We aimed to analyze injury profiles and injury severity in Korean youth soccer players. Data on all injuries that occurred in U-15 youth soccer players during the 2019 season were collected from 681 players of 22 teams through a medical questionnaire. The questionnaire was based on injury surveillance procedures of the Federation International de Football Association Medical and Research Centre and International Olympic Committee, and it comprised questions on demographic characteristics, training conditions, and injury information. Among all players, defenders accounted for 33.0%, followed by attackers (30.7%), midfielders (26.8%), and goalkeepers (7.9%). Most players played soccer on artificial grounds (97.4%). Injuries occurred more frequently during training (56.3%) than during matches (43.7%). Recurrent injury rate was 4.4% and average days to return to full activities were 22.58. The ankle (26.6%) and knee joints (14.1%) were the most common injury locations, and ligament sprains (21.0%), contusions (15.6%), and fractures (13.9%) were the most frequent injury types. In conclusion, Korean youth soccer players have a high injury risk. Therefore, researchers and coaching staff need to consider these results as a key to prevent injuries in youth soccer players and injury prevention programs may help decrease injury rate by providing injury management. Soccer is one of the most popular team sports in the world . In theiBased on the data from previous studies ,14,15,16FIFA founded the FIFA Medical Assessment and Research Centre (F-MARC), which established criteria for epidemiological studies to investigate the characteristics of soccer injuries and to prevent injuries and illnesses in athletes ,18. The According to the TRIPP framework, there is a need to conduct an epidemiological study on youth soccer players to establish injury prevention strategies that are based on the phenomena of youth soccer injuries in Korea. However, although many researchers have investigated the physical performance of players ,24, leadAmong 7838 Korean male U-15 soccer players from 235 teams, we randomly selected 24 teams, which accounted for approximately 10% of the total teams, and requested them to participate in this investigation. A total of 681 male U-15 soccer players from 22 teams accepted our request and agreed to participate. All selected players and teams were officially registered with KFA. Demographic characteristics of all participants are shown in To conduct this retrospective study, eight investigators were trained about exact definitions and procedure of measurement, visited 22 selected teams, and instructed all participants to fill out the questionnaire about injuries diagnosed by medical doctors during the 2019 season. The questionnaire comprised questions on demographic information , training condition , and information about injuries  based on the F-MARC consensus statement  and the An injury was defined as a physical complaint reported by a player experienced during a soccer match or training and included the following two factors: (1) a “medical attention” injury was defined as an injury that required a player to receive medical attention and (2) a “time loss” injury was considered an injury that rendered a player unable to participate in full training or a match .A recurrent injury was defined as an injury that occurred at the same site of the same type after a player’s full participation in a match or training after the index injury .Training was defined as team-based and individual physical activities under the supervision of the team’s coaching or fitness staff. A match was considered a play between teams from different clubs irrespective of a friendly or official match .Injury severity was graded according to the number of absences from full soccer activities such as team training or a match after injury diagnosed by medical doctors: (1) “slight” injury indicating 0 days lost, (2) “minimal” injury indicating 1–3 days lost, (3) “mild” injury indicating 4–7 days lost, (4) “moderate” injury indicating 8–28 days lost, and (5) “severe” injury indicating >28 days lost .An injury was classified based on the IOC ISS . The queAll data obtained from the questionnaires were analyzed using IBM SPSS software version 25.0 . For each category, we calculated the absolute and relative frequency or mean and standard deviation. The injury rate was derived using the following formula: [ × 100] and/or [ × 100].n = 225) of all positions. Regarding dominant legs, the right leg was dominant in 78.1% (n = 532) and the left leg and both legs in 12.9% (n = 88). Only 26% (n = 177) of all players had received education on injury prevention, with approximately 1.6 sessions per year . The most common player position was defender, which accounted for 33.0%  of reported injuries occurred during training and the remaining during a match . Regarding severity, injuries were most commonly moderate , followed by severe  and mild . More than half of the defenders suffered an injury . The recurrent injury rate and surgery rate were 35.1% (n = 144) and 4.4% (n = 18), respectively. The average number of days before return to full activities was reported to be 23, and players paid approximately 130 USD per injury.As shown in n = 315), followed by the upper extremity  and the head and trunk . The top five injured body locations were the ankle , knee , thigh , foot/toe , and wrist , followed by contusion , fracture , muscle strain , and ligament rupture . In addition, noncontact  and overuse  constituted a large portion of injuries.As shown in This retrospective epidemiological study was conducted to identify injury profiles of Korean male youth soccer players. Based on the data collected on injuries of Korean male youth soccer players, we identified that the most frequent type of injury was a new ankle injury of moderate severity with ligament sprain as a result of contact with other players during training. Medical staff members including clinicians, physiotherapists, nurses, and athletic trainers may need to consider these results when managing youth soccer players. The results of this study indicate that most youth soccer players in Korea play soccer on artificial grounds. According to a previous study , the incn = 144) of all injuries occurred repeatedly. This figure is higher than that reported in a previous study, which found a recurrent injury rate of 15.1% in English youth soccer [n = 119) of the total injuries were reported to be “moderate,” indicating an absence from soccer activities for 1–4 weeks. These results are similar to those of previous studies [The recurrent injury rate in this study was 35.1%  and this study is a systematic report about injury profiles of youth soccer players, we expect that these results will be used to develop evidence-based injury prevention programs and education to reduce injury rate in Korean youth soccer.Previous studies ,30,31 reBecause of the study design, we were unable to determine the precise time of AEs to matches and training. Based on the collected data, we were unable to calculate the accurate incidence of injury using data on AEs. Furthermore, the use of questionnaires to acquire information on injuries of Korean youth soccer players may be associated with a recall bias. Therefore, we recommend collecting injury data prospectively to analyze the actual phenomena of injuries in Korean youth soccer. According to our results, Korean youth soccer train and play matches on artificial grounds, which may result in a higher risk of injury. Furthermore, the results of this study showed that the most frequent type of injury was a new ankle injury of moderate severity with ligament sprain caused by contact with other players during training. In addition, we found a higher recurrent injury rate than that reported in a previous study. Researchers and coaching staff should consider these results as a key to prevent injuries in youth soccer players. Furthermore, because injury prevention education may help to decrease the injury rate by educating players on injury management, we suggest developing well-defined injury prevention education involving practical and need-based training.


CO2 diffusion inside the leaf depends directly on the structure of the mesophyll cells and their surrounding airspace, which have been difficult to characterize because of their inherently three-dimensional organization. Yet faster CO2 diffusion inside the leaf was probably critical in elevating rates of photosynthesis that occurred among angiosperm lineages. Here we characterize the three-dimensional surface area of the leaf mesophyll across vascular plants. We show that genome size determines the sizes and packing densities of cells in all leaf tissues and that smaller cells enable more mesophyll surface area to be packed into the leaf volume, facilitating higher CO2 diffusion. Measurements and modelling revealed that the spongy mesophyll layer better facilitates gaseous phase diffusion while the palisade mesophyll layer better facilitates liquid-phase diffusion. Our results demonstrate that genome downsizing among the angiosperms was critical to restructuring the entire pathway of CO2 diffusion into and through the leaf, maintaining high rates of CO2 supply to the leaf mesophyll despite declining atmospheric CO2 levels during the Cretaceous.Maintaining high rates of photosynthesis in leaves requires efficient movement of CO For leaves to sustain high rates of photosynthesis, they must maintain high rates of CO2 supply from the atmosphere to the sites of carboxylation in the leaf mesophyll. The importance of maintaining efficient CO2 diffusion into the leaf is reflected in the evolutionary history of leaf anatomy; leaf surface conductance has increased during periods of declining atmospheric CO2 concentration . Th. Th19]. figure 1a,b) ,34. By r results ,20. Thes stomata ,22 and m stomata ,34. Not gure S3) ,20,40. Wgure S3) . Despitegure S3) , traits ll cells .Figure 2 absorption, we tested whether the effect of genome size extends beyond limiting the sizes and packing densities of cells to influencing the surface area of the mesophyll tissue exposed to the intercellular airspace (SAmes). Genome size was a strong predictor of the total surface area per tissue volume of the mesophyll cells exposed to the intercellular airspace, SAmes/Vmes , which is the anatomically fixed component of the leaf mesophyll that influences CO2 diffusion. Our results suggest that except for a few ferns with small genomes, only angiosperms have been able to build leaves with high SAmes/Vmes (a). To explore this prediction beyond our dataset, we combined new measurements of SAmes/Vmes on the species for which we had microCT images with data extracted from the literature for 85 additional species . The distribution of SAmes/Vmes among clades in our dataset was consistent with the data extracted from the literature and showed that the highest and most variable SAmes/Vmes occurs only among monocots and eudicots, suggesting that anatomical innovations among the angiosperms are responsible for the heightened SAmes/Vmes necessary to support high rates of photosynthesis. To test the prediction that genome downsizing enabled high SAmes/Vmes  via impacts on cell size and cell packing density, we tested whether SAmes/Vmes was coordinated with the sizes and packing densities of cells and tissues throughout the leaf. The packing densities of stomata, veins, and palisade mesophyll cells were all strongly and positively related to SAmes/Vmes (figure 2b–d), while the diameters of stomatal guard cells and of spongy and palisade mesophyll cells were all strongly and negatively related to SAmes/Vmes (figure 2e–g). This whole-leaf trade-off between cell size and cell packing density  further corroborate the role of genome size in determining the sizes and arrangement of cells and tissues throughout the leaf that enable high rates of CO2 and H2O diffusion between the leaf interior and the atmosphere.Because cell surfaces can be in contact with other cells and be unavailable for COmes/Vmes a. To exp(b)2 enters the leaf on the lower spongy mesophyll layer for most terrestrial plants, creating within the leaf opposing gradients of two of the primary reactants in photosynthesis. Within a leaf, the spongy and palisade layers have divergent cell shapes and organizations that are thought to accommodate these opposing gradients by facilitating CO2 diffusion in the gaseous and liquid-phases. Both cell size and porosity can affect SAmes/Vmes and the diffusive conductances (gias and gliq) that are considered targets of selection to increase photosynthesis [mes/Vmes and on modelled gias and gliq, we measured cell diameter, porosity, and SAmes/Vmes for the spongy and palisade layers separately for 47 species in our dataset, encompassing all major lineages of vascular plants.While light is intercepted primarily by the upper palisade mesophyll layer , CO2 entynthesis ,38,41,42mes/Vmes (e–g) suggested that cell diameter would have a greater impact than porosity on SAmes/Vmes. Smaller cells have a higher ratio of surface area to volume, an effect that could propagate up to influencing SAmes/Vmes of the entire tissue. In contrast, we predicted that porosity would not have a consistent impact on SAmes/Vmes because at very low porosities there is very little cell surface area exposed to the airspace while at very high porosities there is very little cell surface area relative to a large volume of tissue. Consistent with these predictions, decreasing cell size led to higher SAmes/Vmes across species and mesophyll layers, and variation in porosity had no consistent effect on SAmes/Vmes . Many species, particularly angiosperms, have palisade mesophyll characterized by small, highly packed cells that allow volumetric gliq to be higher than gias of this tissue 2 and water. Although coordinating changes in veins, stomata, and the mesophyll undoubtedly involves multiple molecular developmental programmes, the simple scaling of genome size and cell size emerged as the predominant factor driving the increases in SAmes/Vmes and gliq that together enabled higher rates of CO2 movement into the photosynthetic mesophyll cells. While the size and abundance of chloroplasts in the leaf will undoubtedly affect photosynthetic rates, the maximum chloroplast surface area available for CO2 diffusion is limited by the surface area of the mesophyll. Because photosynthetic metabolism is the primary source of energy and matter for the biosphere, leaf-level processes are directly linked to ecological processes globally [Our results suggest that the heightened rates of leaf-level gas exchange that occurred predominantly among angiosperms are coordinated with changes not only in veins and stomata ,9,12,13 globally ,45. Our globally ,46, in cglobally ,48. Incoglobally ,49,50. Wglobally –53, seleglobally .. 3(a)Selaginella kraussiana, 17 species of ferns from 12 families, and major groups of gymnosperms, including gnetophytes, cycads and conifers. Leaves were cut at the base of the petiole or of short stem segment, immediately put in a plastic bag with the cut end wrapped in paper towels, and scanned within 36 h of excision.Mature, fully expanded leaves from healthy, well-watered plants were collected from greenhouses, botanical gardens, fields and other outdoor growing locations to represent a broad phylogenetic diversity of C3 vascular plants . We chose representative angiosperms from the ANA grade, magnoliids, monocots, basal eudicots, eurosids and euasterids. We also sampled the lycophyte (b)MicroCT scanning was carried out at the Advanced Light Source , the Swiss Light Source , and the Advanced Photon Source . Samples were prepared less than 30 min before each scan. For laminar leaves, an approximately 1.5 × 15 mm piece of leaf was excised from between the midrib and the leaf outer edge. For needle and non-laminar leaves, a piece approximately 15 mm long was used. Tissue samples were enclosed between Kapton (polyimide) tape to prevent desiccation while allowing high X-ray transmittance. Samples were scanned using the continuous tomography mode capturing 1025  or 1800 (SLS) projection images at 21–25 keV, using primarily 5×  and 10×  objective lenses, or a 40× objective lens . Each scan was completed in 5–15 min.Images were reconstructed using TomoPy  for all (c)dpalisade or dGC), allowing for cell volume to be calculated as [Leaf and mesophyll thickness were measured on cross-sectional slices of the image stack. Cell diameter was measured on at least 10 cells for each mesophyll layer on paradermal slices of the stack, as well as for guard cell length and diameter. For spongy mesophyll cells with lobed or irregular shapes, cell diameter was measured on the lobes of the cells and not on their presumed centres . Some lelated as V=596π were not measured for these traits.Vpores), mesophyll cell volume (Vcells), both summing up to the total mesophyll volume (Vmes), vasculature volume (Vveins) and the surface area exposed to the intercellular airspace (SAmes) were then extracted using published methods [https://github.com/plant-microct-tools/leaf-traits-microct). SAmes/Vmes is less sensitive to leaf thickness than the commonly measured Sm, i.e. SAmes per leaf area . For separate quantification of traits from palisade and spongy mesophyll, segmented stacks were cropped at the interface between tissues or where vasculature was present, in order to accurately characterize SAmes, volumes and cell diameter within those tissues.To extract surface area and volumes, mesophyll cells, airspace, vasculature (combined veins and bundle sheath) and background (including the epidermis) were segmented using published methods ,55 and I methods  with the methods , or usinmes . Overall, lower magnification scans resulted in small (less than 5% for most scans) but significant changes in cell diameter and SAmes . However, reanalysis of scaling relationships reported in Because our sampling included scans made at different magnifications, we tested the effect of magnification on measurements of cell size and SA(d)Zea mays or Vicia faba standards and following standard protocols [Existing 2C genome size (pg) data available in the Kew Plant DNA C-values Database  were matrotocols .(e)gliq and gias 2 projected area , with a total volume of 2d × projected area and a total porosity of 0.186 . Simulating porosity above or below 0.186 was done by changing pore volume and keeping cell volume constant, which modified total lattice volume to represent either a looser cell packing or cells inflated and deformed into each other.To model capsules , with digias, we accounted for tortuosity and diffusive path lengthening as functions of porosity [R2 = 0.21, p < 0.0001; electronic supplementary material, figure S11).Liquid-phase conductance per mesophyll volume was computed  as a funporosity , and mes(f)All analyses, simulations and conductance computations were carried out in R 4.0.3 . StandarClick here for additional data file.


For those on phototherapy, duration and median irradiance were similar. F80 infants experienced reduced: feeding intolerance ; length of stay , maximum weight loss as % birth weight ; decrease in weight Z-score at 10 days . F80 infants regained birthweight earlier  and had no differences in adverse clinical outcomes. We speculate that earlier nutrient supplementation improved jaundice outcomes due to enhanced excretion/elimination of bilirubin.This study aimed to evaluate jaundice outcomes of low-birthweight premature infants commenced on earlier versus later nutrient supplementation . Demographics, feeding regimens, and clinical outcomes data were collected. Infant and maternal characteristics were similar. Earlier nutrient supplementation was associated with multiple improved jaundice outcomes: total (TSBR), unconjugated and conjugated (CSBR) serum bilirubin values (196 ± 46 vs. 228 ± 52, 184 ± 44 vs. 212 ± 50, 12 ± 4 vs. 16 ± 5, respectively, all  The basic etiology of neonatal hyperbilirubinemia is well understood. Neonatal jaundice results from an imbalance between bilirubin production soon after birth and elimination. Factors include: bilirubin formation from the breakdown of heme present in normal red blood cells; conjugation in the liver with glucuronic acid to form a water-soluble form; excretion in the bile ; some hyWhat is not so well understood, despite decades of research, are the influences contributing to hyperbilirubinemia—which, in the neonate, is a multifactorial process, associated with both biochemical and physiological immaturity. In the near term to term infant, risk factors for developing severe unconjugated hyperbilirubinemia include: haemolytic disease; gestational age 35–36 weeks; cephalohematoma; exclusive breastfeeding, particularly if nursing is problematic and weight loss significant; East Asian race; macrosomic infants of a diabetic mothers; male gender [An interrelationship between enteral feeding, the timing of feeds, nutrition and neonatal jaundice has long been recognised. In the 1960s, when 48 h fasting of newborn infants was common, studies showed that babies fed glucose in part saline or water at 4 h of life developed significantly less jaundice than fasting babies . During In our Special Care Nursery (SCN), we manage mostly moderate (32 to 34 weeks’ gestation) to late (34 to 36 weeks’ gestation), low birthweight preterm infants. Previously, we commenced the fortification of expressed breast milk (EBM) or preterm formula at a full enteral feed achievement of 160 mL/kg/day, before moving to a protocol of earlier fortification/preterm formula at 80 mL/kg/day. In a prospective, observational study, we investigated the nutritional requirements of moderate and late preterm infants—an area which has been little researched . We reviDuring the study, we found evidence to suggest a benefit of earlier nutrient supplementation at 80 mL/kg/day on jaundice outcomes—however, due to small sample size, our results did not achieve significance .The aim of this study is to explore the link between an earlier commencement of fortification of enteral feeds and the evaluation of a possible improvement in jaundice outcomes. We hoped to achieve this by retrospectively examining the records of a sufficiently large cohort of infants during the period when the earlier protocol of commencing fortification at 160 mL/kg/day was in place, with a similar sized cohort following the protocol change that commenced fortification at 80 mL/kg/day.This study’s participants were moderately preterm and late preterm LBW neonates admitted to the SCN between January 2012 to December 2018 at The Northern Hospital (TNH) in Epping, an outer suburb of Melbourne, Australia. The catchment area for TNH is a lower-socioeconomic, multicultural, multi-ethnic community with associated concerns such as obesity. We performed a retrospective chart review. Assignment into F80 or F160 groups was done on the basis of chronology. Before 2016, our feeding protocol introduced breastmilk fortification once full enteral feeds had been achieved (160 mL/kg/day). Following this date, we changed our protocol to commence fortification at 80 mL/kg/day. No other changes were made to the protocol. Otherwise, the management of F80 and F160 infants was identical.Inclusion criteria were: birthweight less than 2500 g; gestational age 31 weeks to late preterm; tolerating a total enteral fluid intake of less than 80 mL/kg/day. Infants with gastrointestinal malformations or recognized chromosomal abnormalities were excluded. Infants who did not strictly fit into the commencement criteria of 80 or 160 mL/kg/day enteral fluid intake were also excluded. Ethics approval was provided through the Northern Health Human Research Ethics Committee. (ALR No: ALR 52.2018).Maternal baseline data collected include age, gravity and para, region of birth based on United Nations country grouping, and primary language spoken at home. Maternal clinical data collected include antenatal complications such as gestational diabetes, preeclampsia, and Group B Streptococcus screening result.Baseline data collected on neonates include gestational age, sex, reason for prematurity, mode of delivery, 5-min APGAR score, and birthweight. Data on feeding regimens collected included: day of life at first enteral feed; total fluid intake at first enteral feed; total fluid intake at commencement of enteral supplementation; day of life at commencement of enteral supplementation. F80 neonates were defined as those that were commenced on human milk fortifier (or preterm formula if EBM was unavailable) at total fluid intake of 80 mL/kg/day, while F160 were defined as those that commenced on human milk fortifier (or preterm formula) at total fluid intake of 160 mL/kg/day.The primary outcome was related to hyperbilirubinemia and included: need for phototherapy; maximum number of phototherapy lights required; highest recorded bilirubin value; number of days that phototherapy was required. Anthropometric outcomes included maximum weight loss, days to regain birth weight, and discharge weight. Z-scores were calculated for birthweight, lowest weight post birth and discharge weight using the 2013 Fenton growth charts . AdverseThe use of 2013 Fenton growth charts in this study requires an explanation. Fenton growth charts are based on intrauterine data as opposed to birthweight data used for the more recently developed and internationally widely accepted Intergrowth-21st growth charts. The issue of which type of growth chart is most appropriate has become controversial. While for extremely low birth weight infants, Intergrowth 21st Project growth standards have been shown to be superior to Fenton Growth Charts . Of partDue to a policy of actively promoting and supporting breastfeeding, the majority of premature babies in our SCN are fed breastmilk. All study infants commenced feeding from day 1 at an enteral volume of 60 mL/kg and increased by 20 mL/kg daily as tolerated. The fortification/supplementation regime was standard and straightforward. Human milk fortifier  was introduced into expressed breastmilk (EBM) or preterm formula was commenced if EBM was not available  or S26 low birth weight Formula . FM85 was given at the recommended concentration of 1 g per 20 mL of EBM which boosted the protein content of EBM by 1 g of protein per 100 mL. Preterm formula provided a protein content of 2.9 g/100 mL. Prior to 2016, such fortification was commenced at an enteral volume achievement of 160 mL/kg/day (F160 group). Post 2016, due to a policy change, fortification was commenced once enteral volumes had reached 80 mL/kg/day (F80 group). Fortification or preterm formula was given to all babies up to a weight of 2500 g. Thereafter, cessation was at the discretion of the neonatal consultant and, based on such issues as appropriate weight gaining, extent of feeding intolerance and SGA .p-value. Continuous variables were analysed using the student’s t-test or the Mann—Whitney test where appropriate. A p value of <0.05 was considered significant. Statistical analyses were performed using either SPSS  or NCSS 12 statistical programme . NCSS, LLC. Kaysville, Utah, USA)Results were compared between F80 and F160 infants and differences in categorical variables were assessed using the Chi-square test or Fisher’s exact test where appropriate. Normality for continuous variables was assessed using the Shapiro-Wilk test, with Assuming an 8% change (as indicated from the raw data of our previous study ) in the A total of 215 babies were included in this study, 105 in the F80 group and 110 the F160 group. Infant gestational age ranged from 31 to 36 weeks. Between the F80 group and F160 group, there were no significant differences in birth weight, birth weight Z-scores, APGAR score at 5 min, reason for prematurity and mode of delivery see .Maternal age ranged from 16 to 44 years of age. Approximately half the mothers were born in Australia and New Zealand, with South Asia as the next largest place of maternal birth. English was the predominant language spoken at home. There were no significant differences in maternal characteristics between F80 and F160 groups see .The F80 group experienced less maximum weight loss as a percentage of birth weight compared to the F160 group. Infants commenced on earlier enteral supplementation regained birth weight before infants commenced on later enteral supplementation. Weight gain at 10 days as measured by change in Z-score from birthweight showed a significantly lower decrease in weight percentiles for F80 infants compared to F160 infants see .Multiple positive jaundice outcomes were associated with earlier supplementation of fortified breastmilk or preterm formula. For the F80 group compared with the F160 group, these outcomes significantly included lower total serum bilirubin (TSBR), unconjugated bilirubin (USBR) and conjugated bilirubin (CSBR): less infants below a phototherapy recommended cut off value for LBW infants at 36 weeks gestation of TSBR 250 µmol/L ; fewer iFeeding intolerance was lower for the F80 group. Length of stay (LOS) was significantly shorter for F80 infants than F160 infants. There were no differences in the frequencies of presumed sepsis, hypoglycaemia and respiratory distress syndrome. There was no occurrence of NEC see .As a consequence of changing our feeding protocol to an earlier commencement of fortification (at 80 mL/kg/day rather than 160 mL/kg/day), infants began supplementation of their feeds almost 5 days earlier—on average before day 3. The key finding of our study is that earlier nutrient supplementation of predominantly breastmilk fed babies (mainly by fortification) was associated with multiple beneficial jaundice outcomes such as lower TSBR, USBR and CSBR and lower phototherapy rates and consequently less exposure irradiation. As we have previously reported in some detail , althougIt could be argued that these outcomes are not surprising when viewed against the changes to infant feeding regimes that have occurred over the last half century. One reason for the abandonment of the customary 48 h fasting of newborns in the 1960s was the realization that early hypocaloric feeding, such as glucose, reduced hyperbilirubinemia . This isThere is evidence to suggest that the expression of extrahepatic UDP-glucuronosyltransferase 1A1 (UGT1A1), may play such a role. UGT1A1 is solely responsible for bilirubin conjugation and had been thought to be only present in the liver . In the At a basic level, neonatal jaundice can be thought of as an imbalance in the production and elimination of bilirubin . The proOral glucose has been shown to be an effective inducer of intestinal UGT1A1 without affecting liver UGT1A1 induction and has been suggested as a convenient way to treat neonatal jaundice while continuing the benefits of breastfeeding . The forZinc is another constituent of nutrient supplement that has been demonstrated to improve bilirubin elimination. A recent randomized, double blind trial demonstrated that neonates between 31 and 36 weeks gestation requiring phototherapy, had significantly reduced jaundice within 48 h of receiving oral zinc . These fAnother possible excretion mechanism for bilirubin is bacterial conversion. In adults, bilirubin excreted into the intestinal lumen is very efficiently converted to urobilinogen in the faeces—a natural detoxifying mechanism which aids elimination . In our Some recent studies have associated vitamin D deficiency in neonates with hyperbilirubinemia. TSBR significantly declined in vitamin D treated infants on phototherapy compared to those on phototherapy alone . NeonateWhile the focus of this paper has been on the apparent benefits to jaundice outcomes due to earlier nutrient supplementation resulting from a change in our feeding protocol that commenced supplementation at 80 mL/kg/day rather than 160 mL/kg/day, there were other clear benefits. F80 infants regained birth weight earlier, weight Z-scores were better at 10 days, the incidence of feeding intolerance was less, and LOS was almost 3 days shorter with important cost saving implication for the hospital. There was no difference in discharge weights between either group. This finding is consistent with findings that while phototherapy itself may result in a temporary weight reduction, catch up growth occurs with eventually no weight difference to infants not exposed to irradiation . The redA key limitation of this study is that being retrospective in nature, it has inferior evidence when compared to a prospective study. The convenience sampling inherent in this type of retrospective study may have led to selection bias. Of particular note is the concern of temporal relationships. This study stretched over a 6-year period, yet only a total of 215 infants were included in the study. The reason for this was the strict adherence to the criteria of only including infants commenced on fortification at 80 or 160 mL/kg/day—infants who commenced fortification at any other volume, eg 70 or 150 mL/kg/day were excluded. While this strict selection criteria improved the reliability of outcomes , it lenAnother potential limitation, as discussed in Methods, is that this study used Fenton 2013 growth charts to classify infants as SGA and to determine weight Z-scores. While this allowed a direct comparison with our previous study  and intrOur study has shown that a feeding protocol change that commenced nutrient supplementation at half enteral feed volumes (80 mL/kg/day) compared to full enteral feed volumes (160 mL/kg/day), resulted in predominately breastmilk fed infants commencing supplementation around 5 days earlier. This earlier supplementation was associated with significant clinically important benefits in reducing neonatal jaundice such as lowered levels of hyperbilirubinemia and a reduction in the frequency and extent of phototherapy. There were also additional benefits such as improved growth outcomes, reduced feeding intolerance and an almost 3-day reduction in LOS. We speculate that the improved jaundice outcomes are not related to any change in the dynamics of bilirubin liver production and conjugation, but due to mechanisms promoting more efficient elimination. The earlier nutrient supplementation occurred well before peak jaundice occurred, allowing time for fortification components to induce intestinal UGT1A1 (opposing the effects of β-glucuronidase) as well as promoting bowel motions—thus enhancing bilirubin excretion and elimination and reducing the enterohepatic recirculation of bilirubin.


At birth, the lungs rapidly transition from a pathogen-free, hypoxic environment to a pathogen-rich, rhythmically distended air-liquid interface. Although many studies have focused on the adult lung, the perinatal lung remains unexplored. Here, we present an atlas of the murine lung immune compartment during early postnatal development. We show that the late embryonic lung is dominated by specialized proliferative macrophages with a surprising physical interaction with the developing vasculature. These macrophages disappear after birth and are replaced by a dynamic mixture of macrophage subtypes, dendritic cells, granulocytes, and lymphocytes. Detailed characterization of macrophage diversity revealed an orchestration of distinct subpopulations across postnatal development to fill context-specific functions in tissue remodeling, angiogenesis, and immunity. These data both broaden the putative roles for immune cells in the developing lung and provide a framework for understanding how external insults alter immune cell phenotype during a period of rapid lung growth and heightened vulnerability. Prior to birth, the lung is maintained in a fluid-filled, immune-privileged, hypoxic environment. Upon the infant’s first breath, the lung rapidly transitions to an oxygen rich environment , subjectImmune cells play a central role in the development of many organs, including the kidney , brain  and mammThe prevailing notion is that the neonatal immune compartment is immature . AttenuaIn this report, we combined single cell transcriptomics (scRNA-Seq) with fluorescent multiplexed in situ hybridization (FISH) and flow cytometry to characterize changes in composition, localization, and function of immune cells in the murine lung from just before birth through the first three weeks of postnatal life. At birth, immune cell heterogeneity increased dramatically from an embryonic landscape dominated by immature, proliferative macrophages to a complex landscape comprised of multiple types of macrophages, dendritic cells, granulocytes, and lymphocytes. Dynamic changes in macrophage heterogeneity were particularly striking, both transcriptionally and spatially, including the presence of embryonic macrophages encircling developing vessels prior to birth. After birth, these embryonic macrophages disappear, and numerous unique macrophage populations emerge, each exhibiting unique gene signatures suggesting specific roles in immunosuppression, pathogen surveillance, angiogenesis, and tissue remodeling. Multiple populations of dendritic cells, basophils, mast cells and neutrophils are also present in the postnatal lung, expressing genes important for rapid pathogen response. In contrast, although lymphocytes increase in abundance across three weeks, they remain functionally immature and skewed toward type-2 immunity. Taken together, our data demonstrate a previously underappreciated plasticity of immune cells in the perinatal and neonatal lung suggesting unique and essential roles in regulating immune function and lung structure.To comprehensively define the lung immune landscape at birth, we isolated whole lungs from C57BL/6 (B6) mice at four stages of perinatal development: the early saccular (E18.5), late saccular (P1) early alveolar (P7) and late alveolar stages (P21) , and quaCd68, and distinguished by expression of Gal (Mac I), Itgax (Mac II), Car4 and Itgax (Mac III), C1qa (Mac IV), or Plac8 (Mac V). Dendritic cells (DCs) separated into three clusters, all expressing some amount of Batf3, but distinguished by the expression of Itgae (cDC1), Cd209a (cDC2), or Mreg (mig-DC). We also identified mast cells (expressing Mcpt4), basophils (Mcpt8), and neutrophils (Retnlg). Four lymphoid clusters were found, consisting of B cells (expressing Ms4a1), T cells (Cd3e), natural killer (NK) cells (Gzma), and group two innate lymphoid cells (ILC2) (Areg) (Fifteen cell clusters were identified via Leiden community detection  and veri) (Areg) .We next assessed cluster distribution across time . Mac I cCd68, indicative of macrophages or monocytes (m/m)  . At E18.es (m/m) . After bDab2), which regulates macrophage polarization (e (Plac8), which is related to bacterial immunity  in Mac I included the proliferation markers and Mcm5 . Across and Mcm5 . These dand Mcm5 .Cd68+ cells scattered throughout the lung parenchyma but also, surprisingly, forming almost complete rings around blood vessels of 20–30 μm in diameter found adjacent to large, conducting airways . Perivascular macrophages were found on in situ analysis to express Dab2 but not Plac8 . These studies demonstrated that the majority of Cd68+ macrophages surrounding the vessels were Gal+ (83.1 ± 12.8%)  , indicat± 12.8%) . The Machenotype . Moreovehenotype . Taken tCrispld2, a glucocorticoid-regulated gene previously thought to be restricted primarily to the lung mesenchyme that promotes lung branching , a membrane bound serine proteinase inhibitor and regulator of angiogenesis   (Il1rn and Dapk1)  with a subset of Mac IV  . We alsond Tgm2) . In addind shape , and Hhiignaling . Hhip numphysema . The Macd Mpeg1) , but alsd Dapk1) . A subpof Mac IV  suggestilar wall .Car4, Bhlhe41, Trim29)   , Table 3 Trim29) , includi, Il1rn) , and liptabolism . Tissue ntegrity , Serpine inhibit  or promo inhibit , and Il1modeling . In situen by P7 , confirmC1qa, C1qb, C1qc, C3ar1) and CCR2 ligand Ccl12  . These transcriptional differences within Mac IV are similar to two recently reported interstitial macrophages (IMs) in adult lung that can be differentiated by expression of Mrc1 and MHC II genes   , but witII genes . However2, Cd68)  were dif airways . The chaGal-promoter driven fluorescent reporter will be required to firmly address the fate of the Mac I cells in the postnatal lung.The transient presence of the Mac II cells, and significant overlap with the transcriptomes of Mac I, and III, suggested that Mac II may represent an intermediate population. To determine if cells within these clusters were undergoing gradual transcriptional shifts across time, we performed pseudotime analysis on cells from clusters Mac I, II, and III. Ordering of the cells within the Mac clusters I-III by pseudotime  defined Plac8    , as well1, Lst1) . This duCcr1 and Ccr2) expressed at one end and other genes (e.g. Ace and Slc12a) expressed at the other end of the spectrum         , matrix 1, Vcan) , and red, Prdx6)  in keepi, Prdx6) . Over bof1, Spn) , macroph2, Cd36) , negativ, Lair1) , and inn Treml4) . These dCcr2 or Lair1 in combination with Ifitm6 and Cd68 allowed the identification of early Mac V cells within the distal lung parenchyma . Second, the proportion of IMs expressing H2-Eb1 is higher in their dataset. Third their monocyte cluster is located closer to our Mac V and both express Plac8, indicating a similar cell type. While a few of their cells embedded completely within our Mac V, the majority are in a distinct, yet nearby cluster, possibly reflecting developmental differences. Likewise, although our Mac III and their AMs express Car4 and cluster near one another, they remain in separate clusters, perhaps reflecting either technical or biologic differences. Comparison with Tabula Muris  and Fcer1g (FcεRIγ) were widely expressed by all five clusters, while Fcer1a and Fcer2a were not expressed by any cluster. Expression of Fcgr1, a high affinity receptor for IgG important for the endocytosis of soluble IgG, phagocytosis of immune complexes, and delivery of immune complexes to APC , an Fc receptor with a key role in IgG recycling, was highest in Mac IV cells.Fc receptors serve as an important link between cellular and humoral immunity by bridging antibody specificity to effector cells  and are s to APC  was highult mice . Fcgrt, Zbtb46 , a modulator of lysosomal hydrolase maturation  play multiple roles in the immune system including antigen presentation and regulation of tolerance and can be distinguished from other mononuclear phagocytes by the expression of Zbtb46  and Flt3Zbtb46 . We founZbtb46 –8 expresand Flt3 . Classicor CD103 , which por CD103 , and a nnd Cadm1 . The nexand Irf4 . Althougccharide , and lowy absent . Given tturation . Mreg hans cells ; Fscn1, uring DC ; Ccl5, amph node , therefomph node –8. Quantmph node . cDC2 waCpa3 that could be further distinguished as mast cells and basophils by the expression of Mcpt4 and Mcpt8, respectively  and tryptase (Tspab1)  . Lung re(Tspab1) . Our bas and Osm . We alsoammation , and Stfentation . Our datentation , and thaAreg), NK cells (Gzma), B cells (Ms4a1) and T cells (Cde3) were present in the lung at low frequencies (approximately 2% of immune cells) prior to birth and increased in frequency after birth. By P21, lymphocytes comprised 60% of total immune cells  increased by P21 while other subsets remained constant   . Few CD4=0.0047) .At birth, the lung undergoes marked physiological changes as it transitions from a fluid-filled, hypoxic environment to an air-filled, oxygen-rich environment. How these changes affect immune populations during this transition and the ensuing period of rapid postnatal lung growth remains unclear. Our study demonstrates a rapid increase in immune cell heterogeneity, especially within macrophages and monocytes. We identified five macrophage/monocyte subpopulations, each expressing a specific gene signature, spatial localization, and putative functions. Mac I, the predominant immune cell present just before birth, were highly proliferative, and completely encircled small blood vessels, suggesting a previously unrecognized role for lung macrophages in modulating lung vascular growth or remodeling during development. During the first week of life, a transitory population (Mac II) emerged from Mac I and later disappeared, transitioning into either an alveolar (Mac III) or Mac IV macrophage phenotype. One interstitial macrophage population (Mac IV) expressed complement proteins and other antibacterial molecules. Another interstitial population (Mac V) expressed antiviral molecules and spanned a gradient between two extreme phenotypes, one that expressed high levels of homeostatic genes during early postnatal development, and a second with immunomodulatory functions that resembles previously reported nonclassical monocytes. Lymphocytes increased in abundance from almost zero before birth to more than half of lung immune cells by P21, but maintained a naive phenotype skewed toward type II immunity and with predominantly Cd4- Cd8- T cells.This comprehensive study has far-reaching implications for lung biology. Resident tissue macrophage populations are established during development, wherein progenitors undergo differentiation guided by the tissue-specific microenvironment . HoweverTek, the VEGF co-receptor Nrp1, growth factors  and MMPs . Although the perivascular macrophages we observed in the embryonic lung expressed low levels of Nrp1, they appear distinct from the macrophages that influence retinal and hindbrain angiogenesis, expressing a unique set of ECM remodeling and angiogenic genes, including genes that may modulate vascular tone and permeability. Whether the macrophages form non-contiguous rings around the vessel, or sheets that completely encircle the full length of the vessel is not clear and would require antibody-based immunofluorescence with deep imaging and reconstruction of thick tissue sections. The distinctive location of these macrophages and their gene signature imply a role in vascular development. Furthermore, these encircling macrophages disappeared after birth, suggesting a function temporally restricted to prenatal development. Future studies to selectively target this subpopulation will be required to further establish their function and to delineate the signals responsible for the cessation of the macrophage-vascular interaction after birth.Another key finding in our study was the unexpected presence of embryonic lung macrophages encircling small blood vessels. Vascular growth is a key driver of distal lung growth during the late saccular and alveolar stages of development . MacrophItgae and Cd209a) and novel (Mreg) markers superior to classical markers (Zbtb46 and Flt3) to distinguish dendritic subpopulations in the postnatal lung. Functionally, both Itgae and Cd209a/DC-SIGN can induce T cell immunity  rapidly increase from E18.5 to P21. Given that resident alveolar macrophages are anti-inflammatory and mitigate the airway hyperresponsiveness that characterizes asthma, a perinatal insult that decreases either Mac V or Mac III abundance could possess long term, pro-inflammatory effects. Further investigation into the effect of injurious stimuli on the phenotype, abundance and localization of these discrete immune cell populations is likely to provide deeper insight into the mechanisms underlying pediatric and adult lung disease.In addition, whether alterations in immune cell diversity may underlie the origin of pediatric lung disease remains to be determined. Many of the Mac clusters express genes with seemingly contrasting functions, highlighting a key role for these populations in the fine-tuned regulation of complex biological process such as inflammation and angiogenesis. Stimuli that disrupt this careful balance, might have profound pathologic effects and contribute to diseases such as BPD, pulmonary hypertension and asthma. For example, in the mouse, inflammatory activation of embryonic macrophages disrupts lung branching , and abnental PH . WhetherIn summary, these data highlight the marked increase in immune cell diversity after birth, with a developmental plasticity that provides distinct immune populations to fill specific roles in tissue and vascular remodeling, immunoregulation, and bacterial and viral pathogen defense. Injuries to the developing, immature lung can have profoundly untoward and life-long consequences as a significant component of lung parenchymal and vascular development occurs during late pregnancy and the first few years of postnatal life. Many of these injurious stimuli including acute infection, hyperoxia, and corticosteroids are known to have significant effects on immune cell phenotype and function. Therefore, our data provide a detailed framework that enables a more complete understanding of how disruptions of immune cell phenotype may contribute to altered lung development, both through the induction of pathologic, pro-inflammatory signaling as well as the suppression of essential homeostatic functions. Further, a deep understanding of the diversity of immune cell functions during this important window of postnatal development, and how specific immune cell phenotypes are regulated could allow for the application of immunomodulatory therapies as a novel strategy to preserve or enhance lung development in infants and young children.C57BL/6 mice were obtained from Charles River Laboratories. For studies using E18.5, P1, and P7 murine lungs, pregnant dams were purchased, and pups aged prior to lung isolation. At E18.5, dam was asphyxiated with CO2 and pups extracted. At P1, P7, and P21 pups were euthanized with euthanasia solution (Vedco Inc). Genetic sex of mice at developmental stages E18.5 and P1 was determined by performing PCR amplification of the Y chromosome gene Sry. P7 and P21 mice were sexed through identification of a pigment spot on the scrotum of male mice . For all6 cells per well and stained with Fc block  for 30 min on ice. Cells were surface stained with the endothelial marker CD31 , epithelial marker Epcam , and immune marker CD45  for 30 min on ice. The live/dead dye, Sytox Blue (Invitrogen), was added to cells and incubated for 3 min prior to sorting into 384-well plates  prefilled with lysis buffer using the Sony LE-SH800 cell sorter (Sony Biotechnology Inc), a 100 μm sorting chip  and ultra-purity mode. Single color controls were used to perform fluorescence compensation and generate sorting gates. 384-well plates containing single cells were spun down, immediately placed on dry ice and stored at −80C.Lungs were plated at 1 × 10Complementary DNA from sorted cells was reverse transcribed and amplified using the Smart-Seq2 protocol on 384-well plates as previously described . ConcentSTAR aligner (HTSeq (snakemake was used (https://linnarssonlab.org/loompy/) and cells with less than 50,000 uniquely mapped reads or less than 400 genes per cell were discarded. Of the 4199 immune cells that were filtered, an additional 147 were removed as suspected doublets. Doublets were removed in two ways. First, clusters that expressed epithelial, endothelial or mesenchymal genes were excluded. Clusters showing joint expression of mutually exclusive cell type markers  with Cd45 were manually excluded as well. Counts for the remaining 4052 cells were normalized to counts per million reads. For t-distributed stochastic embedding (t-SNE) (scikit-learn (Leiden (C++/Python implementation) (https://github.com/iosonofabio/singlet) and for specific analyses, custom Python three scripts were used: the latter available at https://github.com/iosonofabio/lung_neonatal_immune/ . Pathway analysis on the differentially expressed genes from the five macrophage clusters was performed on each the top 100 most differentially expressed genes for each cluster in a one-vs-rest comparison and evaluated the gene set enrichment via Metascape  shown in the figure panel. Because this approach assumes the existence of a unique pseudotime potential, it cannot account for a possible nonzero curl in the vector field (e.g. a self-renewing embryonic population). To address this simplification, RNA velocity was performed (https://www.biorxiv.org/content/10.1101/820936v1). To evaluate the global patterns of genes demonstrating low or noisy expression, we calculated a simmetrized k-nearest neighbor similarity graph in PC space and smoothed the count matrix twice over the graph (i.e. reaching second-order neighbors). This approach is similar in spirit but conceptually simpler than MAGIC and other scRNA-Seq imputation algorithms (https://www.biorxiv.org/content/10.1101/820928v2), while manual merging and embedding was performed to compare our data with https://figshare.com/articles/Diverse_homeostatic_and_immunomodulatory_roles_of_immune_cells_in_the_developing_mouse_lung_revealed_at_single_cell_resolution/12043365.Sequencing reads were mapped against the mouse genome (GRCm38) using  aligner  and gener (HTSeq . FZ has was used . Gene ex (t-SNE) , 500 feait-learn . Unsuperntation) . Singletetascape . The moserformed  using scgorithms . It alsogorithms  using thgorithms  with thegorithms  and Changorithms . NorthstEmbryonic and post-natal mice were euthanized as described above. Female and male mice were randomly selected from the litter, and at least two litters were used to source the lung tissue for all validation studies. E18.5 lungs were immediately placed in 10% neutral buffered formalin following dissection. P1, P7, and P21 murine lungs were perfused as described above, and P7 and P21 lungs inflated with 2% low melting agarose (LMT) in 1xPBS and placed in 10% neutral buffered formalin. Following 20 hr incubation at 4C, fixed lungs were washed twice in 1xPBS and placed in 70% ethanol for paraffin-embedding. In situ validation of genes identified by single cell RNA-seq was performed using the RNAscope Multiplex Fluorescent v2 Assay kit (Advanced Cell Diagnostics) and according to the manufacturer’s protocol. Formalin-fixed paraffin-embedded (FFPE) lung sections (5 μm) were used within a day of sectioning for optimal results. Nuclei were counterstained with DAPI (Life Technology Corp.) and extracellular matrix proteins stained with hydrazide . Opal dyCd68+ cells located in the perivascular versus the perivascular space, all stitch-and-tile images containing large areas of parenchyma and at least on small vessel (20–30 μm) were included in the quantification. For all quantification analysis, a cell was deemed positive if it contained >1 puncta per cell for a specific gene. All studies were performed >2 times, with a total of n = 8 E18.5, n = 6 P1, n = 4 P7, and n = 4 P21mice used for the FISH studies.For select RNA scope experiments, the number and location of specific cells were quantified. Adjustments to the brightness and contrast of an image to increase the clarity of the signal was always applied to the entire image and never to separate areas of the image. For the determination of the percentage of P1 and P21 male and female murine lungs were isolated as described above. Cells were blocked for 30 min with CD16/CD32 (Tonbo Biosciences). For intracellular analyses, cells were stimulated with PMA , ionomycin , and GolgiStop (BD Biosciences) for 5 hr, and then surface stained with fluorochrome-conjugated antibodies for 30 min: CD3 , CD4 , and CD8a . Cells were then permeabilized with FoxP3 Fixation/Permeabilization Kit (BD Biosciences) as indicated by the manufacturer, and stained for TBET , GATA3 , FOXP3 , RORγt , IFNγ , IL-4 , IL-10 , and IL-17  for 30 min. Cells were read using an LSRII flow cytometer using FACSDiva software. Flow data was analyzed using FlowJo (Tree Star Inc). Flow cytometry analysis for this project was done on instruments in the Stanford Shared FACS Facility.To identify differentially expressed genes within cell populations, Kolmogorov Smirnov tests on the distributions of gene expression were performed on all genes, and the genes with the largest test statistic  were chosen as the top differentially expressed genes. For the flow cytometry studies of lung lymphocytes, 7–8 mice were in each group. Data are presented as mean ± SD. Differences between groups was determined by Student’s t-test with a P value of ≤ 0.05 considered statistically significant. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This work provides a comprehensive bioinformatic view of the immune cell landscape of the developing mouse lung at an important biological transition from in utero to the neonatal air-breathing period. The data are coupled with well-quantified spatial analysis of some of the most dynamic cell types identified in the analysis. They are also presented clearly in the context of analogous recent studies and provide a biological overview of immune cell evolution in mouse lung perinatal development.Decision letter after peer review: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 Tadatsugu Taniguchi as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Diverse homeostatic and immunomodulatory roles of immune cells in the developing mouse lung at single cell resolution" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Summary:The present study by Domingo-Gonzales et al. is a comprehensive bioinformatic approach, in which they investigate immune cells in the lung perinatally by characterizing changes in composition, localization, and function before birth and through the first three weeks of life. To this end, they combined a transcriptomic analysis (single cell RNA-seq) with fluorescent in situ hybridization. They clearly identified and characterized the localization and dynamics of five macrophage/monocyte populations with very distinct functions in murine lungs. Moreover, they provide strong data on the abundance and distribution of other important immune cells (e.g. dendritic cells) that are central in lung developmental processes, but also in lung diseases. Overall the manuscript is very well written addressing an highly important and disease-relevant topic that has not been elucidated to this extent yet.Essential revisions:These can all be done without additional experiments.+ cells should be scored as Dab2+, Plac8+, both, or neither. In Figure 3 the % macrophages around the vessels versus scattered should be given, the relative % of type 1 versus IV around the vessels and the % of co-labelling with Mki67. Moreover, clarification of the structures seen in Figure 3 would be helpful – are the macrophages arranged in intermittent circles around the diameter of the vessels? Are they forming lines along their length ? If possible, an antibody-based immunofluorescence approach to improve the cellular resolution of the image data would be desirable.1) The quality of the spatial information describing the location of the macrophages/monocytes needs to be improved. A detailed quantification of the in situ hybridisation images is required for clarifying the distribution of perivascular macrophage/monocytes in the tissues e.g. in Figure 2E, F all Cd682) The data in Figure 4A and the respective tables are very interesting, providing new insight in the functional role of the macrophages. Pathway analysis using these respective genes in the supplementary tables would give more information of the pathways and functional regulation of the individual macrophage types.3) Figure 3 and Figure 4 suggest that some macrophage population, e.g. Mac I, might regulate and promote angiogenesis in the lung. These findings coupled with Figure 4 showing genes related to angiogenesis let me query if anti-angiogenic factors might be produced by specific macrophages. That might be of interest in regulating alveolarization, but also in the pathogenesis of lung diseases (PAH or BPD).4) Did the authors relate macrophages or other immune cells, such as dendritic cells or B cells, to the regulation of stemness or lung progenitor cells? They are crucial in the process of alveolarization.5) We recommend including Fc receptor data in the main manuscript. They are important in disease development and often a therapeutic target.6) How do the present mouse data relate to human lung? At birth, murine lungs are in the saccular stage, whereas human lungs are already at the alveolar stage. What might be the main driver of macrophage differentiation: oxygen? mechanical forces? That could be further addressed in the Discussion.7) Discussion: It would be interesting to discuss more in detail how this precise regulation of immune cell differentiation and distribution in the lung during development could underlie the origins of lung disease. For example, there are data showing that BPD is related to activation of macrophages. Could asthma originate from adverse effects on dendritic and T cells during a critical phase of differentiation? That could be discussed.8) To make this manuscript more useful to the community further details need to be provided about the bioinformatic analysis:a) The authors should state exactly how many cells in total were profiled and how many cells passed QC. It will be useful as part of QC metric to see the number of genes per cell state identified.b) The link to scripts, gene count and metadata table provided in the manuscript was inactive.c) The authors should state what is actually displayed as gene expression for all figures – is this expression value on log scale? What does min, mod, high e.g. Figure 4A refer to quantitatively?d) Smart-seq2 is less likely to have doublet contamination but it is not clear if doublet detection and removal during analysis was attempted.e) Was batch correction methods e.g. Harmony implemented? – this would be helpful to assess if findings were impacted by batch effects.f) The observation of Mac I that encircles blood vessels prenatally and disappears after birth is interesting. However, the claims based on using inferred trajectory analysis of scRNA-seq data to suggest Mac I transition into Mac II and III needs to be more cautiously made as definitive lineage tracing or fate-mapping experiments were not done to validate the trajectory inference.g) What pseudotime trajectory method was used?9) DC nomenclature may be confusing as DC1, DC2 and DC3 are recognised subsets of conventional DCs. DCII described by authors are monocyte-derived DC and DCIII identity is unclear. This should be clarified in the text.10) Some integration of this dataset with other existing datasets from embryonic, perinatal and adult immune cells would permit harmonization of the cell states described by the various manuscripts and enhancing the value of this manuscript from a biological advancement and resource value of the manuscript. Essential revisions:These can all be done without additional experiments.+ cells should be scored as Dab2+, Plac8+, both, or neither. In Figure 3 the % macrophages around the vessels versus scattered should be given, the relative % of type 1 versus IV around the vessels and the % of co-labelling with Mki67. Moreover, clarification of the structures seen in Figure 3 would be helpful – are the macrophages arranged in intermittent circles around the diameter of the vessels? Are they forming lines along their length ? If possible, an antibody-based immunofluorescence approach to improve the cellular resolution of the image data would be desirable.1) The quality of the spatial information describing the location of the macrophages/monocytes needs to be improved. A detailed quantification of the in situ hybridisation images is required for clarifying the distribution of perivascular macrophage/monocytes in the tissues e.g. in Figure 2E, F all Cd68+ vs. Plac8+ cells at E18.5 and P7; (ii) the percentage of perivascular vs. parenchymal macrophages at E18.5, (iii) the % proliferating macrophages; and (iv) the % of Mac I vs. Mac IV found around vessels at E18.5. These data are now included in Figure 2E and Figure 3D, E, and G. Whether the macrophages are forming lines along the length of the vessels is an intriguing and important question that is difficult for us to definitively answer with our current images. Although some images that capture both the cross-section and a longitudinal cut of the vessel suggest that the macrophages may also be present along the length of the vessel, we cannot claim this to be true with confidence. We now acknowledge the limitation in the Discussion and state that antibody-based immunofluorescence followed by Z-stack imaging of thick tissue sections would be required to definitively address this important question.We very much appreciate this point from the reviewers. We have quantified the in situ hybridization images to provide the: (i) percent Dab22) The data in Figure 4A and the respective tables are very interesting, providing new insight in the functional role of the macrophages. Pathway analysis using these respective genes in the supplementary tables would give more information of the pathways and functional regulation of the individual macrophage types.We agree. We have performed pathway analysis using the DEGs from the five macrophage/monocyte populations and have included these data as Figure 4—figure supplement 1, and top pathways for each population reported in the Results. Given that many top DEGs have published functions that are not included in the pathway analysis, we also continue to highlight individual genes of interest with the relevant citations.3) Figure 3 and Figure 4 suggest that some macrophage population, e.g. Mac I, might regulate and promote angiogenesis in the lung. These findings coupled with Figure 4 showing genes related to angiogenesis let me query if anti-angiogenic factors might be produced by specific macrophages. That might be of interest in regulating alveolarization, but also in the pathogenesis of lung diseases (PAH or BPD).Serpine1, and Pf4 .We agree with the reviewer that the production of anti-angiogenic factors may be important not only for understanding alveolarization but also relevant to lung diseases such as PAH and BPD. We now also highlight some DEGs that are reported to have anti-angiogenic functions, including 4) Did the authors relate macrophages or other immune cells, such as dendritic cells or B cells, to the regulation of stemness or lung progenitor cells? They are crucial in the process of alveolarization.Wnt, Shh, Tgf-β, and BMP pathways. Of note, the Mac II and Mac IV clusters expressed secreted molecules that regulate these pathways. The Mac II cells express both Tgf-β1 and 2, Nestin 1 and 4 and Hhip, while the Mac IV cells expressed Fgf10, Bmp4, Wnt2 and Wnt5. These data are now detailed in the Results  and included as Figure 4—figure supplement 2.Although it was not our initial focus, we agree that this is a very important point given the role of lung progenitor cells in all stages of lung development. We also looked at the expression of secreted growth factors and other effectors of key pathways that regulate lung progenitor cells including the 5) We recommend including Fc receptor data in the main manuscript. They are important in disease development and often a therapeutic target.Thank you for this recommendation. The Fc receptor data is now included in the main manuscript as Figure 7.6) How do the present mouse data relate to human lung? At birth, murine lungs are in the saccular stage, whereas human lungs are already at the alveolar stage. What might be the main driver of macrophage differentiation: oxygen? mechanical forces? That could be further addressed in the Discussion.We thank the reviewers for this important point. An additional paragraph has been added to the Discussion (fifth paragraph) addressing factors that might be driving immune cell diversity in the developing lung and how these data from the mouse might be compared to similar data from human lungs to better understand whether the physiologic changes at birth, as opposed to differences in the lung immune cell niche at the different stages of development might be the predominant driver of diversity.7) Discussion: It would be interesting to discuss more in detail how this precise regulation of immune cell differentiation and distribution in the lung during development could underlie the origins of lung disease. For example, there are data showing that BPD is related to activation of macrophages. Could asthma originate from adverse effects on dendritic and T cells during a critical phase of differentiation? That could be discussed.We agree with this excellent point. We have addressed this by including an additional paragraph in the Discussion relating our findings to pediatric lung diseases such as bronchopulmonary dysplasia, pulmonary hypertension and asthma .8) To make this manuscript more useful to the community further details need to be provided about the bioinformatic analysis:a) The authors should state exactly how many cells in total were profiled and how many cells passed QC. It will be useful as part of QC metric to see the number of genes per cell state identified.Thank you for this suggestion. Please see the breakdown of the parameters and QC during data ingestion and filtering as outlined in Author response table 1. Please note that the dataset was sequenced in combination with other studies on the same biological samples. Therefore, the number of immune cells at the second to last step is much smaller than the total number of sequenced cells, which include other cell populations processed from the same plates.Author response table 1b) The link to scripts, gene count and metadata table provided in the manuscript was inactive.We would like to thank the reviewers for identifying this error. The link to the scripts was inactive due to a renaming issue which has been corrected. The metadata and gene counts are available on FigShare and on GEO as stated.c) The authors should state what is actually displayed as gene expression for all figures – is this expression value on log scale? What does min, mod, high e.g. Figure 4A refer to quantitatively?Thank you for this request, which we clarified in the revised manuscript. The color scheme of the gene expression tSNE embedding is logarithmic viridis color scales, with a pseudocount of 0.1 counts per million, and normalized to the highest expressing cell. The color scheme of the heatmaps plasma, logarithmic with a pseudocount of 0.1 counts per million, and normalized to the highest expressing population for each gene – i.e. every column will have one or more populations expressing “high”/yellow. The legend refers to 0%, 33%, 67%, and 100% expression  compared to the highest expressing population, i.e. describes equally spaced intervals of expression . These details have been added to the figure legends.d) Smart-seq2 is less likely to have doublet contamination but it is not clear if doublet detection and removal during analysis was attempted.Epcam, Cdh5, Col6a2) with Cd45 were manually excluded as well. 147 out of 4199 cells were removed, which is a slightly conservative filter compared to the expected doublet rate of around 1-2% in 384-well plates on our cell sorter. We have added this information to the Materials and methods .Doublets were removed in two ways. First, clusters that expressed epithelial, endothelial or mesenchymal genes were excluded. Clusters showing joint expression of mutually exclusive cell type markers (e.g. e) Was batch correction methods e.g. Harmony implemented? – this would be helpful to assess if findings were impacted by batch effects.We purposely did not run batch effect correction methods to avoid eliminating potentially relevant biological signals . In our experience, such batch correction methods are primarily needed when mixing different technologies. However, during the feature selection step for calculating neighborhood graphs towards clustering and embeddings, we identified over-dispersed features within each mouse and only kept features found in several mice. This enabled us to maintain a straightforward biological interpretation for the counts (reads per million mapped uniquely) while mitigating technical artifacts in the visualizations.f) The observation of Mac I that encircles blood vessels prenatally and disappears after birth is interesting. However, the claims based on using inferred trajectory analysis of scRNA-seq data to suggest Mac I transition into Mac II and III needs to be more cautiously made as definitive lineage tracing or fate-mapping experiments were not done to validate the trajectory inference.Thank you for this comment. We completely agree, and we have added an additional sentence to the Results stating that definitive genetic lineage tracing studies are necessary to confirm the transition of the Mac I cells to Mac II and III. We also added RNA velocity to Figure 4G, as an additional method supporting the pseudotime analysis.g) What pseudotime trajectory method was used?We apologize for omitting this detail. For the pseudotime analysis we used the default algorithm in Scanpy. This information is included in the Materials and methods. We also added RNA velocity data to the manuscript, and this was performed using scVelo .9) DC nomenclature may be confusing as DC1, DC2 and DC3 are recognised subsets of conventional DCs. DCII described by authors are monocyte-derived DC and DCIII identity is unclear. This should be clarified in the text.We appreciate this comment and agree that the suspected DC identity was not clear in the prior version. We have examined numerous additional genes associated with cDC1 and cDC2 and find that the prior population we called “DCII” are most consistent with cDC2 rather than monocyte-derived DC. We have included these additional t-SNE plots in Figure 8—figure supplement 1, and renamed our DC populations: cDC1, cDC2, and migratory (mig)-DC.10) Some integration of this dataset with other existing datasets from embryonic, perinatal and adult immune cells would permit harmonization of the cell states described by the various manuscripts and enhancing the value of this manuscript from a biological advancement and resource value of the manuscript.Thank you for this excellent suggestion. To this end, we have analyzed our data in concert with two previously published data sets: Schyns et al., 2019, and Tabula Muris, 2018. The results from these harmonized data analyses are included in the manuscript (subsection “Comparison of Mac I-V populations with adult murine lung data sets”), and the data included in a new figure .