Pdx1 expression. Males had a high diabetes incidence that was increased by early overnutrition, characterized by a progressive increase in insulin secretion as well as β cell death, indicated by histological analysis and increased circulating miR-375 levels. Females maintained normoglycemia throughout life. High-fat diet (HFD) increased diabetes incidence in males, whereas low-fat diet was completely protective. This protective effect was abolished in early overnutrition males transiently exposed to HFD in early life. Although Swiss Webster mice are not known to be diabetes-prone, the high diabetes incidence suggests an underlying genetic susceptibility that can be induced by overnutrition and increased dietary fat intake in early life. Thus, the nutritional environment in early life may impact long-term β cell function and increase diabetes risk, particularly in genetically susceptible individuals.Childhood obesity and early rapid growth increase the risk for type 2 diabetes. Such early overnutrition can be modeled in mice by reducing litter size. We investigated the effects of early overnutrition and increased dietary fat intake on β cell function in Swiss Webster mice. On a moderate-fat diet, early overnutrition accelerated weight gain and induced hyperinsulinemia in pups. Early overnutrition males exhibited higher β cell mass but reduced islet insulin content and  Maternal obesity, high energy diet, and hyperglycemia during pregnancy have all been implicated in increased offspring diabetes risk10. Although it is difficult to investigate postnatal risk factors in humans without the confounding effects of in utero environment differences, both human and animal studies have noted increased diabetes risk with increased infant adiposity, early rapid growth, accelerated catch-up growth following intrauterine growth restriction (IUGR), and childhood obesity13. These observations suggest that the early developmental environment can have long-term effects on glucose homeostasis and ultimately contribute to disease. However, the effects of early overnutrition alone on the development of β cells and glucose homeostasis are unclear.A major risk factor for obesity and type 2 diabetes is prolonged overnutrition, particularly with an obesogenic diet. In addition, evidence from epidemiological and animal studies suggests that an adverse 14. A similar pattern of postnatal expansion is observed in mice, with the highest rate of β cell mass expansion between birth and weaning, and peak β cell replication at 9 days of age15. It has been speculated that this early β cell mass expansion during infancy and childhood prepares the individual to cope with the metabolic demands of puberty and adulthood14. Consequently, insults that impair postnatal β cell mass expansion or functional development may reduce the capacity to meet later metabolic demands, leading to diabetes.In humans, β cell mass rapidly increases after birth, with the greatest rate of expansion occurring before 2–5 years of age and the highest rate of β cell replication up to 2 months of age22. However, the effects of early overnutrition and dietary fat in early life on β cell development have not been thoroughly examined. In the Swiss Webster mouse, reduced litter size has previously been shown to increase susceptibility to HFD-induced obesity and insulin resistance20. We hypothesized that overnutrition and increased dietary fat intake during the early postnatal period in Swiss Webster mice would adversely affect β cell development and long-term function.We investigated the effects of early overnutrition on β cells by employing a mouse model of early overnutrition, induced by reducing litter size, which reduces competition for maternal milk. Numerous studies have demonstrated that in rodents, this model results in accelerated body weight gain and adiposity in pups, with some studies showing alterations in glucose homeostasis, although phenotype varies by species and strainIn both male and female pups Fig. , early oBoth early overnutrition males and females maintained a small but significant increase in body weight, relative to their control counterparts, post-weaning and into adulthood Fig. . HoweverIns1), insulin 2 (Ins2), or insulin receptor (Insr) between early overnutrition and control males . Genes that have been shown to be important in β cell lipotoxicity, endoplasmic reticulum (ER) stress, and oxidative stress 27 did not differ between early overnutrition and control islets , suggesting that the loss of insulin staining is not due to dramatic dedifferentiation of β cells.Islets from non-diabetic early overnutrition and non-diabetic control mice generally exhibited similar and normal morphology Fig. , althougion Fig.  in 3 of ion Fig.  or undetion Fig. . Insulinion Fig.  and therion Fig. . We obseTo investigate whether dietary fat content influenced the effects of early overnutrition on β cell function, we compared early overnutrition and control males whose mothers were fed either LFD or HFD throughout lactation with offspring weaned onto the same diet (LFD and HFD groups). In addition, to investigate the effects of increased dietary fat that was limited to the preweaning period, a subset of mice weaned from dams on HFD were switched LFD for the remainder of their life (HL group). Early overnutrition male pups, from dams consuming either LFD or HFD during lactation, exhibited significantly increased body weight relative to their control male counterparts, beginning at P10 through weaning at P21 Fig. . At P50,Pdx1 expression and β cell dysfunction, in the absence of insulin resistance even in HFD-fed mice, that progresses to β cell death. In contrast, female mice maintained normoglycemia throughout life. Since litter size reduction in other rodent strains does not typically induce such profound β cell death, Swiss Webster mice likely have a genetic susceptibility to β cell dysfunction and diabetes. Our findings suggest that the nutritional environment during early life may impact long-term β cell function and increase the risk of diabetes, particularly in genetically susceptible individuals.We have demonstrated that overnutrition and increased dietary fat in early life can promote the development of diabetes. In Swiss Webster male mice, this diabetes is characterized by reduced 22, we observed accelerated weight gain in both early overnutrition males and females with reduced litter size, as well as hyperinsulinemia and hyperleptinemia, relative to their control counterparts. We also observed elevated β cell mass relative to controls in males, even when normalized to body weight. Slightly heavier body weight, higher adiposity, as well as disproportionally higher β cell mass persisted into adulthood in early overnutrition males. Despite higher β cell mass and more islets in adult early overnutrition compared to control males, total pancreas insulin content did not differ, likely due to the reduced insulin content per islet. Although isolated islets from early overnutrition males responded normally to a short-term exposure to high glucose and KCl, the low insulin reserves per islet are likely insufficient to respond to prolonged stimulants, such as chronic HFD, making them more susceptible to β cell failure.As previous studies have shown33. The reason for this sexual dimorphism is unclear; however, estrogens are known to have protective effects against the development of diabetes by increasing β cell insulin content34, increasing insulin sensitivity36, and protecting against β cell apoptosis37. Young, non-diabetic early overnutrition males exhibited increased plasma triglyceride levels, suggesting dysregulated lipid homeostasis. However, glucose tolerance and insulin tolerance did not differ significantly from control males. This suggests that the β cell dysfunction in these mice is not secondary to obesity-induced insulin resistance, as often occurs in mouse models in response to chronic HFD exposure.We observed a high rate of overt diabetes in Swiss Webster males, with higher incidence in early overnutrition compared to control males. Diabetes was not observed in females, despite experiencing a similar acceleration in body growth in early life. A previous study also observed diabetes in male, but not female, Swiss Webster mice, in a colony selectively bred for diabetes susceptibilityPdx1 expression that was observed in young early overnutrition males and the later development of low islet insulin content, suggests a primary β cell dysfunction induced by early overnutrition. This occurred despite the rapid β cell mass expansion observed in early overnutrition pups. Interestingly, the decrease in Pdx1 expression occurred prior to changes in insulin expression. Pdx1 is a key transcription factor required for normal pancreas development40. In humans, mutations in the PDX1 gene can lead to maturity onset diabetes of the young41, and reduced Pdx1 levels in mice have been shown to increase β cell susceptibility to apoptosis and HFD-induced ER stress42. Previous studies found that IUGR results in progressive reduction in Pdx1 levels due to epigenetic silencing, and the increased diabetes incidence in this model could be prevented by treatment in early life with exendin-4 that increases Pdx144, suggesting that reduced Pdx1 during development can induce diabetes. Thus, reduced Pdx1 levels in early overnutrition mice could be an early contributor to β cell dysfunction and render them more susceptible to metabolic stressors. The marginally reduced Neurod1 expression could also contribute to this dysfunction, since both Pdx1 and NeuroD1 act in concert to regulate insulin expression45. Predominantly cytoplasmic localization of Pdx1, as observed in some early overnutrition mice, has previously been observed in rats exposed to high glucose and lipids46, and after 8 weeks of consuming 58% HFD in C57BL/6J mice47. Reduced Pdx1 expression and immunoreactivity were observed at an age when early overnutrition mice did not exhibit differential glucose levels relative to control. Since elevated plasma triglycerides were observed in young early overnutrition males, it is possible that elevated lipid, but not glucose, levels may have contributed to the observed cytoplasmic Pdx1 localization. Although we did not detect increased expression of genes implicated in β cell lipotoxicity in young, non-diabetic early overnutrition mice, it may be that lipotoxic effects occurred at a later age.The reduced 29. The increased cleaved caspase-3 in β cells of diabetic mice suggests that cell death is at least in part due to apoptosis. In addition, the presence of fibrosis in and around islets is likely a consequence of this cell loss. Since mice developed diabetes at different ages, to better understand the progression of diabetes we tracked individual mice relative to diabetes onset. This revealed a progressive increase in insulin and proinsulin levels leading up to diabetes onset, suggestive of worsening β cell function. A progressive decrease in insulin and proinsulin occurred following diabetes onset and was therefore associated with hyperglycemia and increased β cell death. The susceptibility to overnutrition-induced β cell death in Swiss Webster mice makes this strain a useful model to study progression to diabetes, relative to the more commonly used C57BL/6 mice that exhibit relatively low rates of measurable β cell death on HFD despite developing obesity, hyperglycemia, and insulin resistance49. Indeed, a low dose of the β cell toxin streptozotocin is often combined with HFD in C57BL/6 mice to better mimic the β cell death that occurs in humans with type 2 diabetes50.The rise in plasma miR-375 levels observed in Swiss Webster mice prior to diabetes onset is indicative of β cell death, since dying β cells release miR-375 into the circulation51. The HFD we employed had a higher proportion of saturated  and monounsaturated (mainly oleic acid) fatty acids compared to the LFD. Previous studies have observed that β cells are particularly sensitive to prolonged elevations in saturated fatty acids, with chronic palmitate exposure resulting in impaired β cell function including decreased Pdx1 expression and increased ER stress54. Our studies suggest that increased exposure to saturated and/or monounsaturated fatty acids specifically during the early postnatal period may have long-term detrimental effects on β cell function.To investigate the role of dietary fat intake on diabetes susceptibility and its interaction with early overnutrition, control and early overnutrition offspring were raised either on HFD or LFD. HFD-feeding induced diabetes in both control and early overnutrition mice, whereas LFD was completely protective. In fact, early overnutrition mice maintained on LFD throughout life had improved glucose tolerance despite having a heavier body weight than control LFD mice, suggesting that early overnutrition could even be beneficial as long as dietary fat is kept low. HFD exposure only during the preweaning period prevented these beneficial effects of early overnutrition, suggesting that the preweaning period is a critical time of susceptibility to early overnutrition and dietary fat-induced β cell dysfunction. Whereas total fat content of milk remains high regardless of the mother’s level of dietary fat intake, the composition of fatty acids in milk varies to reflect that in the diet20. However, these mice were of different origin from a different vendor , so may not have the same genetic susceptibility or microbiome composition as the mice in the present study. Alternatively, since HFD in the previous study began at 6 weeks of age instead of shortly after birth, moderate to high dietary fat intake during the early postnatal period, as in the present study, is required to see the detrimental effects on β cell function that lead ultimately to β cell death in the absence of insulin resistance. This is supported by the increased diabetes incidence observed in early overnutrition offspring exposed to HFD only during the preweaning period.In a previous study, reduced litter size in Swiss Webster males did not result in overt diabetes and β cell death, and instead mice were more prone to HFD-induced obesity and insulin resistancePdx1 expression eventually leads to β cell death. Our findings may offer insight into potential detrimental effects that could occur in humans, particularly those with a genetic susceptibility for primary β cell dysfunction, resulting in adverse consequences from overnutrition in infancy and/or childhood. A better understanding of the underlying mechanisms mediating the development of β cell dysfunction in the Swiss Webster mouse, and the time frame in which they occur, may help us identify at-risk children that show a similar susceptibility, allowing for early interventions aimed at preventing long-term adverse outcomes.Swiss Webster mice appear to have a genetic susceptibility that predisposes them to diabetes, induced by metabolic challenges such as overnutrition and increased dietary fat in early life, during the period of rapid β cell mass expansion. Despite an early increase in β cell mass induced by early overnutrition, the β cell dysfunction reflected in the reduced islet insulin content and reduced ad libitum access to food and water. Pregnant (16–17d) Swiss Webster mice (Model #SW-F) were purchased from Taconic  and maintained on a moderate fat  standard chow diet . Two days post-delivery, litters were culled to 10 pups for the control group. For the early overnutrition group, litters were initially culled to 6 pups at P2, and then further culled to 3 pups at P5. This stepwise reduction in litter size reduces the chances of insufficient maternal milk production. Groups were assigned such that pup body weights did not differ between control and early overnutrition groups at P2. Pups were weaned onto the same diet as the mother (5LJ5). For the HFD study, pregnant Swiss Webster mice purchased from Taconic were placed on 10 kcal% low-fat diet . Two days post-delivery, dams were either maintained on LFD or switched to 45 kcal% lard-based HFD , with litters culled as above. All offspring were weaned at P21, with mice from the LFD and HFD groups maintained on the same diet as their mother throughout life. To examine long-term effects of HFD that was limited to the preweaning period, a subset of mice weaned from HFD-fed dams was switched to LFD at P21 (HL group).Mice were housed under a 12-hr light, 12-hr dark cycle (lights on at 0700 h), with constant temperature and humidity and Since only male Swiss Webster mice developed diabetes, detailed metabolic assessments in adults were performed only in male mice. Adult tissue collection was performed after a 4 hr fast (0900 h to 1300 h) and all tissues were weighed immediately upon dissection. Pup tissue and trunk blood collection was performed in the AM in non-fasted male mice after isoflurane anesthesia. Tissues for immunohistochemistry and histology were post-fixed in 4% paraformaldehyde followed by paraffin-embedding and sectioning at 5 µm by Wax-It Histology Services Inc. . For total pancreas insulin content, whole pancreas was incubated in −20 °C 0.18 M HCl/70% ethanol, homogenized, then incubated again at −20 °C overnight. Following centrifugation, the aqueous solution was neutralized 1:2 with 1 M Tris, pH 7.5. All procedures with animals were approved by the University of British Columbia Animal Care Committee and carried out in accordance with the Canadian Council of Animal Care guidelines.29. Food intake (averaged over 7 days) was measured in individually housed mice (aged P60 to 80) by weighing food hoppers daily. For glucose tolerance tests, 6 hr fasted mice were given 2 g/kg glucose either by intraperitoneal injection or oral gavage, and blood collected prior to glucose administration and at indicated times after injection into heparin-coated capillary tubes. For insulin tolerance tests, 4 hr fasted mice were injected intraperitoneally with 0.75 U/kg insulin , with glucose measures conducted at 0, 10, 20, 30, 60, 90, and 120 min post-injection. For assessment of circulating miR-375 levels, ~100 µL of blood was collected from the saphenous vein into EDTA-coated capillary tubes. This was performed once per week, beginning at P32, in early overnutrition males maintained on 5LJ5 diet. Plasma was stored at −80 °C prior to RNA isolation.Blood glucose values were determined with a OneTouch Ultra 2 glucometer  measured from saphenous vein blood. Fasting (4 hr) glucose and body weight were monitored weekly beginning at P30-35. Diabetes onset was defined as the first day of two consecutive fasting glucose measures ≥ 20 mM 55 using Type XI collagenase  injected into the pancreatic duct. Following digestion and filtration, islets were picked with a pipette in three rounds to remove any remaining acinar tissue. For islet RNA isolation, ~100 islets per mouse were placed in a 1.5 mL tube and spun down at 200 x g for 1 min. Excess buffer was removed and then islets were homogenized in 600 µL RLT Buffer  using a 1 mL syringe with a 21 gauge 1” needle. Tubes were then frozen on dry ice and stored at −80 °C for RNA isolation.Islets were isolated as previously described38. Briefly, 80 size-matched islets per mouse were perifused with a HEPES-buffered Krebs-Ringer buffer under temperature and CO2-controlled conditions. Perifusate was collected every 5 min under basal glucose (3 mM), high glucose (20 mM), and 30 mM KCl conditions, and insulin secretion analyzed by radioimmunoassay. For islet insulin content, 5 islets per mouse were acid-ethanol extracted as described above and stored at −20 °C.Islet perifusions were performed as previously described2O2 and 10 min PBS wash prior to the blocking step. On day 2, following washes in PBS, slides were incubated in biotinylated anti-guinea pig IgG  diluted in Dako Antibody Diluent, for 1 hr at room temperature in a humid chamber. Slides were then washed in PBS, incubated for 30 min with R.T.U. VECTASTAIN ABC Reagent (Vector Laboratories), washed for 10 min in PBS, then incubated in 3,3′-diaminobenzidine (DAB) Peroxidase (HRP) Substrate solution (Vector Laboratories). Slides were counterstained with hematoxylin and eosin (H&E), dehydrated through a graded ethanol and xylene series, and then coverslipped with Vectamount Permanent Mounting Medium (Vector Laboratories).All primary antibodies and dilutions used for immunohistochemistry are listed in Supplementary Table β cell mass was analyzed in three pancreas sections per mouse, at least 200 µm apart, on insulin-DAB stained sections for P14 pups and immunofluorescent insulin-labeled sections in adults. Image threshold was set such that weak insulin staining within an islet was considered positive. Islet size was quantified as the insulin-positive area per islet. H&E staining and Masson’s trichrome staining on adult pancreas sections were performed by Wax-it Histology Services Inc. .Hprt1 as the reference gene, or TaqMan Fast Universal PCR Master Mix for Ins1 and Ins2 (Applied Biosystems) with mouse β-actin  as a reference gene, in a StepOnePlus real-time PCR system . Transcript levels were quantified by the 2−ΔΔCT method. All primer sequences are listed in Supplementary Table Total RNA was isolated from islets using the Qiagen RNeasy Mini Kit as per the manufacturer’s instructions, and reverse transcribed with iScript cDNA Synthesis Kit . Quantitative RT-PCR was performed using Ssofast EvaGreen Supermix (Bio-Rad) with For miR-375 measurement in the circulation, extracellular RNA was isolated from plasma samples in the presence of an MS2 carrier RNA  using the Qiagen miRNeasy Kit. Reverse transcription was performed with the Universal cDNA Synthesis Kit II . Quantitative RT-PCR was performed using the ExILENT SYBR Green Master Mix (Exiqon) with LNA-based miRNA primers (Exiqon). Relative values were calculated with the ΔCT method using miR-16* as a reference miRNA.Insulin was measured in plasma, pancreas, and islets using the Mouse Ultrasensitive Insulin ELISA . Insulin from perifusions was measured with the Rat Insulin RIA . Proinsulin , leptin , and triglycerides  were measured in plasma.Statistical analyses were performed using GraphPad Prism 7, with significance defined as p < 0.05. For the HFD study, all statistical comparisons were done within dietary condition. Area under the curve (AUC) measurements were conducted with the baseline set to a value of 0. Statistical tests are detailed in figure legends. T-tests were used for normally distributed data . Bonferroni correction was used to correct for multiple comparisons, where applicable. Nonparametric tests (Mann-Whitney) were used for any data that did not pass the Shapiro-Wilk test for normality. Continuous data are represented by line graphs with median ± interquartile (IQR) range, and categorical data are represented by box and whisker plots. Where a panel contains a group with n = 3, floating bars represent the range of values and the line is at the median. For box and whisker plots and floating bar graphs, all individual data points are shown. For line graphs that show no indication of sample size such as a corresponding AUC graph, sample size is provided in the figure legend.Supplementary information


Wnt5a functions as a spatial cue to coordinate cell polarity and cytoskeletal oscillation. These processes diminish tissue rigidity and help cells to overcome the energy barrier to intercalation. YAP/TAZ and PIEZO1 serve as downstream effectors of Wnt5a-mediated actomyosin polarity and cytosolic calcium transients that orient and drive mesenchymal cell intercalations. These findings advance our understanding of how developmental pathways regulate biophysical properties and forces to shape a solid organ primordium.Multiple vertebrate embryonic structures such as organ primordia are composed of confluent cells. Although mechanisms that shape tissue sheets are increasingly understood, those which shape a volume of cells remain obscure. Here we show that 3D mesenchymal cell intercalations are essential to shape the mandibular arch of the mouse embryo. Using a genetically encoded vinculin tension sensor that we knock-in to the mouse genome, we show that cortical force oscillations promote these intercalations. Genetic loss- and gain-of-function approaches show that  Morphogenesis of tissue sheets is well studied, but mechanisms that shape bulk tissues are unclear. Here, the authors show that mesenchymal cells intercalate in 3D to shape the mouse branchial arch, with cortical forces driving intercalations in a Wnt5a-, Yap/Taz- and Piezo1-dependent manner. In particular, exchange of cell neighbours is central to tissue shape change in two dimensions (2D) and involves a limited number of transient multicellular configurations including tetrads (T1 exchange) and rosettes. Actomyosin contractions generate forces that promote cell neighbour exchanges and are oriented, in part, by physical properties of tissue that may be anisotropic in nature9. Some of these principles have been extended to curved epithelial sheets by combining empirical and theoretical approaches10. However, it remains unclear whether similar mechanisms apply to mesenchymal tissues, in part because mesenchymal tissues have been regarded as less confluent due to the presence of abundant extracellular matrix in some contexts and to potentially less adherent cell–cell junctions11. Changes in the viscoelastic properties of tissue are also associated with, and may partly drive, morphogenetic movements15, although the relationship between cellular and tissue scale properties remains incompletely understood and may be context-dependent.Morphogenesis refers to the process of shaping tissue during development, the reproducible nature of which is essential for appropriate pattern formation and function. Most of the recognised principles of morphogenesis concern mechanisms that shape sheets of embryonic tissue16 and mesodermal cell intercalations through junctional remodelling, though the latter takes place in a sheet-like manner18. Another example is elongation of the rod-like skeletal anlage in the vertebrate limb that is attributable to a highly structured columnar arrangement of chondrocytes embedded within abundant extracellular matrix and to oriented rearrangement of nascent daughter cells20. In contrast to these examples, very little is known about how more-or-less isotropic volumes of confluent cell organise to generate morphogenetic movements. For example, although the directional nature of mesodermal cell movements from the lateral plate to the limb bud has been demonstrated23, the cellular basis of those movements, and of volumetric morphogenesis in general, remain largely uncharacterised.Multiple organ primordia such as the branchial arches and limb buds are composed of an internal bulk layer of mesenchyme. In models of multilayered vertebrate tissues such as the frog gastrula, mechanisms of morphogenesis include amoeboid endodermal cell movements24 and cranial mesodermal25 origins of branchial arch mesenchyme are well recognised, mechanisms by which the structure grows outward and acquires shape are less clear. Loss-of-function approaches that were intended to define the roles of various signalling pathways identified cellular processes that are relevant to branchial arch morphogenesis, such as cell survival27, cell proliferation28, and cell migration29. Those studies generated important insights, but were not intended to explain how the branchial arches acquire shape.Early branchial arches are composed of a core volume of mesenchyme that is surrounded by a single cell layer epithelium. Although the neural crest30. The mandibular portion of the first branchial arch generates multiple facial structures including the lower jaw, and some syndromes are associated with structurally common features of malformation such as a short (front to back) and broad (side to side) mandible. One of these, Robinow syndrome, is caused by mutations in components of the noncanonical WNT pathway including WNT5A  and ROR2 (recessive form) which encode a ligand and a downstream receptor tyrosine kinase, respectively33. Wnt5a34 and Ror236 mutant mice that phenocopy many of the features of the human syndrome exhibit a short and broad mandibular arch. Facial anomalies also result from mutations of Dachsous1 and FAT4 in autosomal recessive Van Maldergem and Hennekam syndromes38. These genes encode a receptor-ligand cadherin pair that regulates planar cell polarity (PCP) and are upstream of yes-associated protein (YAP), a transcriptional effector of the Hippo pathway39. Autosomal recessive mutations of piezo type mechanosensitive ion channel component 1 (PIEZO1) that encodes a pore forming subunit of a mechanically activated ion channel also cause facial dysmorphism with hypoplastic features that overlap with those above41. The cellular and biophysical basis of these malformations are not clear, although it has been shown that mutant WNT5A may exhibit neomorphic properties that affect cell polarity and migration in a chick model of human Robinow syndrome42.Craniofacial anomalies are common birth defectsWnt5a-dependent, Yap/Taz-dependent, and Piezo1-dependent manner, implying these regulators spatially fine tune physical cell behaviours.Here we study the mandibular arch as a model of two distinct modes of 3D morphogenesis. We show that cell division and tissue-scale physical properties are important for growth but do not sufficiently explain how the arch primordium acquires a narrow mid-portion and a bulbous distal portion. Our data support a model in which 3D mesenchymal cell intercalations narrow and elongate the mid-portion. Relatively high amplitude cortical force oscillations and cell polarity promote cell intercalations in a The first branchial arch buds at the ventral aspect of the midbrain-hindbrain boundary in the mouse embryo. As shown by optical projection tomography (OPT), the mandibular portion of the first branchial arch remodels from a rod-like structure at the 19 somite stage (~E9.0) to form a narrow central waist and a distal bulbous region between somite stages 21–28 )ted Fig. . Cell dited Fig. , implyinPhysical tissue properties profoundly influence morphogenesis. Using atomic force microscopy (AFM) to measure elasticity Fig. , we perf44 but with the addition of empirical physical properties. Based on prior work, we initially hypothesised that disproportionately increased stiffness of the middle epithelium might resist hoop stress due to growth of the mesenchyme to yield a growth process reminiscent of directed dilation46. However, simulated growth and deformation based on the spatial variations of cell cycle time, elasticity and viscosity predicted an inappropriately short and wide arch , and protrusive activity  with relatively few neighbours in the mid-portion of the mandibular arch , varied along the proximodistal axis of the mandibular arch with the highest values observed in the middle region  exhibited a greater slope for cells in the middle compared to those in the distal region, indicating the former are more directionally consistent over time  revealed that cells in the middle region converged centripetally relative to one another as tissue growed longitudinally Fig. , 13. At 54. F-actin and phospho (p)-myosin light chain (pMLC) were biased parallel to the short, transverse axis among epithelial and mesenchymal cells in the middle waist, but not proximal and distal regions, of the arch -based vinculin tension sensor (VinTS), for which fluorescence lifetime in nanoseconds (ns) is proportional to force in piconewtons (pN)58, into the mouse Rosa locus. We generated two control knock-in strains that should exhibit maximal (donor only VinTFP—no FRET), and minimal  fluorescence lifetime, respectively  to intact mouse embryos and quantified the proportion of X-rhod-1 fluorescence intensity to total cell area (marked by VinTS) over time. X-rhod-1 intensity fluctuated to a greater extent among middle region mesenchymal cells than in distal cells  for both persistence time and angle from the mean compared to cells in WT embryos  locus using Sox2:Cre to drive ubiquitous expression and diminish the Wnt5a gradient in the mandibular arch  or in neural crest, respectively, both of which contribute to branchial arch mesenchyme. Although deletion of all four Yap/Taz alleles caused lethality prior to branchial arch development, removal of any three Yap and Taz alleles with either Cre grossly phenocopied Wnt5a null mutants with respect to mandibular shape , indicating its expression or stability is regulated by a Wnt5a-Yap/Taz pathway. Interestingly, nuclear abundance of YAP was diminished in the absence of Piezo1 , mTmG73 (Jackson Laboratory: Gt(ROSA)26Sortm4(ACTB-tdTomato-EGFP)Luo/J)), CAG::myr-Venus74, full length vinculin tension sensor (VinTS) – see below, vinculin TFP control (VinTFP), vinculin tailless control (VinTL), Wnt5a+/−34, Piezo1+/−75. All mouse lines were outbred to CD1. All animal experiments were performed in accordance with protocols approved by the Animal Care Committee Hospital for Sick Children.Analysis was performed using the following mouse strains: Rosa26 locus, constructs were cloned using the following plasmids: vinculin tension sensor (VinTS) and vinculin tailless (VinTL)58 , and Ai27 . To generate VinTS targeting vector, Mlu1 sites were introduced upstream of the start codon and downstream of the stop codon in VinTS using PCR. To generate VinTL, an Mlu1 site was introduced upstream of the start codon and a stop codon followed by an Mlu1 site downstream of the Venus sequence. To generate vinculin teal fluorescent protein (VinTFP—FRET donor only), an Mlu1 site was introduced upstream of the start codon and a stop codon followed by an Mlu1 site downstream of the mTFP1 sequence. PCR products were digested with Mlu1 and purified using Qiaex gel purification kit (Qiagen). Reverse primers for generating VinTL and VinTFP were identical, so the constructs were distinguished based on size in an agarose gel following Mlu1 digestion and gel purification and bands were cut out accordingly. The inserted construct in the Ai27 Rosa26 targeting vector was removed with Mlu1 and replaced with VinTS, VinTL, or VinTFP. Sequences for all targeting vectors were confirmed through DNA sequencing . The final targeting vectors were constructed as follows: CAG enhancer—FRT—loxP—stop codons—3× SV40 poly(A)—loxP—VinTS/VinTL/VinTFP—WPRE—bGH poly(A)—attB—PGK promoter—FRT—Neo—PGK poly(A)—attP.To target the VinTS, and 2 positive clones from each of VinTL and VinTFP were aggregated with CD1 morula to obtain chimeric mice following standard procedures. Chimeric mice were outbred to CD1 mice to obtain an F1 generation through germline transmission. All procedures involving animals were performed in compliance with the Animals for Research Act of Ontario and the Guidelines of the Canadian Council on Animal Care. The Toronto Centre for Phenogenomics (TCP) Animal Care Committee and Hospital for Sick Children Animal Care Committee reviewed and approved all procedures conducted on animals at TCP and at the Hospital for Sick Children, respectively.Generation of ES cell lines and generation of chimeras were performed by the Transgenic Core at the Toronto Centre for Phenogenomics. Briefly, linearised constructs were electroporated into G4 ES cells and G418-resistent clones were screened by PCR. 4 positive clones from Z/Wnt5a line was generated by targeting a Cre-activated Wnt5a expression vector upstream of the endogenous Ubiquitin-b (Ubb) locus using a similar strategy as described for generating the Z/Norrin expression cassette77 . Expression of the Wnt5a transgene is activated only after Cre-mediated recombination, which removes a LacZ-STOP cassette upstream of the Wnt5a open reading frame. Sox2:Cre78 was employed to drive ubiquitous expression.The 79. Three-dimensional (3D) data sets were reconstructed from auto-fluorescence projection images acquired over a 25 min scan period at an isotropic voxel size of 4.5 µm. The 3D surface renderings of the OPT data were generated by MATLAB software, version R2011b (Mathworks).E9.5 mouse embryos were harvested and fixed in 4% paraformaldehyde overnight at 4 °C. OPT was performed using a system that was custom-built43.Pregnant females were injected first with CIdU intraperitoneally at E9.25 and then with IdU after 2.5 h. Thirty minutes following the second injection, embryos were dissected in cold PBS and fixed with 4% PFA overnight at 4 °C. Whole-mount immunofluorescence against CIdU and IdU was performedMouse embryos were incubated in 50% rat serum in DMEM on a 35 mm dish in which 2% agarose was poured around the perimeter. The mandibular arch was immobilised to the agarose with pulled glass needles pinned through the flank adjacent to mandibular arch. The arch was examined using an AFM  mounted on an inverted microscope (Nikon Eclipse-Ti). AFM indentation tests were performed using a spherical tip (radius: 15 µm) at distinct locations categorised as proximal, middle and distal mandibular arch. Spherical tips were made by assembling a borosilicate glass microsphere onto a tipless AFM cantilever using epoxy glue. The cantilever spring constant was calibrated before every experiment by measuring power spectral density of thermal noise fluctuation of the unloaded cantilever.80 to indent epithelium up to 1 µm in depth to avoid influence from the mesenchyme. The Hertz model for a spherical tip was used to calculate the elastic modulus of the epithelium from the small indentation data. To determine mesenchymal elastic modulus and the overall tissue viscosity, we applied large indentation (depth: 7–10 µm) at indentation rates of 5, 10 and 15 µm/s. The Kelvin–Voigt model was used to fit the large indentation force–displacement data. To extract overall tissue viscosity, the range of data beyond 1.5 µm indentation depth was used, and the elastic range was neglected. The elastic modulus value of mesenchyme was then determined by using experimental force–displacement data and finite element simulation.To determine the elastic modulus of epithelium, a trigger force of 300 pN was consistently applied. For both small and large indentation measurements in each region , 15 locations were measured and repeated in triplicate, amounting to 45 measurements at each region. Because the epithelial thickness is approximately 10 µm, we followed the empirical 10% ruled and the loading force F isE1, v1 are the elastic modulus and Poisson’s ratio of the indenter; and E2, v2 are the elastic modulus and Poisson’s ratio of epithelium. The spherical tip was made of borosilicate glass (elastic modulus and Poisson’s ratio: 63 Gpa and 0.2). The Poisson’s ratio for epithelium was set to 0.481. Using the experimental data from small indentation as well as the above calculation, epithelial elastic modulus E2 was quantified.For small indentation, the Hertz model for a spherical tip was used to fit the experimental force–displacement data and determine epithelial elastic modulus values. The relationship between the indentation depth a is the contact radius, the left-hand side results in total force applied to epithelium and mesenchyme. For a bilayer structure, the contact radius tio t, indentation depth d, indenter radius R. Comparing the elastic modulus of epithelium Eepithelium and the overall modulus , we found the ratio Eepithelium/Emesenchyme) was small (~2). Thus, according to the Hertz model82, v is indentation rate. It is evident that For large indentation, the Kelvin–Voigt model was used to fit experimental force–displacement data. Stress–strain relation in the Kelvin–Voigt model isR2 value (correlation coefficient between the two curves) was greater than the threshold value (0.99).A snapshot of the meshed 2D axisymmetric finite element model with a bilayer structure (epithelium and mesenchyme) under frictionless loading is shown in Supplementary Fig. ves Fig. . MesenchUx = Uy = Uz = 0, Rotation: URx = URy = URz = 0) at the extended proximal end to ensure the boundary condition did not influence growth of the proximal region  image stack was first concatenated and reconstructed using ImageJ, and a 3D model was exported in the STL format. We used MeshLab to isolate the mandibular arch from the embryo. Quadric edge collapse decimation algorithm was employed to reduce the total number of 3D triangular mesh faces to the target number of faces (~5000 without distorting the geometry). The model was then imported into Solidworks in which the 3D mandibular arch model was segmented into two layers (epithelium and mesenchyme) with 24 regions based on the geometric locations of cell cycle time measurements. The finite element model was fixed in all six degrees of freedom  with instantaneous elasticity and two-pair Prony relaxation. We also performed comparative simulations with different viscoelastic values within the range of our AFM measurement and predicted similar tissue shape change with no significant difference.44, and strain values were incorporated in the finite element model for tissue shape prediction. Cell density was considered to be the number of cells per volume. The experimentally measured 3D cell cycle time values were converted to a spatial pattern of strainmentclass2pt{minimH2B-GFP emitted light with relatively high intensity. Each image was processed as following: (a) using a Gaussian deconvolution with a standard deviation roughly the size of one cell, the images were smoothened. (b) Local maxima were sought in square boxes, again, roughly the size of one cell. Each local maximum was marked as a cell centre. This process was repeated for each image in ML-PD plane. The layers of marked cells were then compared against one other. If a point was marked to be the centre of a cell within seven or more consecutive layers in the stack, the middle layer was marked as the RC position of that cell. This process gave us a 3D representation of roughly 6000 cell nuclei in the tissue. Voronoi tessellation was performed using these cell nuclei as nodes for the tessellation algorithm.Using light sheet microscopy, we obtained approximately 1000 2-dimensional images (ML-PD plane) of the mandibular arch, each image about 0.4 microns apart from the next along the perpendicular RC axis. Cell nuclei marked by 223. Time-lapse images were acquired on a Quorum WaveFX-X1 spinning disk confocal system (Quorum Technologies Inc.) at 20× magnification. Images were processed with Volocity software or ImageJ/Fiji. Representative images are shown from at least three independent experiments for each condition, and unless otherwise indicated, from at least three independent cohorts. No statistical method was used to predetermine sample size. Experiments were not randomised. Investigators were not blinded to allocation during experiments and outcome assessment.Live image acquisition was performed by submerging embryos were submerged in 50% rat serum in DMEM without phenol red (Invitrogen) in a 25 mm imaging chamber. Cheese cloth was used to immobilise the embryo and position the mandibular arch directly against the coverglass. Embryos were imaged in a humidified chamber at 37 °C in 5% CO2. Images were acquired using a 20×/1.0 objective with dual-side illumination, and a z-interval of 0.479 µm that was automatically calcuated based on the numerical aperature (1.0) of the objective.. All experiments were imaged in multi-view mode with 3 evenly-spaced views spanning approximately 90 degrees . Images were acquired for 3–4 h with 10 min intervals. Fluorescent beads  were used as fiducial markers for 3D reconstruction and to aid in drift-correction for cell tracking. Multi-view processing was performed with Zen 2014 SP1 software to merge the 3 separate views and generate a single 3-dimensional image. Further analysis and cell tracking were performed using Arivis Vision4D software (Arivis).Three-dimensional (3D) time-lapse microscopy was performed on a Zeiss Lightsheet Z.1 microscope. Embryos were suspended in a solution of DMEM without phenol red containing 12.5% rat serum and 1% low-melt agarose (Invitrogen) in a glass capillary tube. Once the agarose had solidified, the capillary was submerged into an imaging chamber containing DMEM without phenol red, and the agarose plug was partially extruded from the capillary until the portion containing the embryo was completely outside of the capillary. The temperature of the imaging chamber was maintained at 37 °C with 5% CO3D timelapse datasets of cell membranes were processed with the ImageJ macro TissueCellSegmentMovie  to generate membrane segments prior to analysis with Imaris software (Bitplane). Surface objects were created using Imaris and this data was used for 3D cell neighbour analysis. Cell neighbours were manually counted by identfying the target cell and counting all cells, which share an interface within the plane, as well as adjacent cells in the planes above and below. Analysis was performed on three separate areas in both middle and distal regions of the mandibular arch over two independent experiments for each condition.z-stacks at 5 or 10 min intervals. The large drift between images over time and the resolution of the images made it difficult to follow more than 130 cells throughout the entire movie. Only cells that could reliably be identified and followed by eye in at least 33 frames were considered for the random walk analysis.Cell tracks (cell positions tracked over time) were calculated manually. Cell nuclei were followed between image z-stacks for each time point were aligned, and fusion stacks were created to generate z-plane images based on the mean voxel intensity at each voxel. The fused stacks were then imported into Arivis Vision 4D software , and automated tracking was used to generate cell tracks. Tracks were validated manually, and validity annotations were entered into an Excel spreadsheet. Tracks were defined to be valid at a given timepoint if the segment determining the track’s location at that timepoint was centred in the middle of a nucleus. Tracks were defined to be suitable for use in further analysis if time between their first valid timepoint and final valid timepoint was 150 min or greater. Whether a track tracked an epithelial or mesenchymal cell was determined observing the nuclear location of the track relative to the tissue boundary as well as the morphology of the nucleus .For each movie, the To correct for drift, the frame by frame displacement of red fluorescent beads that were embedded adjacent to the embryo within the agarose plug used in the light sheet microscope was calculated. These displacements were imported into MATLAB for initial track concatenation and drift correction. Track and segment information and validity were acquired on Arivis. The concatenation function returned drift corrected tracks that were filtered to only include valid tracks. Tracks could then be plotted, and end to end displacements calculated and plotted.83 for our analysis. In an OU-process, the trajectory of a particle is determined by a relation describing its change in velocity over time. The change of velocity in the model is proportional to its velocity in the past (persistent) plus a randomly generated term . This approach has been used to analyse the properties of cell migrations under many contexts including tumour growth52 and wound repair51.We described cell motions from tracking experiments by stochastic processes. In particular, we adopted the Ornstein-Uhlenbeck (OU) processv is the cell velocity, t is time, D is the diffusion coefficient, vector w describes a Wiener process, and τ is the time scale often referred to as the persistent time. The persistent time, which may be understood as the length of time a given velocity “remembers” itself, describes the time of the velocity auto-correlation functionn is the space dimension of the tracks. The mean squared displacement (MSD) is given byThe OU-process is defined by the Langevin equationτ and the diffusivity coefficient D. Consequently, we were able to simulate cell trajectories by Eq.  was was7) was by Eq.  to furthn = 179 number of cells tracked with a time interval of t = 5 min. over a 3 h period. Following Wu et al.52, we simulated cell trajectories by applying a first-order Euler’s scheme to Eq.  and S is the cell speed with D = S2τ. We applied principle component analysis to observed cell trajectories prior to our analysis so that we could simulate cell displacement in each direction separately. In other words, we diagonalised the correlation coefficient matrix so that off-diagonal components were zero.The wild type dataset that we used for the numerical simulation contained An important characteristic of a random walk model is its mean squared distance (MSD). For an arbitrary trajectory, the MSD as a function of time follows a power-law, with power of 0 representing a truly random trajectory, and power of 2 representing movement in a straight line with no randomness. On a log–log plot, these two extremes translate into lines with slope 0, or a horizontal line, for the random trajectory, and a line of slope 2 for the walk in a straight line. A persistent random walk, however, is characterised by slope 1, the short black line in Supplementary Fig. A rectangular grid of points was superimposed on the first frame of a rostrocaudal-proximodistal plane of a time-lapse light sheet movie of the mandibular arch. The points were followed in subsequent frames by calculating the correlation function of a box around each point in the initial time frame to all the boxes of the same size in the vicinity of the original box in the next frame. The vector that connected the location of the box in the first frame to the location of the neighbouring box with the highest correlation with the original box in the second frame was the displacement vector. The points were moved by this displacement vector in each time frame, and this process was repeated for all 31 time frames.Embryonic day (E) 9.0–9.5 mouse embryos were fixed overnight in 4% paraformaldehyde in PBS followed by three washes in PBS. Embryos were permeabilised in 0.1% Triton X-100 in PBS for 20 min and blocked in 5% normal donkey serum (in 0.05% Triton X-100 in PBS) for 1 h. Embryos were incubated in primary antibody overnight incubation at 4 °C. Embryos were washed in 0.05% Triton X-100 in PBS , and then incubated in secondary antibody (1:1000) for 1 h at room temperature. Embryos were washed , followed by a final wash overnight at 4 °C, and stored in PBS. Images were acquired using a Quorum spinning Disk confocal microscope (Zeiss) at 10×, 20×, or 40× magnification, and image analysis was performed using Volocity software (Perkin Elmer) and ImageJ.; N-cadherin ; Desmoglein ; CIdU ; Idu ; Calpain2 (E-10) ; VANGL2 . All secondary antibodies were purchased from Jackson Immunoresearch and used at 1:1,000 dilutions. Rhodamine phalloidin ; Alexa Fluor 633 phalloidin .Phospho-myosin light chain 2 (Thr18/Ser19) ; PIEZO1 84. Wildtype and mutant littermate embryos were processed identically in the same assay for comparison. The Wnt5a riboprobe was gift from A. P. McMahon.Whole mount in situ hybridisation was performed after fixation in 4% paraformaldehyde followed by dehdration through a methanol series. Embryos were bleached then underwent digestion in proteinase K, hybridisation with probe in formamide, SSC, SDS and heparin, treatment with anti-Digoxygenin-AP, and colourimetric reaction85. Cell interfaces were manually identified, and average fluorescence intensities were calculated for all interfaces and grouped into 15° angular bins, with 0°–15° bin representing interfaces that are parallel with the AP axis (PD interfaces) and 75°–90° bin representing interfaces that are parallel with the PD axis (AP interfaces). Average fluorescence intensity values for each bin were normalised to average fluorescence intensity of PD interfaces (0°–15° angular bin). Error bars indicate standard error of the mean and p values were calculated using Student’s t-test.Single confocal slices taken 2 µm above (for epithelium) and below (for mesenchyme) the basal surface of epithelial cells stained with phospho-myosin light chain 2 (Thr18/Ser19) (pMLC), rhodamine–phalloidin or Alexa Fluor 633 phalloidin were analysed using SIESTA software23. Epithelial tetrad formation and resolution angles were measured by first assigning a proximodistal reference axis taken from a low 10× confocal magnification view of the embryo flank. Tetrads were identified manually, frame by frame. The angle between the long axis of the ellipse outlined by each tetrad and the reference axis was documented at the beginning of a given movie and upon resolution.Metaphase-to-telophase transition angles were measured using a line between the centres of daughter nuclei with respect to a proximodistal reference axis2. Data were acquired over 15 frames at 2 min intervals for 20 min.Fluorescence lifetime microscopy (FLIM) was performed on a Nikon A1R Si laser scanning confocal microscope equipped with PicoHarp 300 TCSPC module and a 440 nm pulsed diode laser (Picoquant). Data were acquired with a 40×/1.25 water immersion objective with a pixel dwell time of 12.1 µs/pixel, 512 × 512 resolution, and a repetition rate of 20 MHz. Fluorescence emission of mTFP1 was collected through a 482/35 bandpass filter. Embryos were prepared in 50/50 rat serum/DMEM for live timelapse microscopy and imaged in a humidified chamber at 37 °C in 5% COn-exponential reconvolution model in SymphoTime software (Picoquant) with model parameters n = 2 for VinTS and VinTL and n = 1 for VinTFP. Fitting was performed to achieve a chi-squared of χ2 = 1.000 ± 0.100.Fluorescence lifetime of mTFP1 was determined using R0 is the Förster radius of donor-acceptor pair, R the donor to acceptor separation distance, TDA the fluorescence lifetime of donor in the presence of acceptor, and TD the fluorescence lifetime of donor only.FRET efficiency (E) was calculated based on the molecular separation between donor and acceptor and the corresponding fluorescence lifetime using the following equation:58.Force was calculated from FRET efficiency based on the calibration results reported by Grashoff et al.86. Cells were cultured in DMEM  supplemented with 2 mM GlutaMAX (Invitrogen), 0.15 mM monothioglycerol, 0.1 mM MEM Non-essential amino acids, 1 mM sodium pyruvate, 50 μg/mL Penicillin/Streptomycin, 1000 U/mL Leukaemia Inhibitory Factor (Chemicon), 15% foetal bovine serum  on a feeder layer of mouse embryonic fibroblasts. Prior to transfection, ES cells were removed from the feeder layer and cultured in gelatin-coated plates for 2 passages. Cells were transfected with Cre recombinase using Lipofectamine 2000, according to the manufacturer’s protocol. YFP-positive cells were sorted by flow cytometry, which was performed in the SickKids Flow Cytometry Facility. Positive cells were recovered and grown on gelatin.Embryonic stem (ES) cell clones of VinTS, VinTL, VinTFP knock-ins were generated by the Transgenic Core at The Centre for Phenogenomics and cultured using standard protocols87.To differentiate cells into embryoid bodies (EBs), cells were diluted in differentiation media  at a concentration of 1000 cells per 10 μL and plated in drops of 20 μL on the lid of a 10 cm plate. Cells were grown in this hanging drop configuration for 6 days to allow for embryoid body differentiation. EBs were harvested and fluorescence lifetime was measured using the LIFA system by Lambert Instruments on an Olympus IX81 equipped with an Li2CAM iCCD with GenIII GaAs intensifier. Spontaneous differentiation of EBs to contracting myocardial cells continued in 24-well plates2, 5% CO2 at 37 oC prior to FLIM experiments that were conducted using the same conditions as above.The effect of inhibitors Y27632 (Sigma #Y0503) and Cytochalasin D on fluorescence lifetime of VinTS were conducted as follows: Embryos were incubated with 20 µM Y27632 or 20 µg/mL Cytochalasin D for 15 min in 50% rat serum with DMEM in roller culture with 40% OCalcium indicators Fluo-8 AM (Abcam #142773) or X-rhod-1 AM (Invitrogen #X14210) at 10 μM were added in 2 mL of 50% rat serum medium and incubated for 30 min at 37 °C. Embryos was permiabilised with 0.1% Pluronic F-127 (Invitrogen #P3000MP) in culture medium for 20 min before X-rhod-1 staining. The culture medium was removed and washed (two times) and replaced with 2 mL culture medium. Live cell calcium imaging was performed on a Quorum Spinning Disk confocal microscope (Zeiss) equipped with a 40× water objective lens or a Nikon A1 confocal microscope (Nikon) equipped with a 20× dry objective lens. Calcium indicators were excited with an argon laser line (488 nm), and emissions were recorded in the green channel (500–560 nm) for Fluo-8 AM (acetoxyomethyl) and red channel (600–700 nm) for X-rhod-1 AM. Images were acquired from a single confocal plane every 2 or 5 min for up to 20 min; image and data acquisition was performed using Volocity (Perkin Elmer) or Image J.2+ concentration data series with zero time-lag. In this study, we defined the percentage of X-rhod-1 AM staining area for each cell in relation to Ca2+ concentration. Assume the first time series (force vs. time) are called x , and the second series are y (Ca2+ concentration vs. time). First we calculated the mean and standard deviation of x and y. Let us refer to the mean of x, µx and standard deviation of x as σx. µy and σy for y. N is the number of data points. We subtracted the means from each series and multiplied data points corresponding to the same time, and summed the results. The formula follows:We calculated the correlation between VinTS lifetime and Cahttps://www.mathworks.com/help/signal/ref/xcorr.html).We analysed the amplitude of normalised correlation by using MATLAB xcorr (This will give us a number between −1 and 1. For values approaching 1, the two series are strongly correlated. Approaching 0, they are not correlated. Approaching −1 implies they are anti-correlated.Further information on experimental design is available in the Supplementary informationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Supplementary Movie 11Supplementary Movie 12Supplementary Movie 13Supplementary Movie 14Supplementary Movie 15Supplementary Movie 16Supplementary Movie 17Supplementary Movie 18Supplementary Movie 19Supplementary Movie 20Supplementary Movie 21Supplementary Movie 22Supplementary Movie 23Supplementary Movie 24Supplementary Movie 25Supplementary Movie 26Supplementary Movie 27Supplementary Movie 28Supplementary Movie 29Supplementary Movie 30Supplementary Movie 31Supplementary Movie 32Reporting SummarySource data


In this paper, we report an unexpected, Microwave “photoelectric effect”, when MW irradiates on the semiconductor materials, leading to generate electric current and electron-holes pairs (e−cb + h+vb), on the semiconductor materials and on the MW catalyst. Further, we show that the action mechanism of Microwave “photoelectric effect” made water adsorbing on the surface of Microwave catalyst transform into hydroxyl radical (∙OH). Thus, this study has revealed the principle of generation Microwave “photoelectric effect” under MW irradiation, and the mechanism of MW catalytic oxidation degradation of organic in the wastewater and the mechanism of MW reduction method for preparation of nano-particle metal supported catalysts. Our findings challenge the classic view of MW irradiation only as heating method, which cannot excite to produce electric current and electron-holes pairs (e−cb + h+vb). Our findings will open new field to use MW technology for MW catalytic oxidation degradation of organics in the wastewater, and for MW reduction method of metal supported catalysts preparation.Generally, photon of Microwave (MW) electromagnetic waves have long been thought to be lower energy, which could not excite metals or semiconductor materials to generate electric current and electron-holes pairs (e The application of microwave technology in the wastewater treatment is a new development16. The microwave plays the role of inducement, enhancement, and assistance degradation in the microwave induced catalytic degradation (MICD)17,the microwave-enhanced catalytic degradation (MECD)20, and the microwave assisted catalytic degradation method (MACD)24, Zhang et al.24. reported under MW irradiation to form powerful oxidizing •OH (and other ROS), which can non-selectively attack the organic contaminants, and possible principle on the degradation of parathion in A-TiO2/AC/MW and R-TiO2/AC/MW is that TiO2 can be excited by hot-spots to generate electron-hole pairs, and then, to form hydroxyl radicals (•OH) in aqueous solution. But to date it is still not clear that the nature of MW interact with MW catalyst for the wastewater treatment. El-Shall et al.25 has reported Microwave synthesis of supported Au and Pd nanoparticle catalysts. However, it is not well understand the mechanism of this method26. Herein, we found that microwave “photoelectric effect” exists when MW irradiates on the semiconductors and microwave “photoelectric effect” made water on the surface of Microwave catalyst to transform into hydroxyl radical (∙OH).Our finding revealed the mechanism of microwave catalytic oxidation reaction for degradation of organics and reduction of metal ion into metal on the oxide support under MW irradiation.Microwave (MW) refers to the electromagnetic waves of 300 MHz-300 GHz, wavelength in meters (excluding 1 m) to 1 millimeter. Microwave technology has already been applied widely in many fields2,CuO,CeO2 and Mn2O3 as acceptor of MW photon, our observation is that the electrical conductivity of semiconductor CuO,CeO2 and Mn2O3 increased greatly and electric current produced under MW irradiation, whereas there are no phenomena without MW irradiation. The conductivity of semiconductor materials CuO,CeO2 and Mn2O3 could change with changing MW irradiation power (PMW), and the produced electric current could change with changing PMW  to prepare TiO2/AC, CuO/AC,CeO2/AC and Mn2O3/AC catalysts, when MW irradiates on these catalysts, we observe the same phenomenon. We found that the conductivity of TiO2/AC,CuO/AC,CeO2/AC and Mn2O3/AC catalysts suspension in the aqueous solution could change with changing PMW . Our experiments found that Microwave “photoelectric effect” exists under MW irradiation.MW irradiation on semiconductor experiments were conducted using semiconductor TiOMW Table -2. AlthoMW Table . HoweverMW Table –5, and t2 to generate electric current and electron-holes pairs. Our experiments observation in fact conform that Microwave “photoelectric effect” phenomenon exists when MW irradiation on semiconductor.However, MW refers to the electromagnetic waves of 300 MHz–300 GHz, so the energy of MW photon is much lower than that of UV-light wave photon. Generally, MW photon could not excited metal or semiconductor such as TiO2,CuO,CeO2,Mn2O3 and SiC could be excited to generate electric current and electron-holes pairs (e−cb + h+vb), whereas Al2O3 could not be excited to generate electric current and electron-holes pairs. So generation of Microwave “photoelectric effect” also depends on the dielectric properties of materials under MW irradiation. Generally, semiconductor materials have excellent dielectric properties and result in strong microwave absorption, for example, the relatively high loss tangent of CeO2,TiO2, and ZnO result in strong microwave adsorption26, low dielectric loss materials like Al2O3 almost do not interact with MW and do not absorb MW. Only when the energy of MW photon is higher than electric work function of materials, can the microwave “photoelectric effect” be produced.MW irradiation will lead to greatly reduce electric work function of semiconductor or MW catalysts.MW irradiation decreasing the electric work function of semiconductor catalysts depends on the properties of materials and its dielectric properties.The above results also indicated that semiconductor TiO2/AC,CuO/AC and Mn2O3/AC (φ). In spite of the lower energy of MW photons, the influence of MW irradiation will lead to reduce electric work function of MW catalysts. MW irradiation decreasing the electric work function of MW catalysts depends on the properties of materials and its dielectric properties. MW irradiation can greatly decreased the electric work function of MW catalysts, only when hν > φ, could the microwave “photoelectric effect” be produced.To reveal the nature/mechanism of Microwave “photoelectric effect” phenomenon, we give the explanation and elucidation for the principle of generation Microwave “photoelectric effect” under MW irradiation. The microwave is an electromagnetic wave and the corresponding irradiation is an electromagnetic wave irradiation. Based on the principle of quantum theory, photons of MW electromagnetic wave should/must interact on the microwave catalyst definitely under irradiation and thus there should be/existing the microwave “photoelectric effect”. However, photons of MW electromagnetic wave could not directly excite catalyst to produce electrons because generally the energy of MW photons (hν) is much lower than electric work function of materials such as MW catalyst TiO27. Moreover, the quantum energy of MW could help to decrease the activation energy by making the vibration of molecules28. On the other hand, MW catalytic activity was mainly attributed to the microwave activation owing to dipolar polarization, conduction mechanism and interfacial polarization. Dipolar polarization contributing to most of MW heat energy results from intermolecular inertia. When the dipole is subjected to a high-frequency alternating electric field of the MW, rotation of the dipole can’t completely follow the rate of change of direction of the electric field. This leads to a time delay, causing abundant energy to be spent29.Based on the presented experiments, we consider that MW radiation can decrease the electronic work function of semiconductor from these views: On the one hand, owing to MW irradiation, MW energy could be absorbed by semiconductor rapidly depending on the dissipation factor of the semiconductor (loss tangent). Dissipation factor is the ratio of relative loss factor (ε″) to the permittivity (ε′). Permittivity is a relative measure of the MW energy density in the material and the relative loss factor is responsible for the internal loss mechanisms, such as the quantity of MW energy that is consumed in the semiconductor as heat energy. Therefore, a lossy material with a high relative loss factor can easily absorb MW energy30,as long as its frequency is higher than the limit frequency , and MW is not an exception. Interestingly, although the frequency (ν = 20 KHz–300 MHz) of ultrasonic wave is much smaller than the frequency (ν = 300 GHz–300 MHz) of microwave, ultrasonic wave can also excite to produce electron31. This indicates the microwave electromagnetic wave with a higher hν value is easier to excite to produce electron than ultrasonic wave.Theoretically, any kind of electromagnetic wave is able to induce photoelectric effect32.Consequently, semiconductor can be excited to generate electron-hole pairs under MW irradiation.Therefore, once catalyst with high relative loss factor and dielectric permittivity was subjected to MW irradiation, the electron hole pairs could be produced. This was probably attributed to the mechanisms of dipolar polarization, conduction mechanism and interfacial polarization. Dipolar polarization contributing to majority of MW heat energy results from intermolecular inertia. When the dipole is subjected to a high-frequency alternating electric field of the MW, rotation of the dipole can’t completely follow the rate of change of direction of the electric field, leading to a time delay and causing a substantial quantity of energy to be spent. MW can produce a lot of energy, leading to electrons in the high-energy state. Because electrons is instability in the high-energy state, which is in an active state and will produce transition. MW can produce a lot of energy, and high MW energy can directly effect on lowering the electric work function of semiconductor. Similarly, thermal energy or ultrasonic energy can also excite electrons. Accordingly, the electronic work function of the MW catalyst can be decreased by abundant thermal energy. Moreover, the quantum energy of MW can induce the vibration of molecules, which will help to decrease the activation energy of the reaction2/AC,CuO/AC,CeO2/AC and Mn2O3/AC could be heated to a higher temperature due to the effect of microwave thermal, producing a lot of thermal energy also lead to lower the electric work function of semiconductor MW catalysts. These two aspects effect above made the electric work function of semiconductor or MW catalyst to greatly decrease. So when the energy of photons are greater than electric work function of semiconductor or MW catalyst (hν > φ), MW catalyst could be excited directly to produce electrons under MW irradiation, and therefore generating electric current and electron-hole pairs (e−cb-h+vb) on the semiconductor or MW catalyst as like UV-light radiate on the metal to exist photoelectric effect. MW irradiation on semiconductor or the MW catalyst generating electric current and electron-hole pairs (e−cb-h+vb)could be called as MW “photoelectric effect”.So the principle of generation microwave “photoelectric effect” could be revealed and elucidated as follow. On the one hand, the electric polarization effect of microwave electromagnetic field under MW irradiation lead to the dipole ordering result in conductivity rising, and then lowering the electric work function of semiconductor MW catalyst; MW irradiation could exist coupling effect of external electric field with internal electric field to lead to lowering the electric work function of semiconductor MW catalyst; On the other hand, MW energy partly transforms into materials interior energy level, and semiconductor or the whole MW catalyst such as TiO33 studied the influence of electromagnetic fields on conductivity of aqueous NaCl solution at microwave frequency, and their results indicated that microwave energies were partly transformed to intermolecular energies of cluster, which made conductivity of aqueous NaCl solution interrelated with intensity of microwave. Their reported work33 also provides evidence on the change of conductivity under MW irradiation.Interestingly, Huang2O3/AC can be excited to produce electron-holes pairs, furthermore, the holes of generation electron-holes pairs on the semiconductor catalyst could react with H2O and OH to generate ∙OH for degradation of organics in wastewater.Based on the principle of our finding Microwave “photoelectric effect”, MW irradiation can directly excite semiconductor or MW catalyst to produce electron-holes pairs, the strong oxidizability of holes of generation electron-holes pairs under MW irradiation could be used for the oxidation reaction. Under MW irradiation, semiconductor catalyst such as Mn−cb + h+vb) on the semiconductor catalyst by Microwave “photoelectric effect”, we use the principle of generation Microwave “photoelectric effect” to conduct the experiments for Microwave catalytic oxidation degradation of PNP in waste water using Mn2O3/AC as catalyst. In present of MW catalyst Mn2O3/AC, without adding oxidation agent such as H2O2, MW irradiation could catalytic oxidation degradation of organics in the wastewater and mineralize to CO2 and H2O  because of the lower energy of photons. MW irradiation can greatly lower the electric work function of MW catalysts. So when the energy of photons are greater than electric work function of MW catalyst (hν > φ), MW catalyst could be excited directly to produce electron-hole pairs (e−cb-h+vb) under MW irradiation. The holes with strong oxidation ability react with the H2O molecules or OH− on the surface of Mn2O3/AC particles, and electrons react with the O2 molecules dissolved in aqueous solution, respectively, producing the hydroxyl radicals (∙OH) and super oxygen radical anions (∙O2−). At last, unstable ∙O2− also becomes ∙OH through series of chemical reactions. The generation process for ∙OH was shown in Fig. 2, H2O and other inorganic substances finally (Eq. (10)). Among them, Eqs (1–3) are the main process, and Eqs (4–9) are secondary, because the dissolved oxygen in reaction solution are limited. Obviously, as elucidation above, our finding MW “photoelectric effect” revealed clearly that mechanism of MW catalytic oxidation degradation of organics. Our previously work35 reported the MW catalytic oxidation degradation method (MCOD) for organic wastewater, and it is suggestion that hydroxyl radical (∙OH) generated under MW irradiation is the main oxidation agent. These results provide the evidence that hydroxyl radical (∙OH) generated by MW “photoelectric effect”. Quan et al.36 also observed that generation of hydroxyl radical in aqueous solution by microwave energy using activated carbon as catalyst also provides the evidence. Thus our finding MW “photoelectric effect” revealed the mechanism of hydroxyl radical (∙OH) generated in the MW catalytic oxidation degradation method.According to the above results, ∙OH generated in the reaction process under MW irradiation is probably the main oxidant for the oxidation degradation of 4-NP. What is the mechanism for the generation of ∙OH under MW irradiation? Generally photons of MW electromagnetic wave could not directly excite catalyst to produce electron-hole pairs , on the semiconductor materials and on the MW catalyst. We found that microwave “photoelectric effect” exists when MW irradiates on the semiconductors and microwave “photoelectric effect” made water on the surface of Microwave catalyst to transform into hydroxyl radical (∙OH). This study has revealed the principle of generation Microwave “photoelectric effect” under MW irradiation, and the mechanism of MW reduction method for preparation of nano-particle metal supported catalysts. Our findings challenge the classic view of MW irradiation only as heating method, which cannot excite to produce electric current and electron-holes pairs (e−cb + h+vb). Our findings will open new field to use MW technology for MW catalytic oxidation degradation of organic in the wastewater, and for MW reduction method of metal supported catalysts preparation.In summary, we report an unexpected, Microwave “photoelectric effect”, when MW irradiates on the semiconductor materials, leading to generate electric current and electron-holes pairs  nanoparticle were prepared with a coprecipitation method.10 g of Cu(NO3)2·3H2O, Ce(NO3)3·6H2O, 50%Mn(NO3)2 Solution, Ti(SO4)2, Al(NO3)3·9H2O was dissolved in ethanol, respectively, and then 1 ml polyethylene glycol was added dropwise to the above mixture under continuous vigorous stirring. The resulting solution was adjusted the pH to 10 by the addition of NaOH and then irradiated under microwave (the working power of 214W) in a pulsated way for 10 min. After filtration and being washed with deionized water and ethanol,the solid obtained was dried at 80 °C for 12 hours and calcination at 500 °C for 3 hours.MeOx/AC were prepared with an impregnation method. Activated carbon  was pretreated with the boiling deionized water, and then dried at 80 °C for 12 h. The desired amount of AC was impregnated with aqueous solution of Mn(NO)2, Ce(NO3)3, Ti(SO4)2, Cu(NO3)2, respectively, at room temperature for 12 h followed by drying at 80 °C for 12 h. The MeOx/AC samples were obtained after calcining at 250 °C for 2 h. The characterization results of MeOx/AC were listed in Figs MeOx powder was placed in a silica crucible and pressed with silica wool, and then, the silica crucible or a carborundum plate, connected with two wire at the bottom both sides, was irradiated under various MW power levels for a certain time. The wire was winded with aluminum foil to eliminating microwave interference. The resistance and current of the MeOx was measured by a Fluke AVOmeter.Microwave single-mode experiments was carried out in a HY-SG1500 microwave tubular furnace (Hunan Hua’e Microwave Technology Co. Ltd), as shown in Fig. x/AC or AC was added to 100 mL of deionized water, and then, the suspension was irradiated under various MW power levels for a certain time under continuous magnetic stirring. The resistance and current of the suspension was measured by a Fluke AVO meter which connected with two wires from the bottom of 3 mouth flask. The wire was also winded with aluminum foil to eliminating microwave interference.Microwave multi-mode experiments was carried out in a COOLPEX-E Microwave apparatus (Shanghai Yiyao technology) of 2450 MHz frequency, as shown in Fig. 2O3/AC as MW catalyst was conducted in detail seeing supporting information section II.The experiments for Microwave catalytic oxidation degradation of PNP in waste water using MnSupplementary Information