all three ages (category BBB or AAA) for the Healthy Food and the Traditional Food pattern. This indicates tracking of dietary pattern and was further supported by the fact that the observed number of children in category AAA and BBB was higher for all three dietary patterns than the theoretically calculated numbers expected if no tracking was present.The number of children in each category of development in dietary patterns is shown in 3.4.Transition Food pattern. The BMI z-scores at 36 mo were higher for children with lower adherence to the Transition Food pattern than average at 18 and 36 mo irrespectively of intake at 9 mo  compared to children with higher adherence to the Transition Food pattern than average at 36 mo   and FMI (p = 0.006) at 36 mo were significantly different between the eight categories of development in the AA, BBA) , however3.5.Healthy Food pattern. Significant differences in cholesterol or LDL were seen when the categories of development in the Healthy Food pattern varied for 9 and 18 mo irrespectively of the intake at 36 mo. This trend was also observed at 9 mo for height and IGFBP3 irrespectively of intake at 18 and 36 mo. This is based on the following findings. Total cholesterol and LDL were higher for children eating less healthy than average at all three ages (category BBB) compared to children eating more healthy than average at the first two registrations, 9 and 18 mo and less healthy than average at the last registration, 36 mo (category AAB) , IGFBP3 (p = 0.04), total cholesterol (p = 0.02) and LDL (p = 0.03) at 36 mo were significantly different between the eight categories of development in the ory AAB) . The heiory AAB) . IGFBP3 ory AAB) . The Hea3.6.Traditional Food pattern.None of the tested outcomes  were associated with the 4.Transition Food, Healthy Food and Traditional Food. When categorising children according to the course of development in each of these three dietary patterns over ages, the most common course among the children was to stay either above or below the average of the cohort at all three ages. BMI z-score and FMI at 36 mo were significantly different between categories of development in the Transition Food pattern, while total blood cholesterol, LDL, height z-score and IGFBP3, at 36 mo differed significantly between the categories of development in the Healthy Food pattern. Especially, groups of children with lower adherence to the Transition Food pattern or lower adherence to the Healthy Food pattern than average at two or all three ages had higher BMI, FMI and metabolic risk markers and thereby seem to represent undesirable development in dietary patterns for toddlers.Three dietary patterns were identified: 4.1.Healthy Food pattern  than expected if the individual score values were independent over time . This wa4.3.Transition Food pattern primarily showed an age gradient in the development of diet from baby to family food. However, this pattern might be more than an age gradient because it was also associated with risk markers for later obesity. The unfavourable findings of higher BMI z-scores and FMI for children with lower adherence to the Transition Food pattern than average at both 18 and 36 mo  are difficult to interpret but might be related to a less diversified dietary intake for these children. This is supported by a higher mean intake of Porridge in category ABB and BBB for the Transition Food pattern. Porridge is the only food group with low loadings at the Transition Food pattern which is eaten by most of the children at 18 and 36 months and therefor relevant here. Moreover, when looking at the cohort in general, children with a higher intake of Porridge at 18 and 36 months, compared to the cohort median, had higher mean BMI z-score at 36 months than children both with a lower intake of Porridge and the whole cohort. Another possibility is a higher degree of under-reporting for children with highest BMI and FMI. However, the estimated proportion of under-reporters is low compared to other studies The 4.4.Healthy Food pattern and the potential metabolic risk markers of later cardiovascular diseases, LDL and total cholesterol. Intake during the early period  tended to be most important for these associations because categories AAB and BBB differed at 9 and 18 mo, but not at 36 mo of age. The association between higher total cholesterol and LDL, and potentially unfavourable dietary patterns, found in SKOT I, was also seen in a Finnish study of children 3–18 years old Healthy Food pattern and might contribute to lower LDL levels, while saturated fat and sugar are highly present in foods with lowest loadings (SugaryDrink and SweetsCake) and might contribute to higher LDL levels. In addition, the Finish and Chinese studies also found associations between dietary patterns and triacylglycerol, insulin, glucose and HDL, while we did not find any such associations.To our knowledge, no studies compare dietary patterns with biomarkers in children below 5 years, as was also pointed out in a recent review 4.5.Healthy Food pattern and height in SKOT I cannot directly be confirmed by other studies examining dietary patterns, as only few studies have looked at this. SWS did not find any association between dietary patterns and height at 12 mo Healthy Food pattern milk has a positive although small loading (Healthy Food pattern also indicated importance of 9 mo patterns. IGF-I is important for linear growth during childhood Healthy Food pattern and IGFBP3, which may reflect a more general effect on the IGF system. To our knowledge, we are the first to investigate associations between IGF-1, the binding protein, and dietary patterns in childhood.The association between the  loading , which m4.6.The main strengths in our study are the longitudinal approach, not only looking at cross sectional dietary patterns, and the use of seven days' food diaries with thorough portion size estimations. Including longitudinal data in one PCA ensures that score values from different time points are compared precisely on the same dietary pattern; this approach is widely accepted in other fields using PCA It is a general limitation that PCA is not independent of subjective decisions when selecting content and number of food groups even though it is a data-driven method. This is especially worth noticing when condensing the dietary complexity extensively as we do in this study both across the whole diet and across different ages. We ended up with relatively few and broad dietary variables in the PCA to ease the visualisation and interpretation of data. Our relatively precise portion size estimation of different food groups is probably as important as differentiating between very specific food groups when looking at associations with body composition and risk markers of later diseases. Moreover, the categorisation method for development in dietary patterns used in this paper is not a validated method, which might be considered as another limitation and should be further investigated. The categorisation suggested here is one way of simplifying types of developments. Other more model based approaches including linear mixed models can be applied to address tracking 5.Development in dietary patterns can, in an informative manner, be characterised by PCA and related to toddlerhood body size, body composition and metabolic risk markers. The explorative method applied in this analysis seems to be able to handle some of the complexity in the understanding of child nutrition. The tracking of dietary patterns already from 9 mo for some children, together with changes in the course of development in dietary patterns for other children and the association with risk markers, even within a high socioeconomic and relatively homogeneous population, emphasize the need for early and sustained promotion of healthy diets in the first years of life.Tracking of dietary patterns was observed already from 9 mo for a relative large group of children, together with changes in the course of development in dietary patterns for other children.Development of certain dietary patterns from 9 to 36 mo was associated with potential risk markers of later obesity and cardiovascular diseases.These findings, even within a high socioeconomic and relatively homogeneous population, emphasise the need for early and sustained promotion of healthy diets in the first years of life.


Central sleep apnea (CSA) and periodic breathing are unusual findings described in pediatric patients with congestive heart failure. However, CSA has not been reported in children with pulmonary hypertension. We hereby report on a 10-year-old girl with idiopathic pulmonary arterial hypertension who had frequent central events in a periodic breathing fashion seen in her polysomnography, which was normalized following medical treatment leading to improvement of the pulmonary pressures. Idiopathic pulmonary arterial hypertension (IPAH) is an uncommon disorder in childhood with an incidence of 0.48 cases per million children per year and a prevalence of 2.1 cases per million, based on the United Kingdom registry [Central sleep apnea (CSA) and periodic breathing (PB) have occasionally been described in adults with pulmonary arterial hypertension (PAH) . Cheyne-We describe in this report a child with IPAH who had evidence of PB on an overnight in-lab polysomnography (PSG) which was normalized following the improvement of the pulmonary pressures.To our knowledge, this is the first report describing a complete resolution of PB in a child with IPAH after optimal pharmacological management.The patient, a 10-year-old previously healthy female, presented with a progressive decline in her exercise tolerance and episodes of shortness of breath with activity as well as rest and presyncopal events over 3-4-month period. Her symptoms were New York Heart Association (NYHA) functional class III, and she had a low exercise capacity with 380 m total distance achieved in the 6-minute walk test (6MWT). She was diagnosed with IPAH after a thorough work-up. The transthoracic echocardiogram (ECHO) showed evidence of significant pulmonary hypertension with a right ventricular systolic pressure (RVSP) estimated to be suprasystemic, dilated right ventricle (RV), septal flattening in systole, and diastole with dilated main pulmonary artery (MPA) and excluded the presence of congenital heart disease . The ele2 followed by a rapid transient increase in the respiratory rate (up to 38 breaths per minute) for about 30–90 seconds, leading to a drop in the baseline etCO2 (20–22 mmHg). Those events were terminated by central events occurring in a PB fashion . After the sleep study, she was discharged home on 3 liters/minute of oxygen via nasal prongs during sleep. Further neurological evaluation after the sleep study including a brain MRI and EEG was within normal limits and excluded any neurological reason for the frequent central events.Given the frequency of the central events as well as the severity of the SpO2) therapy, was empirically initiated as a continuous subcutaneous infusion. At 5-month follow-up, 6MWT increased to 660 m and the patient was in NYHA class I. The ECHO showed a significant reduction in the pulmonary pressures from previous and estimated at <1/2 systemic RVSP as the septum was mildly flat in systole, RVSP (TR) 50 mmHg (2 improved to 95% and there were significantly fewer desaturations throughout the night (desaturation index = 0.5/hr). The CAHI was within normal limits at 0.5/hr (The planned right heart cardiac catheterization was deemed not safe to pursue at the time of the diagnosis given the hemodynamic instability. Thus, treprostinil, a synthetic analog of prostacyclin (PGI 50 mmHg . The NT- 50 mmHg . The bast 0.5/hr .PB during sleep is characterized by periods of central pauses in respiration separated by normal breathing pattern . It coul2 below the apnea threshold giving rise to central events [The mechanism of PB and CSA in IPAH has not yet been fully understood; however, in our case it might be similar to that described in CHF. First, the cardiac output will likely be reduced leading to a prolonged circulation time, thus hypoxemia. Second, the hypoxemic milieu would stimulate the peripheral chemoreceptors leading to hyperventilation and this change in ventilation would drive the PaCOl events .Few studies have reported the prevalence of central sleep apnea and PB in adult patients with PAH , 5, 9. PThis case supports the importance of PSG in pediatric patients with IPAH, not only to exclude OSA as a potential cause but also to assess for the presence of PB. We also show that the presence of PB might be a sign of disease severity and can be a marker of response to medical treatment. Prompt diagnoses and management of the IPAH would improve SDB in this vulnerable population and might lead to a favorable prognosis. However, more studies are needed to evaluate the extent of PB in children with IPAH.


Skin fragility and recurrent wounds are hallmarks of hereditary epidermolysis bullosa (EB). Treatment options to accelerate wound healing are urgently needed. Oleogel-S10 contains a betulin-rich triterpene extract from birch bark. In this study, we tested the wound healing properties of topical Oleogel-S10 in patients with dystrophic EB. We conducted an open, blindly evaluated, controlled, prospective phase II pilot trial in patients with dystrophic EB (EudraCT number 2010-019945-24). Healing of wounds treated with and without topical Oleogel-S10 was compared. Primary efficacy variable was faster reepithelialization as determined by 2 blinded experts. The main secondary outcome variable of the study was percentage of wound epithelialization. Twelve wound pairs of 10 patients with dystrophic EB were evaluated. In 5 of 12 cases, both blinded reviewers considered epithelialization of the intervention wounds as superior. In 3 cases, only one reviewer considered Oleogel-S10 as superior and the other one as equal to control. Measurements of wound size showed a trend towards accelerated wound healing with the intervention but without reaching statistical significance. Our results indicate a potential for faster reepithelialization of wounds in patients with dystrophic EB when treated with Oleogel-S10 but larger studies are needed to confirm significance. Epidermolysis bullosa (EB) comprises a heterogeneous group of inherited skin diseases characterized by skin fragility leading to recurrent wounds , 2. CutaUp to now, no causal therapy in the sense of prevention of blister formation or improvement of skin stability is available. Treatment of EB mostly remains symptomatic  with optBetula species) is a medical plant. Its bark has been used as a natural remedy for skin diseases and wound care for centuries [ Betulae cortex (birch bark) and refined sunflower oil (SFO) without the need for further excipients [Birch .The effects of Oleogel-S10 have already been investigated in vivo on different types of wounds where this treatment seemed to promote wound healing and was very well tolerated –35. AddiThe results of these different reports were in keeping with our own experiences from using Oleogel-S10 for wound care of few individual patients suffering from different subtypes of EB outside of a study setting .In the present study, we investigated the effect of Oleogel-S10 on wound healing in patients with dystrophic EB. We hypothesized that Oleogel-S10 in combination with standard nonadhesive wound dressings would lead to faster wound healing as opposed to the use of standard wound care alone. Furthermore, we assessed the feasibility of conducting a larger trial in this patient group.This study was designed as an open-label, prospective, controlled, blindly evaluated, monocentric phase II pilot trial to compare intraindividually the efficacy and tolerance of Oleogel-S10 in combination with nonadhesive wound dressing versus nonadhesive wound dressing alone . The intThe study (EudraCT number 2010-019945-24) was conducted in compliance with IEC, informed consent regulations, and ICH and GCP guidelines and was approved by the ethics committee of the University of Freiburg.2 and 200 cm2  were recruited for this study. We differentiated wounds that had appeared less than 6 weeks earlier and those with no tendency to heal for at least 6 weeks. Children were deliberately included in this study, as they represent a large proportion of our patients and optimized wound healing would be of particular interest to them.Patients of any age beyond infancy and with an immunochemically and/or genetically proven diagnosis of hereditary EB and at least 1 wound between 10 cmPatients were excluded from this study if they had been on a systemic treatment with corticosteroids within the last 30 days or if they had taken any investigational drugs within 3 months before screening. They were also excluded if they suffered from uncontrolled diabetes mellitus, diabetic ulcers, or other diseases or conditions that could interfere with the assessment of safety, tolerance, or efficacy of Oleogel-S10 during the study.Patients or their legal representatives had to provide written informed consent before participation in the study. For this proof-of-concept study, we aimed to include 10 patients.2 to 200 cm2) eligible for study treatment, it was divided into two halves which were then allocated to either the intervention or the treatment arm. This method ensured a comparison between wound areas in a similar anatomic location and within identical wound healing phases. Alternatively, if there were two comparable lesions (≥5 cm2 each) of similar size and shape, these were selected and considered one wound pair. Wounds located at sites of major trauma  and circumferential wounds were not included.Patients of all ages presenting at the EB-Centre Freiburg for regular follow-up visits were screened according to the study protocol. If the investigator identified a skin lesion  and covered by a nonadhesive wound dressing ; the unwounded skin next to this half as well as the border between the wound halves was marked to ensure that Oleogel-S10 was always applied to the same part of the wound. In other prior studies, it had been shown that the product did not spread to other areas than the ones it was applied to. Each wound half was measured and the data were entered into a wound surface-measuring program developed at our wound care clinic  to ensure a comparable initial wound size. To serve as control, the other half of the lesion was covered with a nonadhesive wound dressing only . If the investigator had identified two comparable wounds, these were treated correspondingly. The intervention and control treatment were applied in an open-label fashion.Dressings were changed based on the study flow chart every 24 to 48 hours until the end of treatment at day 14. In case of delayed wound healing (wound present > 6 weeks at initiation of study treatment), the treatment period was prolonged until day 28.Before the start of treatment and at every dressing change, photographs of the respective wounds were taken. We used a Canon camera, EOS Digital SLR series, and our in-house wound measuring system, which allowed outer tracing of wound borders and calculation of wound surface.2) was recorded on day 0 and at each dressing change until the end of the treatment period at day 14/day 28. These data enabled us to calculate the percentage of reepithelialization. Two independent experts evaluated treatment efficacy in a blinded fashion based on comparing chronological series of cropped wound photographs taken before the start of treatment, during wound dressing changes, and at the end of treatment on day 14/day 28 and choosing the series with signs of better reepithelialization. Remote photographic analysis has been shown to be a reliable method of wound evaluation [The images were uploaded into the electronic Case Report Form (eCRF), cropped to remove pen markings at the treatment side, and coded for blinding. The (open) wound surface area  and assessment of tolerability by the investigators and patients and/or their legal representatives.p = 0.05 against the null hypothesis of no difference between treatments. For the secondary outcome variable of the amount of reepithelialization, we applied Wilcoxon signed-rank test.A sample size calculation was not performed, as the planned number of patients eligible for enrollment into this pilot study was limited. Statistical analyses on the primary outcome parameter of faster reepithelialization were performed using a two-sided exact binomial test with a significance level of  COL7A1 were determined as summarized in 10 patients were enrolled in the study. As 2 patients participated in a second cycle of treatment, 12 wound pairs were evaluated. None of the patients was lost to follow-up or dropped out : all pat2 in the intervention group (7.3–45 cm2) versus 17.7 cm2 in the control group (6.3–45 cm2). In 7 cases, large wounds were chosen and divided into two halves for comparative treatment. In the other 5 cases, 2 comparable wounds were identified, where one was treated with Oleogel-S10 and the other with nonadhesive wound dressing alone ( both areas for the duration of 3 dressing changes towards the end of the study due to a suspected superinfection.Nine of 12 wound pairs (75.0%) analyzed in the study were present in less than 6 weeks. 3 of 12 wound pairs (25%) were chronic, showing no tendency to heal for more than 6 weeks. The mean wound size was 17.5 cmng alone . All patp = 0.063, binomial test) in favor of Oleogel-S10 compared to standard-of-care treated wound halves.In 5 of 12 cases (41%), the intervention wound was rated as better epithelialized unanimously by both independent reviewers. In another 3 of 12 cases, 1 reviewer rated epithelialization of the intervention wound as superior whereas the other expert considered the wounds as healing equally well. In the remaining 4 cases, either epithelialization was considered as equal or the result was controversial . Thus, tp = 0.21 on day 7; p = 0.33 on day 14, Wilcoxon test, Measurement of wound sizes suggested a trend towards faster closure of wounds that had been treated with Oleogel-S10 both on day 7 and on day 14. On day 7 (±1), the median epithelialization of the initial wound surface was 69.7% (intervention) versus 57.4% (control) and on day 14 was 87.7% (intervention) versus 79.2% (control)  or acceptable (2.6%) in the intervention and as good in 100% of the control group. Patients reported treatment with Oleogel-S10 more frequently as efficient (75.3%) than with standard dressings alone (53.3%). The application of Oleogel-S10 was easy. The substance was tolerated very well without any complaints of stinging or burning.We did not find any relevant differences in the perception of touch sensitivity, itch, and amount of exudate between intervention and control arms.In this phase II pilot study, we investigated the effects of Oleogel-S10 on wounds of patients suffering from dystrophic EB. Both the evaluation of two blinded experts and the percentage of epithelialization measured by the investigators were consistent and suggested a trend towards faster healing of wounds when treated with Oleogel-S10 combined with nonadhesive dressings versus standard wound care with the dressings alone. The number of healed wounds  was higher and the mean time needed for wound closure was shorter in the intervention than in the control group. Likely owing to the relatively low number of 10 patients, however, these results did not reach statistical significance.The results of this pilot trial showed feasibility of study procedures and call for larger randomized controlled trials to confirm the promising effects of Oleogel-S10 application in EB wounds.Recruitment of patients was achieved within the scheduled period and patients were compliant, cooperative, and motivated to fulfil this study. Also, the duration of the study between 2 and 4 weeks was feasible for all patients. None of them dropped out or was lost to follow-up, which proved the good tolerability of the product and the great need for new therapeutic measures to treat wounds in patients with EB. However, healing of large wounds exceeded the anticipated 4 weeks, which led to a rather low number of fully epithelialized wounds after the completed study period of 2 and 4 weeks.Tolerability of the study medication was very good. We did not experience any local or generalized irritability or contact sensitization towards either of the two components of Oleogel-S10. Certainly, any medically active ingredient has the potential for allergic sensitization, especially when applied onto open wounds. Therefore, in patients with known sunflower seed allergy, Oleogel-S10 should be used with caution.According to the study protocol, we included the first 10 patients willing to participate in the study. As the majority of patients who present at our center suffer from dystrophic EB, we included only this EB subtype in our study. This allowed a more homogenous study population sharing characteristic traits of this rare disorder. However, it is possible that our results are not applicable to other EB types.Dystrophic EB causes major morbidity for the affected individuals leading to multiple secondary complications such as anemia and deficiencies of iron, zinc, vitamins, and micronutrients that impair wound healing. Therefore, the intraindividual control, comparing wounds or wound halves of the same individual, was a suitable approach, eliminating confounding factors.When planning the study, we chose to compare Oleogel-S10 to standard treatment instead of using the vehicle, respectively, placebo, as control. Apart from betulin extract, the only other ingredient of Oleogel-S10 is sunflower oil (SFO) which, other than Oleogel-S10, does not stay in the allocated wound half due to its liquid consistency. Also, in recent studies, a significantly better effect on wound healing of Oleogel-S10 as opposed to both SFO and SFO with ethyl cellulose has been demonstrated . The useOleogel-S10 is a topical gel made of pure sunflower oil and a 10% triterpene extract from birch bark. We used Oleogel-S10 on acute and chronic wounds (>6 weeks) in 10 patients with dystrophic EB.The results of the present study were promising and provided reason for further investigations of the effects of Oleogel-S10. Larger studies using Oleogel-S10 for wounds of patients with all different forms of EB are needed to clarify the effectiveness and safety in EB in general.Concurrent to additional clinical studies with Oleogel-S10, further insights into the anticarcinogenic effects of betulin on human SCC cells, ideally coming from EB patients, are needed. The anticarcinogenic effect, if proven to be real, in combination with wound healing effects would make Oleogel-S10 an ideal substance to use in EB wounds at early stages, especially in severe generalized RDEB, the subtype with the highest number of aggressive and metastasizing SCCs.The above-mentioned additional studies would help to identify the optimal indications for use of Oleogel-S10 and to clarify whether it can stand up to its promises.


HECATE (HEC) genes in regulating developmental trajectories of shoot stem cells in Arabidopsis thaliana. Our work reveals that HEC function stabilizes cell fate in distinct zones of the shoot meristem thereby controlling the spatio-temporal dynamics of stem cell differentiation. Importantly, this activity is concomitant with the local modulation of cellular responses to cytokinin and auxin, two key phytohormones regulating cell behaviour. Mechanistically, we show that HEC factors transcriptionally control and physically interact with MONOPTEROS (MP), a key regulator of auxin signalling, and modulate the autocatalytic stabilization of auxin signalling output.Plant meristems carry pools of continuously active stem cells, whose activity is controlled by developmental and environmental signals. After stem cell division, daughter cells that exit the stem cell domain acquire transit amplifying cell identity before they are incorporated into organs and differentiate. In this study, we used an integrated approach to elucidate the role of  Unlike animals, plants continuously generate new organs that make up their body. At the core of this amazing capacity lie tissues called meristems, which are found at the growing tips of all plants. Meristems contain dividing stem cells. The daughters of these stem cells pass through nearby regions called transition domains. Over time, they change – or differentiate – to go on to become part of tissues like leaves, roots, stems, shoots, flowers or fruits.Stem cell differentiation has a direct impact on a plant’s architecture and eventually its reproductive success. For crops, these factors determine yield. This means that understanding this aspect of plant development is central to basic and applied plant biology. Many factors required for shoot meristem activity have been identified, with a focus so far on the processes that control the identity of the cells produced.Arabidopsis thaliana – the shoot apical meristem. Gaillochet et al. found that HECATE genes (or HEC for short) control the timing of stem cell differentiation by regulating the balance between the activities of two plant hormones: cytokinin and auxin. These genes promote cytokinin signals at the centre of the meristem, and dampen auxin response at the edges. This acts to slow down cell differentiation in two key transition domains of the shoot meristem.Now, Gaillochet et al. have asked which genes are responsible for controlling when stem cells in meristems differentiate. The analysis focused on the meristem that makes all the above ground parts of model plant These new findings provide a molecular framework that now can be further investigated in crop plants to try to improve their yield. The findings also lay the foundation for studies of animals that may define common principles shared among stem cell systems in organisms that diverged over a billion years ago. The evolutionary success of multicellular organisms is based on the diversification of cellular identities and the division of labour among cell types. To orchestrate this diversity, complex signalling systems have evolved to guide stem cell differentiation based on hard-wired developmental programs and environmental signals , BARELY ANY MERISTEM (BAM) receptors system (ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, which encode for negative feedback factors in cytokinin signalling. In essence, WUS acts to sensitize the cellular environment to cytokinin, which in turn promotes WUS expression thus creating a positive feedback loop that helps to establish and maintain the OC , the stem cell system responsible for the generation of all above ground structures, two major fate transitions can be identified: From stem cells in the central zone (CZ) to transit amplifying cells in the peripheral zone (PZ) and further on into organ primordia, which will give rise to fully differentiated lateral structures, such as leaves or flowers (reviewed in ). At thes system . The ress system . In paras system . Transcrn the OC . Cytokinpression .HECATE1 (HEC1), which encodes a bHLH transcription factor that redundantly functions with its closest paralogs HEC2 and HEC3 in various developmental contexts  and LATERAL ORGANFUSION (LOF) genes and loss of their function results in extensive fusions of lateral organs with the active SAM . Local tive SAM .WUS/CLV3 feedback loop by negatively regulating ARR7 , we found that their SAMs were smaller and displayed a reduced cell number, in line with our previous results  domain from rat via a flexible linker. The resulting HEC1-linker-GR protein remained trapped in the cytoplasm and its activity could be induced by treatment with the steroid dexamethasone (dex). Given the limited capacity of HEC-linker-GFP for lateral cell-to-cell movement in the SAM  and CLAVATA3 (CLV3) by fluorescent markers, which faithfully label niche (pWUS:3xYFP-NLS) and stem cells (pCLV3:mCherry-NLS). Interestingly, the increase in SAM size caused by stem cell-specific activation of HEC1 was accompanied by a transient enlargement of both the niche, or organising centre (OC), and the CZ  a local increase of stem cell proliferation; (2) re-specification of early PZ cells into stem cells; and (3) a reduction in the differentiation rate between stem cells and the PZ. To discriminate between these scenarios, we developed a novel imaging tool combining a fluorescent timer protein (pKNOLLE:fast-mFluorescentTimer-NLS) with an ubiquitously and homogeneously expressed GFP (pUBQ10:3xGFP-NLS). The timer protein was exclusively expressed during cytokinesis and then slowly matured from a blue form to a form that exhibits red fluorescence. Therefore, the ratio of the fading blue to stable green signal could be used as a readout for time passed since the last division, allowing us to assess the age of cells and thus served as a proxy for division frequency. Interestingly, we observed that although the SAM expanded, cell division activity after stem cell specific induction of HEC1 was decreased at the centre of the SAM and mostly confined to the PZ as in mock-treated plants  despite the fact that HEC1 protein is largely unable to move from cell to cell, suggesting that cell proliferation in the periphery was stimulated non-cell autonomously  either by stable pCUC2:HEC1-linker-GFP or transient pCUC2:HEC1-linker-GR expression ´´´. Whil; n > 16). Hence,  results . ImportaHEC1 gain-of-function experiments in the CZ .pTCSn:erGFP cytokinin output reporter  treatment or removal of AHP6, a negative component of cytokinin signal transduction   . Treatme to 0.06). While at apices .HEC function was sufficient and required to promote cytokinin signalling, which subsequently affected SAM size. This also suggested a mechanism for the expansion of the OC and CZ after stem cell specific induction of HEC1: A non-cell autonomous stimulation of cytokinin signalling by HEC1 could trigger the activation of WUS, which in turn would promote stem cell fate.Together, these results showed that R2D2 and pDR5v2:3xYFP-NLS reporters , and the output reporters for cytokinin and auxin, pTCSn:erGFP and pDR5v2:3xYFP-NLS, respectively . However, in contrast to the SAM, auxin promotes stem cell fate and cytokinin signalling marks the entry into differentiation (reviewed in ). Since  the RAM   (p<0.05)  . We wereccessful  (Schusteccessful . Surprisccessful . Howeverccessful .TRANSPORT INHIBITOR RESPONSE 1 (TIR1)   Lau et . Interes1 (TIR1) ,  . Further1 (TIR1) . Taken tGiven the strong but slow negative effect of HEC1 on auxin transcriptional output, we hypothesised that HEC1 could interfere with the positive feedback in auxin signalling  either bp16:HEC1-linker-GR plants. In contrast to the set of early targets, we found that transcript levels of essential components for primordia initiation, such as MP itself, but also PID, a kinase required for proper polar localisation of PIN auxin transporters, were substantially reduced in this dataset  just after bolting and assessed developmental responses at the SAM . Thus,  the SAM .hec1,2,3 plants under low light, demonstrating that HEC genes are required for SAM adaptation to environmental changes  with a 33 aa Serine-Glycin linker and GR tag into pDONOR221 vector and recombined in pGreenIIs constructs  in a pGreenIIs vector containing 2.1 kb of genomic sequence upstream of the KNOLLE start codon. The CUC2 promoter used correspond to the 3.2 kb genomic sequence upstream the ATG. pCLV3:HEC1-linker-GFP, pCUC2:HEC1-linker-GFP, pCUC2:3xGFP-NLS and p35S:HEC1-linker-GFP were cloned using the Green Gate system (nstructs . pKNOLLEe system . N7-NLS e system .NotI (BiFC complementation) (For Yeast-two-Hybrid and Bi-Fluorescence complementation (BiFC) assay, HEC1, HEC2, HEC3, MP, BDL and PEP CDS were PCR amplified using Phusion Taq-polymerase  and subsequently cloned by Gibson assembly in pGILDA/pB42AD (Yeast-two-Hybrid)  or ligatntation) A detailed list of primers used in this study can be found in Supplementary file 2.Agrobacterium tumefaciens ASE strain by floral dipping according to standard protocols. All HEC1-GR inducible lines are homozygous for a single T-DNA insertion and were subsequently crossed to the corresponding reporter lines.Plants were transformed with pWUS:3xYFP-NLS_pCLV3:mCherry-NLS  with LED lights or white lights  at approximately 200 µmol m−2 s−1 until flowering transition. As soon as the first flower primordia were observed, plants were transferred to low light intensity regimes (approximately 15 µmol m−2 s−1) and kept for 14 days before imaging.For the light shift experiments, plants were grown under white light at 200 µmol mFor dexamethasone treatment  of the shoot apices, a solution of 10 µM dex, 0.01% ethanol and 0.015% Silwet were manually sprayed and applied on top of the inflorescence meristem of 25–30 DAG plants. Mock treatment (0.01% ethanol and 0.015% Silwet) was conducted similarly. For 1-Naphtalenacetic acid  treatment, 1 mM was applied according to previous studies . Shoot aCytokinin treatment was performed by treating inflorescences with a solution of 50 µM 6-Benzylaminopurine  supplemented with 0.015% Sylwet once every 5 days. Inflorescence meristems were analysed after 8 days.The cumulated silique number was measured by counting over time the total number of flower above stage 15 emerging after plant bolting. The inflorescence plastochron was then obtained by calculating the average time separating the emergence of 2 successive siliques. The number of flower primordia at a given time point were counted up to flower stage 2.For root meristem analysis, plants were grown vertically on 0.5x MS , 0.8% Phytoagar plates under long day conditions (16 hr light/8 hr dark) with white lights . 3DAG seedlings were subsequently transferred and grown vertically on media supplemented with ethanol (0.01%) and with or without dex (10 µM).The EGY48 yeast strain (-Ura) was cotransformed with the combination of pGilda and pB42AD constructs under study or with the corresponding empty vector or negative control. Positive colonies were selected on solid media  and verified by PCR. Induction for testing protein-protein association and colorimetric assays on plates were assayed as decribed in Agrobacterium strain AGL-0. Nicotiana benthamiana leaves were infected and YFP fluorescence assayed 72 hr after inoculation under a Nikon Eclipse TE2000-U epifluorescence microscope. The reciprocal assays for all the BiFC interactions shown in this study were performed obtaining the same results as presented in Agrobacterium strain expressing the viral suppressor p19 , on individually micro-dissected and pooled inflorescence meristems .For qRT-PCR, cDNA was prepared using cDNA synthesis kit after DNase treatment , qPCR was performed using SYBR Green kit .For RNA-seq and Chip-seq, libraries and next-generation sequencing were performed according to standard protocols . For RNA-seq, biological triplicates were analysed. Raw data has been deposited at NCBI GEO under the series number GSE94311.All confocal pictures were acquired using Nikon  A1 Confocal with a CFI Apo LWD 25x water immersion objective. For time series analysis, settings were established at the first day on mock samples and were kept during the course of the experiment. Shoot meristems were manually dissected by cutting of the stem, removing the flowers and were counterstained with 1 mg/ml DAPI. Root meristems were counterstained with propidium iodide  at 0,1 mg/ml. Individual populations of 5 to 15 plants were analyzed daily.pWUS:3xYFP-NLS, pCLV3:mCherry-NLS, pTCS:erGFP intensity plot profiles, we used Fiji software , P2 and P3 and crossing the meristem at its centre.pUBQ10:3xGFP-NLS) was used for imaging. To count numbers of CLV3-expressing cells, this reporter construct was combined with a respective stem cell-specific transcriptional reporter pCLV3:mCherry-NLS.For quantifying the number of cells in the SAM, pre-processing, segmentation and data analysis was done using a customized workflow for the KNIME Image Processing platform (KNIP). 3D visuFor image processing, meristem image volumes were background subtracted and segmented by a 3D seeded watershed algorithm provided by the KNIP package . Using dpUBQ10:3xmCherry-NLS channel were used to obtain mean intensities in the CLV3 channels. Cells were considered to be positive for CLV3 if their respective mean nuclear intensity was higher than 35% of the maximum mean value.To quantify numbers of CLV3-expressing stem cells nuclear segments from the pKNOLLE:fast-mFluorescentTimer-NLS) and combined with a ubiquitously expressed reporter (pUBQ10:3xGFP-NLS). Nuclear segments were obtained as described before and mean intensities for the timer (blue channel) and the GFP channel were measured for each segment. The ratio between the blue and the green mean intensities were normalized and assigned to four different classes . Cells with the highest ratio (Class4) represent young cells with a very recent cell division .To analyse cell proliferation, a cell cycle-regulated transcriptional reporter was constructed  and outer  domain of the meristem a sphere of radius r was fitted through the centroids of the L1 cells of the meristem summit  using a Matlab function. To adjust the size of the inner  domain to overall meristem size all cells with a distance to P smaller then 0.33 * r were considered to belong to this central domain (defined from the size of CLV3 domain in other plant lines), whereas cells with a distance larger then 0.33 * r were classified as peripheral cells.Root cortex cell number was quantified as described in . Cumulathttp://www.bioinformatics.babraham.ac.uk/projects/fastqc).The quality of the sequence files quality was first confirmed using FastQC .To obtain the position of G-boxes and ARE across the genome, bed files were generated using IGV . To measTo assess the percentage of HEC1 target genes carrying G-box, ARE or both and the distribution of these motifs in open chromatin regions, respective bed files were first used to generate lists of annotated genes using the homer function ‘annotate.peaks.pl’. List of genes regulated by HEC1 (p<0.05) were next used and intersected with the list of genes carrying G-box, ARE or both in open chromatin regions using Microsoft Excel . SignifiWe developed a cell population model that takes into account the simplified geometry of the SAM. The model describes the evolution in time of different SAM structures, i.e., CZ, incipient primordia and unspecified PZ cells. Cells are continuously displaced towards the periphery by the divisions of cells more centrally located, with cells located at the outer boundary of the CZ transiting to PZ, depending on local WUS concentrations. Incipient primordia are initiated near the central boundary of the PZ and primordia separate from the meristem at fixed time after their initiation. PZ cells that do not contribute to primordia contribute to longitudinal growth of the plant. The following key processes were considered: (i) proliferation of cells in CZ and PZ, (ii) fate transition from the CZ to the PZ and from the PZ to the organs, (iii) initiation of incipient primordia, (iv) separation of primordia from the meristem and (v) contribution of meristem cells to longitudinal growth.Different feedbacks were included in the model. As suggested by our in vivo data, the initiation frequency of incipient primordia and the transition from the CZ to the PZ, both depend on the CZ cell number. The model is formulated using a system of ordinary differential equations, which number may vary in time. A detailed description of the model is found in  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor.Thank you for submitting your work entitled "Control of plant cell fate transitions by transcriptional and hormonal signals" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that this version of your work will not be considered further for publication in The reviewers thought the work was well written and interesting, and the experiments performed were of high technical quality. However, all three felt that current data are not sufficient to draw the conclusions made and that alternative interpretations could lead to other explanatory models. The reviewers have each made a number of suggestions for improvement and their detailed reviews are appended below.eLife, but if the authors can provide experimental data to address these issues, the editor and reviewers strongly encourage submission of an updated manuscript. As this is likely to take more than the 2 months typical for "revision", a new submission would be required, but the editors will be made aware that the submission was encouraged.In summary, there are several concerns that would need to be addressed to make this suitable for publication in Priority concerns1) The "cell-non autonomous" claim for HEC1 needs to be backed up with experimental evidence appropriate for the tissues being monitored in this study (see reviewer 2 comment 1).2) Many claims are made using only gain of function/ectopic expression experiments that are sufficient to show that HEC family members can interfere with a normal process, but not that they normally control it. Experiments done in mutant backgrounds or by reducing HEC expression in tissue specific ways were suggested by all reviewers.3) Work presented here contradicts previous work  that concluded that HEC1 inhibits CK signaling during control of the SAM. Although the data concerning CK signaling in this new manuscript are convincing, the authors should discuss this (and other) discrepancies with previous work.4) The mechanism of "HEC factors directly modulate auxin signal transduction by physical interaction with MONOPTEROUS" is overstated and only one such possible way that HEC1 could influence auxin. Additional data supporting this mode and discussing alternatives are needed.5) More functional evidence for the connection between HEC, auxin and cytokinin is needed, as well as better modeling.Reviewer #1:Arabidopsis shoot meristem and investigate the transitions cells make from slow dividing behaviors in the CZ of meristem out through the peripheral zone (PZ) which is more of a transit amplifying cell population, and into developing organ primordia. Starting with the counterintuitive phenotype of smaller CZ but faster organ initiation in hec1/2/3 mutants, they use live imaging and modeling to describe the paths cells take along this transition, and manipulate the system to investigate the roles of well-known hormones (Auxin and cytokinin) and regulatory factors (WUS/CLV3) in the process. They also include a clever "timer" protein to see how quickly cells divide during manipulations. To my knowledge this hasn't been used in plants before, but I think it would be a valuable tool for the community.In this well written and interesting article, the authors go beyond current models of stem-cell maintenance in the Priority concernsA) "HEC function appeared not only required and sufficient to control stem cell to PZ, but also mediated PZ to organ fate transitions" This line of reasoning was based on ectopic expression of HEC in the PZ, which says that it can interfere with a normal process, but not that it controls it. I would like to see an amiRNA (or other LOF) experiment to substantiate this conclusion. Also, the expression patterns of HEC1/2/3 are never described in this manuscript. I imagine since they cite 4 prior publications, the patterns are known. This information would be useful included in this manuscript (at least in text). The same issue hold true for the root experiments. Induction of HEC1 can perturb the system, but does it actually work here?B) Reporters for CK and auxin are considered to be evidence for direct regulation of these pathways. However, I wonder whether these reporters might be very downstream. For example, could pTCS-GFP signal report that cells are in a proliferative state and have high hormone response because of that state, rather than the other way around? To really nail the idea that HEC1/2/3 work through auxin or cytokinin, wouldn't doing these overexpression experiment in a background mutant for ARRs or ARFs be more direct?C) The ChIP-seq is done on overexpressed HEC1 and the targets include typical bHLH sites. Could this just be generic binding? It would be nice to take the published bHLH ChIP-seq datasets  and see that the HEC1 sites are not always overlapping with any bHLH.hec1/2/3 might affect a cellular behavior to result in the observed SAM phenotypes. Such models could be very helpful in testing the proposed auxin signaling feedback loops, as I felt this part of the manuscript was veering into "just so stories" about how things could work, but that alternative models could also explain the data.D) I think modeling can be a useful addition to developmental studies and was used well in Reviewer #2:Arabidopsis shoot meristem. The authors perform experiments to demonstrate that HEC1 both inhibit the progression from Central Zone (CZ) identity to Peripheral Zone (PZ) identity and subsequently inhibits the transition from PZ to primordium. The experiments have been carried out using cell-type specific inducible expression of HEC1 in both the CZ domain (under CLV3 promoter) and in the Boundary Zone (BZ) (under CUC2 promoter). The authors next move to investigate the interaction with auxin and cytokinin (CK) hormone dynamics and propose a model for this. In parallel they analyse HEC1 ChIP-seq and RNA-Seq data to reveal potential target genes and identify the Auxin Response Factor MONOPTEROUS (M) as a potential HEC1 interacting factor.This manuscript by Gaillochet et al. describes the function of the HECATE1 (HEC1) gene in regulating cell fate in the There are several interesting aspects of the work, which is also technically well-performed. However, it seems to me that the current data are not sufficient to draw the conclusions that the authors do and that alternative interpretations could lead to other models to explain them.Specific issues:1) It is throughout the manuscript assumed that HEC1 can mediate its effect non-cell autonomously. This is based on the observation in Schuster et al., 2014, where the authors show that HEC1 expressed in the CZ inhibits WUS expression in the OZ via repression of CK signaling. The situation in this manuscript is different: firstly, the cells where HEC1 is supposed to function non-cell autonomously are not the OZ, but the PZ and secondly in Schuster et al. concludes that HEC1 repress CK signaling whereas here they demonstrate that HEC1 stimulate CK signaling (see further comment on this below). In order for the authors to make the non-cell autonomous claim, they will need to create an immobile version (e.g. HEC1-3xGFP) and show the effects are still there.2) The authors list three scenarios for how HEC1 can increase the cell number of the meristem and use elegant inducible systems in specific domains of the SAM. This part is very complicated and I am not convinced about the authors' interpretation. For example, they conclude that HEC1 does not increase stem cell division locally (scenario 1). In order to draw such a conclusion, they should carry out the analysis in a zone-specific manner  and within 2 days, since CLV3 is downregulated at 4 days. Moreover, to eliminate scenario 2 they argue that the constant ratio of CLV3 positive cells over total SAM cells means that PZ cells are not re-specified to stem cells. In order to make this conclusion, they need a marker for PZ and show that this would not 'turn off' upon induction of HEC1 activity. Finally, based on the elimination of scenarios 1 and 2 (which I am not convinced about), they conclude that scenario 3 must be correct without providing experimental evidence for this, but suggest it is supported by showing that the subsequent transition from PZ to primordia is regulated similarly.hec1/2/3 background to test what aspects of this triple mutant is rescued (or not) by the transgene. Without this, it is hard to conclude what the effect of HEC1 would be on these processes in a WT situation.3) Generally, the experiments using ectopic inducible expression of HEC1 under cell-type specific promoters would be much stronger if they were done in the 4) In the Abstract and in the text, it is stated that data in this manuscript "show that HEC factors directly modulate auxin signal transduction by physical interaction with MONOPTEROUS […]" This overstates what the experiments actually show and more work would be required to validate this. The authors have shown that 1) MP is one among 12 ARF genes that are expressed in the SAM and which are direct targets of HEC1, 2) that many HEC1 targets contain both G-boxes (predicted HEC1 recognition sites) and Auxin Response Elements (AREs) and 3) that HEC1 can interact with MP in yeast and in BiFC. However, in order to make a convincing case, they need to provide genetic evidence for this interaction and establish if HEC1 actually does modify MP-mediated auxin signaling. Moreover, there are 12 other relevant ARFs according to the authors (i.e. expressed in the SAM). Does HEC1 also interact with them? Modify their activity?p16:HEC1-linker-GR roots with no attempt to show how auxin signaling response is affected. At the same time, the authors linked the leaf phenotype of the ectopic HEC1 expression to changes in auxin signaling without any prove nor attempting to understand possible crosstalk with cytokinin. The following lists issues particularly regarding this part:5) The authors aim to address whether HEC genes can control cell fate transition in other developmental contexts, such as the root apical meristem (RAM) and leaves, through the regulation of auxin/cytokinin crosstalk. The conclusions that the authors draw from their experiments are again overstated, since they only analysed response to CK signaling in – It's not clear whether HEC1 governs meristem size by controlling CK signalling or whether it is the result of a feedback mechanism operated by auxin. How (when and where) is the auxin response affected after HEC1 induction?p16:mCherry-linker-GR-NLS is expressed in the division zone of the root meristem. Does p16:mCherry-linker-GR-NLS mark the stem cell niche and/or the transition zone?– It would be helpful if the author could better describe in which zones/tissues p16 is expressed in the RAM? From – The control of CK on root meristem size occurs only from the vascular tissue at the transition zone. Is the activity of HEC1 sufficient to regulate root meristem size operating from the transition zone (could use the RCH2 promoter to address this)? Does the TCS expression increase before the meristem size starts diminishing?p16:HEC1-linker-GR we observed a reduction of RAM size, which correlated with increased cytokinin signaling at the transition zone" referring to – Generally, 16hrs of CK treatments are sufficient to shrink the meristem. To address whether HEC1 controls root meristem size by recruiting cytokinin signalling, two days of DEX treatment is too long. Moreover, the description of how the authors treated their roots in these experiments is unclear; in the text it is stated "one day after induction of – From – In the text "HEC1 induction in seedlings " should 6) The conclusion from a previous paper from this group  concluded that HEC1 inhibits CK signaling during control of the SAM by inducing ARR7/15. Here, they describe the opposite. Although the data concerning CK signaling in this new manuscript are convincing, it would be desirable if the authors could discuss this discrepancy.7) It is concluded that induction of HEC1 leads to PIN1 polarization defects referring to Reviewer #3:TCSn::erGFP and DR5v2:3xYFP in loss and gain of functions of HEC they show that HEC induces cytokinin activity and represses the auxin one. They also demonstrate that HECs can control cell fate transition via auxin and cytokinin in the root as expression of HEC1 in p16 domain decreases the root meristem length and increases cytokinin activity. To conclude, they show that HEC1 binds auxin responsive elements and physically interacts with ARF5 and BRAHMA to modulate auxin signalling.In this manuscript Gaillochet and colleagues address the interesting question of how cell fate transitions are coordinated in meristems. Utilizing a combination of computational/genetic/molecular approaches they show that a family of BHLH transcription factors called HECATE (HEC) is central in this coordination. Indeed, they demonstrate that HEC transcription factors regulate cell non-autonomously cell fate transition from stem cells to transit amplifying cells and from transit amplifying to organ primordia. Subsequently, they investigate whether HEC might regulate cell transition via phyto-hormones and they focus on cytokinin and auxin. Via analysis of I really enjoyed reading Gaillochet et al. manuscript, nevertheless the connection of HEC with hormones looks to me not extremely robust, as functional evidences were not reported. I could not understand whether the modulation of auxin and cytokinin is necessary and sufficient to mediate HEC function and this point should be address to validate their model.Also, in previous report from the same group  it was shown that HEC1 promotes type A-ARR expression, negative regulators of cytokinin activity. The authors should discuss with care this point in the discussion.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your revised article has been favorably evaluated by Christian Hardtke (Senior editor), three original reviewers (one of whom is a member of our Board of Reviewing Editors), and one new reviewer.Thank you for resubmitting your work entitled "Control of plant cell fate transitions by transcriptional and hormonal signals" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Overall the reviewers found this to be an engaging read and an interesting conceptual advance. Some softening of bold statements  and a bit more testing of how HEC could modulate transitions in a non-mutant condition, would make this a valuable addition to the plant development literature. Below are 4 essential issues to address in a revision, and in a response to reviewers.1) A critical issue is whether HEC part of the normal regulatory circuit controlling meristem size, and is hard to really be sure of this when using mutants and mis-expression. Recently, however, there were some interesting studies  about cell cycle length and cell size in the meristem where changing nutritional status or light allowed them to make meristems of different sizes. If HEC is part of a homeostatic mechanism, then there should be some clear predictions of reporter/mutant behavior under these different conditions. These experiments would not require any new materials, but could be very helpful to define HECs normal function.2) We urge caution in interpreting the effects of HEC expression in the RAM by looking just at root length, as length can be changed by altering cell behaviors in different ways.P16::HEC-GR seedlings when compared to wt, or (2) to score the number of cells from QC to first differentiated cell as described , and/or progression of cell size .Several more precise methods are commonly used to capture the kinetics of transition from proliferation to expansion, and one or more of these should be employed. For example, (1) evaluation of cell division frequency at the root meristems of Once this finer dissection of the phenotype is performed, you should reconsider whether HEC has different effects in shoot and root or not.hec mutant, loss of HEC activity might result in higher CLV3 expression, which suppresses WUS. Attenuation of WUS would lead to high ARR7, ARR15 expression and suppression of CK signalling. In the CLV3::HEC -GR enhanced HEC activity correlates with increase of WUS expression which when followed by attenuation of ARR7, ARR15 expression ultimately enhances CK signaling. Hence I am not sure one can exclude that CK signaling (monitored by TCS) is under WUS control.3) The distinction between HEC-direct and WUS-mediated effects on CK is not clear. Reviewers were not sure that the pathway as presented is sufficiently supported by the current evidence, In particular, how can HEC direct versus WUS mediated effect on the CK signaling be distinguished? In the To address this, in the PZ zone, the effect of HEC on CK could be measured without the complication of WUS by visualizing the TCS reporter in this background.4) The finding of G-boxes and AREs in common regions is not terribly surprising given the high frequency of these motifs in the genome. It would add substance to this section if (1) the global analysis  [Editors’ note: the author responses to the first round of peer review follow.]Reviewer #1:[…] Priority concernsA) "HEC function appeared not only required and sufficient to control stem cell to PZ, but also.mediated PZ to organ fate transitions" This line of reasoning was based on ectopic expression of HEC in the PZ, which says that it can interfere with a normal process, but not that it controls it. I would like to see an amiRNA (or other LOF) experiment to substantiate this conclusion.HEC1, HEC2 and HEC3, these approaches were unfortunately not successful. Furthermore, we found that stem cell specific expression of MIGS against mCherry caused silencing in the entire shoot meristem, precluding the use of this system to create true domain-specific loss-of-function. To better fit the presented data, we modified the text accordingly.We fully agree that domain-specific knock-down experiments would be the optimal strategy to investigate the role of HEC factors in controlling cellular fate transitions. Therefore, we previously worked hard on trying to use domain-specific MIGS or amiRNA, but given the difficulty to knock down simultaneously Also, the expression patterns of HEC1/2/3 are never described in this manuscript. I imagine since they cite 4 prior publications, the patterns are known. This information would be useful included in this manuscript (at least in text). The same issue hold true for the root experiments. Induction of HEC1 can perturb the system, but does it actually work here?We have clarified HEC expression patterns by including a schematic and citing relevant previous publications. We have also included gene expression data from the root meristem, showing that HEC genes are not expressed in this tissue.B) Reporters for CK and auxin are considered to be evidence for direct regulation of these pathways. However, I wonder whether these reporters might be very downstream. For example, could pTCS-GFP signal report that cells are in a proliferative state and have high hormone response because of that state, rather than the other way around? To really nail the idea that HEC1/2/3 work through auxin or cytokinin, wouldn't doing these overexpression experiment in a background mutant for ARRs or ARFs be more direct?We mostly used reporters as readout for hormonal signalling and do not propose a detailed mechanism from their analysis, we further clarified this in the text. Regarding the TCS reporter, we showed that stem cell specific induction of HEC1 reduces the mitotic index at the centre of the SAM while promoting cytokinin signalling in this domain, excluding that this reporter would merely report the proliferative state of those cells.arr1,10,12, or 35S:CKX, log septuple, or ahk2,3,4 are not possible, since all promoters for SAM specific expression strongly respond to cytokinin signalling status (we have tested p16 and pCLV3).We also agree that it would be important to further uncover the molecular players acting in these hormonal pathways downstream of HEC1. To bridge this gap, we mostly focused our analysis of the interaction between HEC and MP by showing that HEC1 indirectly represses MP expression, leading to reduced MP-GFP accumulation and pin-like inflorescences. Unfortunately, the experiments overexpressing HEC in the mutants interfering with cytokinin signalling, such as C) The ChIP-seq is done on overexpressed HEC1 and the targets include typical bHLH sites. Could this just be generic binding? It would be nice to take the published bHLH ChIP-seq datasets  and see that the HEC1 sites are not always overlapping with any bHLH.We analysed the overlap between HEC1 binding peaks and those obtained from three bHLH and two structurally unrelated transcription factors. We observe that a large proportion of peaks are not shared between these bHLH factors, demonstrating the specificity of HEC1 DNA-binding pattern.D) I think modeling can be a useful addition to developmental studies and was used well in We are happy that the reviewer found the combination of experimental and modelling approaches useful to understand HEC loss-of-function phenotypes. We also agree that using a model that would integrate gene networks could greatly help in testing multiple regulatory scenarios. However, although our current model can be used to phenomenologically describe cell population dynamics, its structure does not allow us to implement a complex gene-regulatory network. We feel that building a new model including gene regulatory networks would go beyond the scope of this manuscript.Reviewer #2:[…] There are several interesting aspects of the work, which is also technically well-performed. However, it seems to me that the current data are not sufficient to draw the conclusions that the authors do and that alternative interpretations could lead to other models to explain them.We are happy that the reviewer found our work interesting and technically well executed. We have carried out more experiments to support our current model and further discussed alternative hypothesis.Specific issues:1) It is throughout the manuscript assumed that HEC1 can mediate its effect non-cell autonomously. This is based on the observation in Schuster et al., 2014, where the authors show that HEC1 expressed in the CZ inhibits WUS expression in the OZ via repression of CK signaling. The situation in this manuscript is different: firstly, the cells where HEC1 is supposed to function non-cell autonomously are not the OZ, but the PZ and secondly in Schuster et al. concludes that HEC1 repress CK signaling whereas here they demonstrate that HEC1 stimulate CK signaling (see further comment on this below). In order for the authors to make the non-cell autonomous claim, they will need to create an immobile version (e.g. HEC1-3xGFP) and show the effects are still there.pCUC2:HEC1-linker-GFP and pCLV3:HEC1-linker-GFP in pCLV3:3xmCherry-NLS, we observe that HEC1-GFP protein does not move long distance laterally, supporting our statement that HEC function in the CZ mostly acts on the PZ cell non-autonomously.We fully agree that using HEC1-3xGFP fusions would allow us to prove the non cellautonomous activity of HEC1 and we thank the reviewer for this suggestion. However, we previously prepared HEC1-GFP-GR and GR-GFP-HEC1 proteins that proved to be nonfunctional, as increasing protein size interfered with HEC1 functionality. Unfortunately, this technical limitation would make the result of the suggested experiment impossible to interpret. To this address this question, we chose an alternative approach and analysed HEC1-GFP protein mobility by driving its expression under various domains of the SAM. We previously published that HEC1-GFP was restricted to the WUS domain when driven under its promoter  and fully cell-autonomous when driven from the pML1 promoter . Consistently, we now find that 2) The authors list three scenarios for how HEC1 can increase the cell number of the meristem and use elegant inducible systems in specific domains of the SAM. This part is very complicated and I am not convinced about the authors' interpretation. For example, they conclude that HEC1 does not increase stem cell division locally (scenario 1). In order to draw such a conclusion, they should carry out the analysis in a zone-specific manner  and within 2 days, since CLV3 is downregulated at 4 days. Moreover, to eliminate scenario 2 they argue that the constant ratio of CLV3 positive cells over total SAM cells means that PZ cells are not re-specified to stem cells. In order to make this conclusion, they need a marker for PZ and show that this would not 'turn off' upon induction of HEC1 activity. Finally, based on the elimination of scenarios 1 and 2 (which I am not convinced about), they conclude that scenario 3 must be correct without providing experimental evidence for this, but suggest it is supported by showing that the subsequent transition from PZ to primordia is regulated similarly.We added experimental data and changed the text accordingly to support and clarify our imaging analysis. First, we quantified the mitotic index in the centre and at the periphery of the SAM independently, as suggested by the reviewer. In line with our previous observations, we found that HEC function does not locally promote cell division in the CZ. To analyse early PZ cell re-specification, the reviewer suggested analysing the expression of a PZ marker after HEC induction. From our experience, no such reporter clearly delimits CZ from PZ domains, making this suggested experiment impossible to perform (we previously tried the UFO promoter which displayed a patchier expression pattern than expected). Therefore, we analysed the ratio between the number of CLV3 cells and the total number of cells, which should increase  if PZ de-differentiation would take place. By showing no dramatic changes in this value, we excluded this scenario. Importantly, computational modelling simulations were used to test all the results from those experiments.3) Generally, the experiments using ectopic inducible expression of HEC1 under cell-type specific promoters would be much stronger if they were done in the hec1/2/3 background to test what aspects of this triple mutant is rescued (or not) by the transgene. Without this, it is hard to conclude what the effect of HEC1 would be on these processes in a WT situation.pCUC2:HEC1-linker-GFP in hec1,2,3 mutant background and observed similar phenotypes than in the WT background, supporting our described function of HEC factors within distinct domains of the SAM. This result was in line with our previous experiments using pCLV3:HEC1 in hec1,2,3 mutants, which showed SAM expansion comparable to WT background .We have now applied domain specific rescue by 4) In the Abstract and in the text, it is stated that data in this manuscript "show that HEC factors directly modulate auxin signal transduction by physical interaction with MONOPTEROUS […]" This overstates what the experiments actually show and more work would be required to validate this. The authors have shown that 1) MP is one among 12 ARF genes that are expressed in the SAM and which are direct targets of HEC1, 2) that many HEC1 targets contain both G-boxes (predicted HEC1 recognition sites) and Auxin Response Elements (AREs) and 3) that HEC1 can interact with MP in yeast and in BiFC. However, in order to make a convincing case, they need to provide genetic evidence for this interaction and establish if HEC1 actually does modify MP-mediated auxin signaling. Moreover, there are 12 other relevant ARFs according to the authors (i.e. expressed in the SAM). Does HEC1 also interact with them? Modify their activity?mp mutants and pCUC2:HEC1 mutants and that MP is the only ARFs that we found significantly regulated after 14 hours of HEC1 induction, we focused on characterizing the interaction between HEC and MP during the initiation of primordia. However, we agree that additional ARFs could play a role. We now show that HEC1 can reduce MP-GFP levels at the PZ and that HEC activity represses MP expression at the RNA level. We have also included new data that show that this repression can be rescued by auxin application, demonstrating that HEC function acts by modulating MP expression and auxin signalling during primordia initiation.We thank the reviewer for this comment and now better discuss alternative hypothesis regarding HEC mode of action in regulating auxin responses in the Discussion section. Given the similar pin-like inflorescence phenotypes of 5) The authors aim to address whether HEC genes can control cell fate transition in other developmental contexts, such as the root apical meristem (RAM) and leaves, through the regulation of auxin/cytokinin crosstalk. The conclusions that the authors draw from their experiments are again overstated, since they only analysed response to CK signaling in p16:HEC1-linker-GR roots with no attempt to show how auxin signaling response is affected. At the same time, the authors linked the leaf phenotype of the ectopic HEC1 expression to changes in auxin signaling without any prove nor attempting to understand possible crosstalk with cytokinin. The following lists issues particularly regarding this part:p16:HEC1-linker-GR roots exclusively aims at testing whether HEC function is sufficient to regulate cytokinin/auxin responses and in turn impact cell fate acquisition in another developmental context, rather than characterizing in depth HEC function at the root meristem. We further clarified this in the text and removed data presented at the seedling stage to avoid driving the reader away from the main focus of our manuscript.We included information on HEC activity in the root meristem and now show that these factors are not expressed in this region. Therefore, our analysis of – It's not clear whether HEC1 governs meristem size by controlling CK signalling or whether it is the result of a feedback mechanism operated by auxin. How (when and where) is the auxin response affected after HEC1 induction?We now analysed DR5v2 24 hours after induction and found a reduced intensity at the transition zone, demonstrating that HEC1 can act on auxin and cytokinin signalling in the root. Given the absence of endogenous HEC activity in the root, we feel that determining the chain of events would contribute little to the main story of our manuscript.– It would be helpful if the author could better describe in which zones/tissues p16 is expressed in the RAM? From We modified the figure accordingly and have marked the transition zone.– The control of CK on root meristem size occurs only from the vascular tissue at the transition zone. Is the activity of HEC1 sufficient to regulate root meristem size operating from the transition zone (could use the RCH2 promoter to address this)? Does the TCS expression increase before the meristem size starts diminishing?Although it would be interesting to understand the fine spatio-temporal dynamics of HEC1 activity in the root meristem, we feel that this experiment would contribute little to understanding how HEC1 acts in the SAM, where it is expressed. We have used the root only to test whether the effects on auxin and cytokinin could be transferred to a tissue devoid of endogenous HEC activity.– Generally, 16hrs of CK treatments are sufficient to shrink the meristem. To address whether HEC1 controls root meristem size by recruiting cytokinin signalling, two days of DEX treatment is too long. Moreover, the description of how the authors treated their roots in these experiments is unclear; in the text it is stated "one day after induction of p16:HEC1-linker-GR we observed a reduction of RAM size, which correlated with increased cytokinin signaling at the transition zone" referring to We included pictures of meristems 24 hours after HEC induction.– From Good point, we included a magnified picture of the ectopic periclinal division.– In the text "HEC1 induction in seedlings " should For reasons outlined above, we felt that this experiment was driving the reader away from the core message of our manuscript and for clarity we decided to remove it.6) The conclusion from a previous paper from this group  concluded that HEC1 inhibits CK signaling during control of the SAM by inducing ARR7/15. Here, they describe the opposite. Although the data concerning CK signaling in this new manuscript are convincing, it would be desirable if the authors could discuss this discrepancy.We thank the reviewer for this comment and further explain the causes of this discrepancy in the Discussion section (short term vs. long term effects).7) It is concluded that induction of HEC1 leads to PIN1 polarization defects referring to We included magnified pictures to better observe the defects in PIN1 polar localization after HEC1 induction.Reviewer #3:[…] I really enjoyed reading Gaillochet et al. manuscript, nevertheless the connection of HEC with hormones looks to me not extremely robust, as functional evidences were not reported. I could not understand whether the modulation of auxin and cytokinin is necessary and sufficient to mediate HEC function and this point should be address to validate their model.We are delighted that the reviewer appreciated our work, and we have included new data to support the functional interaction between HEC factors and auxin and cytokinin signalling pathways.Also, in previous report from the same group  it was shown that HEC1 promotes type A-ARR expression, negative regulators of cytokinin activity. The authors should discuss with care this point in the discussion.We agree that this is an important point and we have further clarified it in the Discussion section.[Editors' note: the author responses to the re-review follow.][…] 1) A critical issue is whether HEC part of the normal regulatory circuit controlling meristem size, and is hard to really be sure of this when using mutants and mis-expression. Recently, however, there were some interesting studies  about cell cycle length and cell size in the meristem where changing nutritional status or light allowed them to make meristems of different sizes. If HEC is part of a homeostatic mechanism, then there should be some clear predictions of reporter/mutant behavior under these different conditions. These experiments would not require any new materials, but could be very helpful to define HECs normal function.We agree that studying how plants integrate environmental cues and relay these signals to stem cell systems are of high relevance to further define the mechanisms driving plant developmental plasticity. Surprisingly, the regulatory mechanisms driving SAM size adjustment and the regulatory function of key stem cell factors under various environmental cues remain poorly characterized, although some studies have paved the way by mostly describing the SAM developmental response at the cellular level .-2 s-1) . By monitoring SAM size, we revealed that hec1,2,3 mutants could not adjust their meristem in response to low light conditions, demonstrating the relevance of HEC function for SAM homeostasis. Interestingly, cytokinin signalling was still responsive to the low light treatment in the HEC loss-of-function mutants, suggesting a complex re-wiring of the regulatory system.In this context, we tested whether HEC function could be part of the regulatory system controlling the adjustment of the SAM size to environmental perturbations by growing plants under low light conditions  We urge caution in interpreting the effects of HEC expression in the RAM by looking just at root length, as length can be changed by altering cell behaviors in different ways.Several more precise methods are commonly used to capture the kinetics of transition from proliferation to expansion, and one or more of these should be employed. For example, (1) evaluation of cell division frequency at the root meristems of P16::HEC-GR seedlings when compared to wt, or (2) to score the number of cells from QC to first differentiated cell as described , and/or progression of cell size .Once this finer dissection of the phenotype is performed, you should reconsider whether HEC has different effects in shoot and roo, or not.We thank the reviewers for their helpful comments. We have now further analyzed HEC activity at the cellular level in the RAM:1) By measuring the number of cortex cells after HEC induction .2) By calculating the cumulative cortex cell length 2 days after HEC induction.3) By qualitatively assessing cell division using the Fluorescent Timer .p16:HEC1-linker-GR induction, we observed that the number of cortex cells decreases, whereas neither cell length nor cell proliferation are altered in the relevant root regions. These data fully support the idea that HEC function would promote RAM cell differentiation by modulating the auxin/ cytokinin balance.Together with the shortening of the RAM length upon 3) The distinction between HEC-direct and WUS-mediated effects on CK is not clear. Reviewers were not sure that the pathway as presented is sufficiently supported by the current evidence, In particular, how can HEC direct versus WUS mediated effect on the CK signaling be distinguished? In the hec mutant, loss of HEC activity might result in higher CLV3 expression, which suppresses WUS. Attenuation of WUS would lead to high ARR7, ARR15 expression and suppression of CK signalling. In the CLV3::HEC -GR enhanced HEC activity correlates with increase of WUS expression which when followed by attenuation of ARR7, ARR15 expression ultimately enhances CK signaling. Hence I am not sure one can exclude that CK signaling (monitored by TCS) is under WUS control.To address this, in the PZ zone, the effect of HEC on CK could be measured without the complication of WUS by visualizing the TCS reporter in this background.We agree that given the lack of information on the directness of the interaction between HEC and WUS or cytokinin in our genomic data and given that WUS and cytokinin signaling cross-regulate in a positive feedback manner, both scenarios could explain our imaging data and thus required further clarification.pCUC2:HEC1-linker-GR and monitoring TCSn:erGFP . Thus, HEC function indirectly represses CK at the periphery by down-regulating MP and by blocking the formation of lateral organs.pCLV3:HEC1 and clv3-7 genetically interact to produce larger shoot meristem than individual single mutants . This argues against a full epistatic relation between HEC and WUS and thus indicates that HEC function acts partially independently of WUS to control SAM size. Along the same line, we did not observe PZ cell re-specification when inducing pCLV3:HEC1-GR as observed when inducing WUS-GR in the CLV3 domain, further dissociating HEC and WUS function in the SAM.Second, we previously observed that Finally, when ectopically expressed in the root meristem, HEC can promote cytokinin signalling in the absence of WUS (where it is not expressed), showing that in this context their function on cytokinin signalling can be uncoupled from WUS.Together we think that these evidences support the idea that HEC function can promote cytokinin signalling partially independently of WUS. As our data cannot fully clarify the network topology between HEC, WUS and cytokinin, we toned down our conclusions and included this point in the Discussion section.4) The finding of G-boxes and AREs in common regions is not terribly surprising given the high frequency of these motifs in the genome. It would add substance to this section if (1) the global analysis We fully agree with the reviewers, and apologize for the lack of clarity in the description of our bioinformatics approach. The presented data  was analAdditionally, we assessed the abundance of ARE and G-box or both motifs at the promoter of HEC1-response genes compared to the genome-wide scale . We foun4) The finding of G-boxes and AREs in common regions is not terribly surprising given the high frequency of these motifs in the genome. It would add substance to this section if (1) the global analysis We fully agree with the reviewers, and apologize for the lack of clarity in the description of our bioinformatics approach. The presented data  wasanalyzed by filtering only for euchromatic regions . We now further described our analysis in the method section.Additionally, we assessed the abundance of ARE and G-box or both motifs at thepromoter of HEC1-response genes compared to the genome-wide scale . We foun