HDM) has shown that Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) species are abundant bacteria within the HDM microbiome. Therefore, our aim was to investigate whether HDM are carriers of bacterial antigens leading to IgE sensitization in patients suffering from atopic dermatitis.IgE reactivity to antigens from Gram‐positive and Gram‐negative bacteria is common in patients suffering from respiratory and skin manifestations of allergy, but the routes and mechanisms of sensitization are not fully understood. The analysis of the genome, transcriptome and microbiome of house dust mites (AD (n = 179) were analysed for IgE reactivity to a comprehensive panel of microarrayed HDM allergen molecules and to S. aureus and E. coli by IgE immunoblotting. Antibodies specific for S. aureus and E. coli antigens were tested for reactivity to nitrocellulose‐blotted extract from purified HDM bodies, and the IgE‐reactive antigens were detected by IgE immunoblot inhibition experiments. IgE antibodies directed to bacterial antigens in HDM were quantified by IgE ImmunoCAP™ inhibition experiments.Plasma samples from patients with AD sensitized to HDM than in AD patients without HDM sensitization. S. aureus and E. coli antigens were detected in immune‐blotted HDM extract, and the presence of IgE‐reactive antigens in HDM was demonstrated by qualitative and quantitative IgE inhibition experiments.IgE reactivity to bacterial antigens was significantly more frequent in patients with HDM) may serve as carriers of bacteria responsible for the induction of IgE sensitization to microbial antigens.House dust mites ( ADatopic dermatitisDer pDermatophagoides pteronyssinusHDMhouse dust mitesIgEimmunoglobulin EIgGimmunoglobulin GISACimmuno solid‐phase allergen chipISUimmuno solid‐phase allergen chip standardized unitskU/Lkilo units per litreA/LkUkilo units antigen per litreMeDALLmechanisms for the development of allergiesODoptical densitySCORADscoring atopic dermatitis1Staphylococcus aureus (S. aureus) were found to be associated with severe forms of asthmaS. aureus.Already in 1932, Cooke suggested that allergic sensitization to bacteria may play a role in asthma.S. aureus fibronectin‐binding proteinEscherichia coli (E. coli) antigens and other bacteria of the gut has been detected in about one‐third of patients with AD.S. aureus typically induces Th1 or Th17 rather than Th2 immune responses, and nonpathogenic E. coli is associated with immune tolerance rather than with allergic sensitization.The occurrence of IgE to bacterial antigens was also observed in atopic dermatitis (AD) patients as early as 1981.Staphylococcus aureus (S. aureus) is present in the skin of patients with AD who frequently suffer from S. aureus superinfections, and it has therefore been suggested that sensitization occurs via the skin.E. coli and other bacteria  which have been reported as sources for IgE‐reactive antigensDermatophagoides farinae comprises a variety of Gram‐positive and Gram‐negative bacteria, among them S. aureus, E. coli and Enterobacteria.S. aureus and E. coli, in a group of patients with AD from Sweden where sensitization to HDM is expected to be less common, as a result of the cold climate.S. aureus and E. coli.Here, we hypothesized that HDM may have another hitherto unknown role in allergic sensitization. In fact, we investigated whether HDM are carriers of microbial antigens that could cause a Th2 type adaptive immune response to bacterial antigens. To study this hypothesis, several lines of investigations were pursued. First, we studied the occurrence of IgE sensitization to HDM and bacterial antigens from 22.1Dermatophagoides pteronyssinus, purchased from Allergon AB . Purified mite bodies were purchased to minimize the effect of faecal contamination in our analysis. Protein extracts were prepared by adding 5 mL of Laemmli sodium dodecyl sulphate (SDS) sample buffer  β‐mercaptoethanol; 10% glycerol)g, and supernatants were stored at −20°C until use.An allergen extract from HDM was prepared using purified bodies of S. aureus strain ATCC25923, and E. coli strain ATCC25922, were obtained as live cultures from the Leibniz Institute German collection of microorganisms and cell cultures . Isolates were grown overnight at 37°C in 500 ml Brain Heart Infusion Medium and then centrifuged at 3220 × g to harvest bacterial cells. Pellets were washed twice in phosphate‐buffered saline (PBS) and inactivated by 3 cycles (one minute each) of alternate heating (95°C) and freezing (liquid nitrogen). Inactivated bacterial cells were resuspended in 5 mL PBS and sonicated using an Ultra Turrax (IKA Labortechnik). Protein concentrations in the extracts were analysed by the Pierce™ BCA Protein Assay .Clinical isolates of 2.2S. aureus extract and E. coli extract. The cohort consisted of 179 patients with moderate to severe AD (001AD‐179AD), 43 patients with seborrhoeic eczema (01S‐43S) and 97 nonatopic individuals without clinical symptoms of skin disease or history of allergy (01N‐97N). Participants were recruited at the Dermatology and Venereology Unit at Karolinska University Hospital, Stockholm, Sweden,Plasma samples from a cohort of 319 Swedish adults aged between 18 and 65 years (median age 28 years)For IgE immunoblotting and IgE inhibition experiments requiring large volumes of serum, we included additional samples from well‐characterized allergic patients (A1‐A6) Table  with knoAll work was conducted in compliance with the Declaration of Helsinki code of ethics for studies on human subjects.2.3E. coli or S. aureus were raised by subcutaneous immunization of rabbits with 3 injections, each containing 400 μg bacterial proteins (S. aureus or E. coli). Complete Freund's adjuvant was used in the first injections, followed by 2 subsequent booster injections in incomplete adjuvant after 4 weeks and after an additional 3 weeks, respectively.Rabbit immunizations were conducted at the Charles River Laboratories in France, internationally accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Immunization procedures were carried out in compliance with the directive 2010/63/EU of the European Parliament and of the council of 22 September 2010, on the protection of animals used for scientific purposes. Antisera specific for E. coli was obtained from Rockland Immunochemicals . For purposes of this work, the anti‐E. coli antiserum raised from rabbit immunized with E. coli extract is referred to as anti‐E. coli.1, and the purchased anti‐E. coli antiserum is referred to as anti‐E. coli.2. Preimmune serum was not available for the  anti‐E. coli.2.In addition, a rabbit antiserum capable of detecting over 300 low to high molecular weight proteins from Rabbit antisera specific for nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 11, rDer p 14, rDer p 21, rDer p 23 or clone 16 were obtained by immunization with the purified allergens2.4S. aureus and E. coli were detected by immunoblotting.IgG and IgE antibodies specific for E. coli.2 antiserum was diluted 1/1000 in gold buffer. Bound rabbit IgG was detected with I125 donkey anti‐rabbit IgG antibodies (1/10 000) , and signals were visualized by autoradiography.For detection of rabbit IgG binding, strips were washed, blocked and incubated with rabbit preimmune or immune sera diluted 1/5000 in gold buffer. The anti‐2HPO4; 8 mmol/L NaH2PO4; 0.5% bovine serum albumin (BSA); 0.5% tween 20; 0.05% NaN3) and incubated overnight at 4°C, with plasma diluted 1/10. After the incubation with plasma, strips were washed, and bound IgE was detected with I125 labelled anti‐human IgE antibodies at a 1/10 dilution . Signals were visualized by autoradiography. Positive IgE reactivities were defined by the presence of at least one clearly visible band on the autoradiograph, distinct from any background bands seen on the strip incubated with plasma/sera from nonallergic subjects and buffer alone (buffer control).For detection of human IgE binding, strips were washed and blocked in gold buffer  and the cut‐off for IgE sensitization was set at 0.1 kUA/L.IgE reactivity to a comprehensive panel of purified Der p allergens  was analysed with the customized allergen chip  using ImmunoCAP ISAC (MeDALL) technology as previously described.Total plasma IgE was quantified by the total IgE ImmunoCAP™ . Samples with total IgE levels above 5000 kU/L were diluted in ImmunoCAP sample buffer to obtain the actual total IgE levels.2.5E. coli extract or for control purposes, with 0.5% bovine serum albumin and then tested for IgE reactivity to nitrocellulose‐blotted HDM or E. coli extract.E. coli extract was expressed as % inhibition of IgE binding.In IgE immunoblot inhibition experiments, 1/10 diluted plasma/serum samples were preincubated overnight (4°C) with 200 μg of E. coli extract in 50 μL PBS or with 50 μL of PBS alone overnight at 4°C. Preadsorbed patient samples were then analysed for Der p‐specific IgE reactivity on the ImmunoCAP™ 250 auto analyser . Resulting IgE levels were corrected for dilution (factor 1.5). The percentages of inhibition of IgE binding to the Der p ImmunoCAP™ were calculated by comparing results from samples preincubated with E. coli extract (inhibited) with results from samples preincubated with PBS only (uninhibited), using the formula: (uninhibited‐inhibited)/uninhibited × 100.Quantitative ImmunoCAP™ inhibitions were performed by preincubating 100 μL of plasma/sera with 100 μg of 2.6P) values were two‐tailed, and P values <.05 were considered statistically significant. Venn diagrams were generated using the Biovenn Web application http://www.cmbi.ru.nl/cdd/biovenn/.Statistical analysis was performed using GraphPad Prism version 6.07 for Windows, GraphPad Software, La Jolla, CA, USA. Contingency data were analysed by Fisher's exact test, and the Mann‐Whitney test was used for comparison of group medians. Odds ratio and risk ratio estimates, differences in proportions, differences in medians and the corresponding 95% confidence intervals were calculated. All calculated probability  or in nonatopic individuals (n = 97). Total plasma IgE levels ranged from <2 kU/L to 390 kU/L for the seborrhoeic eczema (SE) patients, up to 230 kU/L for the nonatopic controls and up to 15 100 kU/L among the patients with AD. Sixty‐one per cent of the patients with AD (n = 110) had IgE levels comparable to the control groups .Figure S. aureus and E. coli extracts as exemplified in Figure S. aureus and E. coli was observed in 21% (n = 38) and 25% (n = 45) of the 179 patients with AD, respectively. Thirty‐three per cent of the patients with AD (n = 59) showed IgE reactivity to at least one of the bacterial extracts, and 13% (n = 24) had IgE to both bacteria. Sixty‐three per cent of the participants who had IgE to S. aureus also had IgE to E. coli and 53% of those who had IgE to E. coli also had IgE to S. aureus. We thus noted that the frequency of IgE sensitization to these bacterial antigens was lower in the Swedish AD patients  as compared to that observed for Austrian and German AD patients earlier .Patients with AD showed IgE reactivity to several different bands in 3.2The frequency of IgE sensitization to HDM allergens was 25% among the Swedish AD patients, when measured by the allergen chip Table .28 SensiAge and gender distribution did not differ significantly between Swedish AD participants with HDM allergy and those without HDM allergy Table A. Howeve3.3S. aureus and E. coli,S. aureus and E. coli, respectively, whereas only 15% and 14% of AD patients without HDM sensitization showed IgE reactivity to S. aureus and E. coli. IgE reactivity to S. aureus and/or E. coli was present in 67% of the HDM‐sensitized patients but only in 22% of those without HDM sensitization. The odds ratios for having IgE antibodies against E. coli, S. aureus or one of the two were 8.3, 3.8 and 7.2 in the HDM‐sensitized vs the HDM‐nonsensitized AD patients, respectively  associated with rhinitis   associated with severe AD compared to AD patients without IgE antibodies against S. aureus antigens  Figure B. In par24 P = .006 associs Figure C. Intere24 P = .006 associs Figure A.3.5S. aureus extract, E. coli extract or E. coli proteins, to detect S. aureus and E. coli antigens by immunoblotting , and against low to high molecular weight E. coli proteins (anti‐E. coli.2), reacted with distinct bands between 20 and 250 kDa in blotted HDM extracts  for reactivity to the nitrocellulose‐blotted HDM extract, s Figure A. No rels Figure B,C.3.6E. coli extract can inhibit IgE binding to blotted HDM extract , 23 kDa, 35 kDa, 55 kDa and 90 kDa (A3), 35 kDa (A4) and 12‐13 kDa  which were inhibited with 3.7E. coli extract and the remaining IgE towards HDM was determined by Der p ImmunoCAP™ measurements. We found that preadsorption of plasma/serum samples with E. coli extract inhibited IgE binding to HDM to varying extents and reached almost 50% (percentages inhibition: 0%‐49%). For control purposes, we included a plasma sample from a patient (010AD) who was positive for IgE binding only to S. aureus but not to E. coli , and some patients with relatively low total IgE levels had IgE to bacteria . Elevated total plasma IgE is therefore not a prerequisite for IgE binding to bacterial antigens.In a previous study from our group,S. aureus, has been shown to be clinically relevant in respiratory allergy and AD.H. influenzae) may also have protective activity,E. coli has not been investigated so far. It is, however, possible that IgE against E. coli may play a role in gastrointestinal manifestations that mimic food allergy, and such gastrointestinal disturbances are quite common in patients with AD.IgE sensitization to bacterial antigens, particularly from In summary, the results of our study demonstrate that HDM can act as a carrier for antigens from bacteria which colonize the skin, the respiratory tract and the gut and thus may cause sensitization to bacterial antigens. This could explain the frequent occurrence of IgE reactivity to bacterial antigens in respiratory and skin manifestations of allergy.Rudolf Valenta has received research grants from Biomay AG, Vienna, Austria, and Viravaxx, Vienna, Austria. He serves as a consultant for Biomay AG, Viravaxx, Fresenius Medical Care, Bad Homburg, Germany, and for Boehringer Ingelheim, Biberach, Germany. The other authors have no conflict of interest to declare.SD, IM, YR‐M performed experiments, analysed data, wrote manuscript and read the manuscript; SV, MN, and AMH, analysed data and read the manuscript. GW and LL contributed with clinical data and samples from the Swedish cohort and read the manuscript; CJ and AS analysed data, wrote parts of the manuscript and read the manuscript; RV designed and supervised experiments, analysed data, wrote manuscript and read the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Binding of allergen-specific IgE to its primary receptor FcεRI on basophils and mast cells represents a central event in the development of allergic diseases. The high-affinity interaction between IgE and FcεRI results in permanent sensitization of these allergic effector cells and critically regulates their release of pro-inflammatory mediators upon IgE cross-linking by allergens. In addition, binding of monomeric IgE has been reported to actively regulate FcεRI surface levels and promote survival of mast cells in the absence of allergen through the induction of autocrine cytokine secretion including interleukin-3 (IL-3). As basophils and mast cells share many biological commonalities we sought to assess the role of monomeric IgE binding and IL-3 signaling in FcεRI regulation and cell survival of primary human basophils. FcεRI cell surface levels and survival of isolated blood basophils were assessed upon addition of monomeric IgE or physiologic removal of endogenous cell-bound IgE with a disruptive IgE inhibitor by flow cytometry. We further determined basophil cell numbers in both low and high serum IgE blood donors and mice that are either sufficient or deficient for FcεRI. Ultimately, we investigated the effect of IL-3 on basophil surface FcεRI levels by protein and gene expression analysis. Surface levels of FcεRI were passively stabilized but not actively upregulated in the presence of monomeric IgE. In contrast to previous observations with mast cells, monomeric IgE binding did not enhance basophil survival. Interestingly, we found that IL-3 transcriptionally regulates surface levels of FcεRI in human primary basophils. Our data suggest that IL-3 but not monomeric IgE regulates FcεRI expression and cell survival in primary human basophils. Thus, blocking of IL-3 signaling in allergic effector cells might represent an interesting approach to diminish surface FcεRI levels and to prevent prolonged cell survival in allergic inflammation. On these cells, FcεRI is expressed as a hetero-tetramer2. IgE binding occurs asymmetrically via two binding sites in the extracellular part of the FcεRI α-subunit (FcεRIα)3. The membrane tetra-spanning β-chain (FcεRIβ), and the two identical disulfide-linked γ-chains (FcεRIγ) are not involved in the IgE interaction but are essential for receptor maturation, receptor surface transport, and the propagation of FcεRI-mediated signaling pathways4. Owing to the high-affinity interaction of IgE:FcεRI complexes basophils and mast cells are permanently sensitized with IgE and are thus ready to immediately respond to allergen challenge. Antigen-induced co-aggregation of receptor-bound IgE stimulates the release of pre-stored and de novo synthesized mediators that induce classical allergy symptoms including bronchoconstriction, vasodilatation, and increased mucus production5.Binding of allergen-specific immunoglobulin E (IgE) to its high-affinity receptor FcεRI expressed on basophils and mast cells is a central step in the induction of allergic hypersensitivity reactions6. These findings have sparked the hypothesis that IgE might be involved in the regulation of FcεRI expression on allergic effector cells. More recently, in vivo studies using the therapeutic monoclonal anti-IgE antibody omalizumab have confirmed that reducing serum IgE level in atopic patients goes along with decreased FcεRI levels on human basophils and mast cells9. In addition, various studies reported that monomeric IgE is directly involved in the regulation of surface FcεRI expression in murine bone marrow-derived mast cells and human umbilical cord blood-derived mast cells13. Binding of monomeric IgE to FcεRI has furthermore been described to promote survival of mBMMC in the absence of allergen17. Interestingly, the mechanisms underlying IgE-mediated cell survival suggested in these studies greatly differed. Although one study attributed prolonged mBMMC survival to the induction of autocrine cytokine secretion and elevated expression of the antiapoptotic Bcl-2 family member Bcl-XL15, the other  study specifically excluded both of these possibilities14. Subsequent investigations have suggested that particularly the induction of autocrine interleukin-3 (IL-3) secretion is responsible for the enhanced mast cell survival upon monomeric IgE binding18. How the monomeric interaction between IgE and FcεRI can induce intracellular signaling cascades in mast cells remained obscure until it was found that there are so-called highly cytokinergic (HC) IgE clones20. Most of these HC IgE clones exhibit polyreactivity to autoantigens or display self-reactivity23. They may therefore induce FcεRI aggregation in the absence of allergen, which could lead to the induction of cytokine production including IL-324. For basophils and mast cells, IL-3 is a well-characterized proliferation and survival factor27. Thus, the enhanced survival that has initially been attributed to monomeric IgE binding might be owing to IgE aggregation on the cell surface in the absence of allergen. Whether HC IgE is present in vivo and if it has a role in the regulation of mast cell survival remains elusive.In 1978, researchers have observed that serum IgE levels of atopic as well as non-atopic subjects positively correlate with surface FcεRI levels on primary human basophils® protein (bi53_79)30, that we used as a tool to actively remove IgE from the cell surface under physiological conditions. Our results demonstrate that removal of IgE leads to loss of FcεRI on the cell surface, whereas FcεRI-bound IgE stabilizes receptor levels. However, the presence and amount of monomeric IgE did not influence the survival of in vitro cultured primary human basophils or change the number of measured blood basophil numbers in vivo. Further, our results provide strong evidence that IL-3 signaling but not monomeric IgE binding regulates FcεRI levels on the basophil cell surface.Despite controversial findings with mast cells, we aimed to investigate the effect of monomeric IgE binding and IL-3 signaling on FcεRI regulation and cell survival of human primary blood basophils in this study. We have recently generated a novel disruptive anti-IgE DARPin31, these experiments demonstrate that the addition of monomeric IgE reduces the loss FcεRI on basophils from both low and high serum IgE level donors.To investigate the effect of IgE on the surface expression of FcεRI we isolated primary human blood basophils from donors with low ( ≤ 32 kU/L) and high serum IgE levels ( > 32 kU/L). Basophils were either incubated with medium, exogenous recombinant human IgE, bi53_79 to remove endogenous IgE or with bi53_79 prior to the addition of exogenous recombinant human IgE. The FcεRI and IgE cell surface levels were determined over a period of 7 days. Compared with incubation in medium the addition of exogenous IgE reduced the loss of FcεRI on basophils from both low Fig.  and high15. For this purpose, we compared the ex vivo survival of isolated human basophils and assessed the effect of IgE removal with bi53_79 versus addition of exogenous IgE. Surprisingly, we did not observe a difference in cell survival over the period of 72 h 32. Addition of murine IL-3 but not monomeric IgE prolonged the survival of these cells as assessed by flow cytometry and western blot  or high ( > 32 kU/L) IgE serum levels by flow cytometry. There was no significant difference in the blood basophil counts between these two groups  by flow cytometry. These cell numbers were compared to the basophil counts of littermates lacking both the murine and the human FcεRIα (huFcεRIα−/−)  Fig. . As an aice Fig. . The incex vivo27. As we did not observe an active upregulation of FcεRI levels on basophils by monomeric IgE, we asked whether IL-3 might be involved in receptor regulation. Thus, we performed cell culture assays with isolated primary human basophils in the presence or absence of recombinant human IL-3. Upon incubation of the cells with IL-3 we detected a time-and dose-dependent increase of FcεRI receptor levels by flow cytometry . Collectively, our findings support the notion that IgE does not directly regulate FcεRI expression but rather prevents FcεRI internalization by stabilizing it through binding on the cell surface31. The exact molecular mechanism of receptor stabilization, however, is still unknown and needs further investigation.In this study, we have evaluated the effect of monomeric IgE and IL-3 on FcεRI regulation and cell survival in primary human basophils. To carefully dissect the role of IgE in this context, we took advantage of the disruptive anti-IgE DARPin40. We further detected a moderate increase of FcεRIγ expression. Surprisingly, the transcription levels for FcεRIα upon IL-3 incubation decreased, emphasizing the pivotal role of the β- and γ-chain in FcεRI surface expression on allergic effector cells. Nevertheless, it is important to note that of all FcεRI subunits the α-chain showed the highest baseline expression of transcripts. Thus, our findings support previous data reporting that pre-synthesized FcεRIα can be stored intracellularly in the endoplasmatic reticulum until it associates with the homodimeric FcεRIγ alone or the FcεRIβ together with the homodimeric FcεRIγ to be transported to the cell surface via the Golgi compartment41.Interestingly, we identified IL-3 as a new transcriptional regulator of FcεRI expression on human primary basophils. Incubation of basophils with IL-3 resulted in a rapid accumulation of surface IgE receptor within 24 h. The receptor increase was associated with a transcriptional upregulation of FcεRIβ, which has previously been described to promote the maturation and surface transport of hetero-tetrameric FcεRI complexes17 we did not observe any pro-survival effect of monomeric IgE binding on primary human basophils. Furthermore, there was no difference in blood basophil counts between low and high serum IgE donors. Nevertheless, it has previously been reported that pathologically high levels of IgE correlate with increased numbers of circulating basophils in patients with hyperimmunoglobulinemia E syndrome or in germ-free mice34. However, as suggested in their study the accumulation of basophils is due to an IgE-mediated upregulation of the IL-3 receptor on basophil progenitor cells in the bone marrow, which leads to increased proliferation and differentiation of the cells. Here, we focused on fully differentiated peripheral basophils, for which we could not detect any correlation between donor serum IgE levels and cell surface IL-3 receptor expression (data not shown).In contrast to various studies using mast cellsTaken together, our data provide strong evidence that IL-3 signaling but not monomeric IgE-binding regulates FcεRI levels and cell survival of primary human basophils. As IL-3-mediated receptor upregulation correlated with initial FcεRI levels of the individual donors, we assume that genetic and environmental factors during basophil hematopoiesis have an additional role in the regulation of FcεRI surface levels. Overall our data suggest that targeted blocking of IL-3 signaling on human blood basophils might represent a promising strategy to prevent or treat allergic inflammation.tg and huFcεRIα−/− on a mixed C57BL/6 J - C57BL/6 N background were obtained from Prof J.-P. Kinet32. Wildtype mice on a C57BL/6 J background were purchased from Janvier Labs . All animal experimentation was approved from cantonal committee on animal experimentation (BE48/15).Human primary basophils were isolated from whole blood of allergic and healthy volunteers with approval from the local ethics committee. Informed consent was obtained from all donors in accordance with the Helsinki Declaration. huFcεRIα® protein bi53_79 was previously characterized29. DARPin molecules were expressed in E.coli and purified from cell lysates with metal-affinity chromatography according to the manufacturer’s manual . The proteins were further purified by size exclusion chromatography with a HiLoadTM 26/60 Superdex-75 prep grade column .Human Sus11-IgE  and NIP-specific human JW8-IgE  were used. Both IgE clones are monomeric as assessed by the provider Fig. . Recombi35. Dextran-sedimented supernatants from voluntary blood donors were used for Percoll density centrifugation and purified with basophil isolation kit II . Purity of the cells was assessed by flow cytometry using anti-CD123 and anti-CD193 antibodies. Flow cytometry data were analyzed with FlowJo . Purified basophils were cultured in 96-well U-bottom plates in complete Roswell Park Memorial Institute medium -1-piperazineethanesulfonic acid (HEPES), 100 μg/ml streptomycin, and 100 U/ml penicillin) at 37 °C, 5% CO2.The threshold of 32 kU/L serum IgE to separate between low and high serum IgE donors was chosen arbitrarily. Human primary basophils were isolated from whole blood as previously described4 isolated human primary basophils from low ( ≤ 32 kU/L) and high serum IgE donors ( > 32 kU/L) were incubated in complete RPMI medium and IgE was removed by incubation with 5 μM bi53_79 overnight. Cells were washed 3 × times with 200 μl PBS to remove dissociated IgE and DARPin® proteins from the supernatant. 50 µl RPMI containing 10 ng/ml IL-3 was used to culture the cells. IgE resensitization and IgE saturation was achieved upon addition of 100 nM human Sus11-IgE for at least 60 minutes. Surface levels of IgE and FcεRI were measured every 24 h using anti-IgE and anti-FcεRI antibodies by flow cytometry.For desensitization experiments, 2.5–5 × 104 isolated human primary basophils from low ( ≤ 32 kU/L) and high serum IgE donors ( > 32 kU/L) were incubated overnight in complete RPMI medium in the presence or absence of bi53_79 (5 µM). IgE removal was determined by flow cytometry. The cells were incubated in medium alone and resensitized or saturated with Sus11-IgE. Basophil survival was monitored with Annexin V and popidium iodide (PI) staining and measured every 24 h. Viable cells were identified as Annexin V/PI double negative cells.In total, 2.5–5 × 105 cells from three different donors were treated under the same conditions as described before to perform western blot analysis. Proteins from every individual donor were precipitated using 10% trichloroacetic acid, pooled and separated on a NuPAGE 12% Bis-Tris gels (Thermo Fisher) and transferred to a polyvinylidene difluoride membrane .A total of 1 × 10+, CD193+) cell number by flow cytometry.Dextran-sedimented supernatants from voluntary blood donors were used for Percoll density centrifugation. The basophil cell layer was collected and cells were stained with anti-CD123 and anti-CD193 antibodies. Addition of CountBright beads  allowed us to quantify the basophil , anti-murine CD200R3 , and anti-murine CD4 . Data from flow cytometric measurement were further processed using the FLOWJO software  and normalized to total cell number per ml blood.Blood from huFcεRIα4 isolated human basophils were incubated 24 h. The cells were cultured in presence and absence of IL-3, IL-5, GM-CSF, and recombinant IL-3 receptor to block IL-3 binding to CD123. In addition, IgE was removed from the cells o/n with bi53_79 and incubated with or without Sus11-IgE (100 nM) and IL-3, IL-5, GM-CSF, and recombinant IL-3. FcεRI expression and receptor-bound IgE was monitored every 6 h with Flow cytometry, as previously described. Results were normalized to the geometric mean fluorescence measured at T0 untreated. As the signal for the FcεRI receptor slightly increase upon removal of the IgE, we suggest that the staining antibody sterically interferes with receptor-bound IgE. Results were thereby corrected by subtracting a mean staining artifact (MSA). The artifact was defined as the difference in geometric mean between the untreated and the bi53_79-treated group. The artifact of every measurement was added and divided throughout the total amount of measurements to generate the MSA.A total of 2.5 × 10Human primary basophils were purified from whole blood of six donors. Total RNA was isolated using the RNeasy Mini kit (QIAgen) following manufacturer instructions. The isolated amount of RNA was quantified on a NanoDrop (Thermo Scientific) and tested for quality using a bioanalyzer (Agilent). RNA fulfilling quality criteria (RIN ≥ 6) of basophils from three different donors were pooled and used for whole-genome array (Agilent 44k) measurements. Two individual arrays were performed for each condition. Data were analyzed and mined using the R project for statistical computing. Measured spot intensities were normalized within and across arrays. Values were log2-transformed. For several genes of interest the relative log2 fold change (RLFC) between medium treated cells and IL-3-treated cells was calculated.4 cells per well in medium in the presence and absence of IL-3 for 18 h. The cells were then sensitized with NIP-specific JW8-IgE for 30 min at 37 °C and washed twice with 150 µl of PBS. For activation experiments, sensitized cells were challenged with NIP-(7)-BSA (0.01-100 nM) from Biosearch Technology  or anti-FcεRI from Bühlmann Laboratories for 25 min at 37 °C. Basophil activation was determined by measuring surface expression of basophil activation marker CD63 with FlowCAST from Bühlmann Laboratories. IL-3-mediated FcεRI upregulation correlation was performed comparing initial FcεRI level with IL-3-treated FcεRI level. Total surface IgE upon endogenous JW8-IgE sensitization of IL-3- and non-IL-3-treated basophils was compared.Purified human blood basophils were seeded at 2.5 × 10tg mouse were flushed with complete RPMI medium. The isolated bone marrow was filtered through a 70 μm cell strainer. Subsequently, the cells were washed, resuspended in 25 ml BMMC culture medium  and seeded in a T75 flask. The cells were cultured for 4 weeks at 37 °C, 5% CO2 and medium was changed twice a week.Femors and tibiae of a huFcεRIαt-test or one-way analysis of variance. P values of < 0.05 were considered statistically significant.Statistical analysis was carried out with Prism 6.0 software . All data are shown as means ± SEM. Comparisons between different treatments were analyzed by using Student’s Supplementary figuresSupplementary Information


Drosophila embryo with high temporal resolution, allowing time-specific and reversible manipulation of morphogen signalling. We find that Bicoid transcriptional activity is dispensable for embryonic viability in the first hour after fertilization, but persistently required throughout the rest of the blastoderm stage. Short interruptions of Bicoid activity alter the most anterior cell fate decisions, while prolonged inactivation expands patterning defects from anterior to posterior. Such anterior susceptibility correlates with high reliance of anterior gap gene expression on Bicoid. Therefore, cell fates exposed to higher Bicoid concentration require input for longer duration, demonstrating a previously unknown aspect of Bicoid decoding.Morphogen gradients provide essential spatial information during development. Not only the local concentration but also duration of morphogen exposure is critical for correct cell fate decisions. Yet, how and when cells temporally integrate signals from a morphogen remains unclear. Here, we use optogenetic manipulation to switch off Bicoid-dependent transcription in the early DOI:http://dx.doi.org/10.7554/eLife.26258.001 Morphogens are molecules distributed in spatial gradients that provide essential positional information in the process of development . By actiThe temporal pattern of morphogen interpretation has been demonstrated in several vertebrate developing systems. Harfe et al. first proposed that the length of time of morphogen signalling is critical for correct cell fate specification. They found that during mouse limb development, cells exposed to Sonic Hedgehog (Shh) morphogen for longer time develop into digits of more posterior identity . SimilarDrosophila melanogaster  patterns the cells along the antero-posterior axis in the early embryo of the fruitfly, nogaster . Bcd is nogaster . This lonogaster . Quantitnogaster . It has nogaster . Howevernogaster .Drosophila embryo with high temporal resolution. Using this tool, we provide a detailed dissection of the temporal components of Bcd decoding in vivo. The results unveil an unexpected temporal pattern of Bcd action – cell fates determined by higher Bcd concentration require Bcd for longer duration, while cells experiencing lower Bcd dosage commit to correct fates at earlier developmental stages. Further combining temporal perturbation with quantitative analyses, we find that the differential expression kinetics of target genes can partly explain the mechanism underlying such temporal interpretation.To address this question, fast and reversible temporal manipulation of Bcd activity is required. In this study, we have developed an optogenetic tool to control Bcd-dependent transcription in the early We built a light-responsive construct to switch off Bcd-dependent transcription by fusing the optogenetic cassette cryptochrome 2 (CRY2) together with mCherry (mCh) to the N-terminus of Bcd  Figure . We usedbcd mutant background , giant (gt), krüppel (kr) and knirps (kni) – are reminiscent of wild-type embryos when the light-responsive Bcd construct is expressed in an otherwise ckground . These eckground . In compckground , resemblhenotype . Illuminhenotype . Further regions . While Cpression , we saw pression . It suggpression . We furtpression  and obsepression , showinglow and CRY2high, respectively  during the hour prior to gastrulation. We used the Even-skipped (Eve) pattern, where Eve stripes 1 and 2 are Bcd-dependent and more posterior stripes are Bcd-independent as a readout of Bcd activity . In a WTr tested .bcd null background  and illuminated them before the initiation of gastrulation for time windows ranging from 10 to 60 min  and reverting them later to dark conditions to recover Bcd-dependent transcription. We find that a 30 min illumination window spanning n.c. 11 ~ 13 results in severe patterning defects  and analyzed the expression of Engrailed (En) and various Hox genes. The most anterior morphological features that can be observed at GBE are the clypeolabrum lobe at the very tip of the embryo and the invagination of stomodeum  results in deviations of the Hb anterior pattern  and Kni (45–37% EL) is subjected to fine-tuning by Bcd-dependent transcription (in combination with Hb) . We findDrosophila embryo development, with models identifying critical components in the gap gene network required for forming robust gene expression boundaries  anterior shift in Kr anterior boundary; (ii) anterior shift in anterior boundary of Kni posterior domain; (iii) reduction in Kni anterior expression; (iv) reduction in Gt anterior expression; (v) anterior shift of posterior boundary of Hb anterior domain and reduction in anterior Hb expression. We identified 9 out of 21 parameter sets that qualitatively agreed with at least three of the above criteria. We compared how the average parameter values of this subset differed from the mean parameter values of all 21 parameter sets,  on persistent Bcd-dependent transcription may explain our previous observations that inhibiting Bcd transcriptional activity both at very late or early stages impairs the proper formation of the anterior-most segments . Any shoSeveral recent studies have used elegantly designed optogenetic systems to unveil the temporal role of signalling pathways in embryonic cell fate induction . As a coThe following results helped us to narrow down the possible mechanisms underlying light-responsive manipulation of Bcd transcriptional activity. (1) Illumination does not affect the spatial distribution of the protein , suggestThe continuous transitions between cell states in a developing embryo are governed by both the transcriptional landscape and the transcriptional history of the cells. Therefore, not only the local concentrations of the combinatorial transcription factors but also the timing and duration of their presence are essential for the correct cell fate decisions. Bcd in the early fruit fly embryo provides the very initial positional cues along the antero-posterior axis, differentiating cells spanning ~10 embryonic segments . The graConsistent with previous studies, we find that the time window ranging from n.c. 13 to early n.c. 14 is indeed most critical for Bcd-dependent patterning of downstream genes; deprivation of Bcd transcriptional activity during this period leads to severe patterning defects in all the Bcd-dependent embryonic regions, from the most anterior to mesothoracic segment . On the bona fide gene expression in the very anterior domains, suggesting that cells are no longer competent to adopt anterior-most cell fates. Whether Bcd primes cell competency by chromosomal remodeling to increase accessibility  or the activation potency  of the Bcd protein  and high (CRY2high) levels (mCherry fluorescent intensity) were employed throughout our analyses. Transcription and mRNA localization in the transformed flies was expected to recapitulate those of endogenous bcd.A CRY2::mCh::Bcd fusion construct was generated by replacing the eGFP sequence in the P vector  by the C] vector  using prbcd knockout fly line was generated by CRISPR-mediated insertion of a MiMIC cassette  transgenic was recombined with the bcd knockout allele to establish a stable line. All experiments were carried out with this fly line unless otherwise stated.A cassette  . The GFP sequence is excised and replaced by mCherry using the Gibson Assembly strategy (NEB). The construct pCaSpeR4-pNOS-NoNLS-MCP-mCh-αTub3’UTR was randomly inserted into the fly genome using P-element transformation (BestGene). A line with II chromosome insertion was crossed to CRY2-mCh-Bcd on the III chromosome to establish a stable line Additionally, the fly line nanos>Gap43::mCh  was usedi.e. blocking blue light). To induce the conformational change of the CRY2 protein, we illuminated the embryos on a Nikon LED light base at 488 nm wavelength. The light intensity was measured with an intensity power meter. All experiments were carried out at 4.0 mW unless otherwise stated. For temporally patterned illumination, blastoderm stage embryos were selected in the dark condition and exposed to light. The illumination treatment of each embryo was recorded. The illumination duration was timed from the moment of light exposure until the onset of gastrulation. When the illumination treatment was followed by dark recovery, we continued the timing by observing embryos with a light source covered with amber paper until gastrulation initiated.Duration of syncytial nuclear cycles were evaluated by imaging embryos on a bright-field stereomicroscope at 25°C. The blastoderm stage (from fertilization to the onset of gastrulation) spans 2.5 hr. The last syncytial cycle (n.c. 14), demarcated by the last division wave and the first sign of gastrulation, lasts about 45 min. The penultimate cycle (n.c. 13) lasts 15 min, while the duration of the previous three cycles (n.c. 10–12) is about 30 min in total. To maintain dark condition, embryos were observed or imaged with all light sources covered by amber paper (AB_2650480), rat anti-Caudal (1:100), guinea pig anti-Hb (1:2000), rabbit anti-Gt (1:800), guinea pig anti-Kr (1:800), guinea pig anti-Kni (1:800), guinea pig anti-Eve (1:800), guinea pig anti-Otd (1:800), mouse anti En , rabbit anti-Dfd (1:100), mouse anti-Scr , mouse anti-Antp , mouse anti-Ubx  and mouse anti-Abd-B  RRID:AB_528061).Primary antibodies were detected with Alexa Fluor-labelled secondary antibodies . Embryos were co-stained with Phalloidin conjugated with Alexa Fluor for staging purpose. Embryos were mounted in AquaMount  and imaged on a Zeiss LSM710 microscope with a C-Apochromat 40x/1.2 NA water-immersion objective. Cad antibody was kindly provided by Eric Wieschaus. Hb, Gt, Kr, Kni and Eve antibodies were gifts from Johannes Jaeger. Otd was kindly given by Tiffany Cook. Last, Dfd antibody was a gift from Thomas C. Kaufman.Embryos at the desired stages were dechorionated by household bleach and fixed in heptane saturated by 37% paraformaldehyde (PFA) for 1 hr. The vitelline membrane was subsequently manually removed. Prior to incubation with primary antibodies, embryos were blocked with 10% BSA in PBS. Image-iTFX signal enhancer was used as blocking reagent instead of 10% BSA for Cad staining. Antibodies used were rabbit anti-mCherry . Bcd nuclear intensity plots were created after background subtraction using morphological opening. Profiles fitted as described in Liu .Images were projected using a maximum intensity projection and then nuclei segmented using Ilastik . Nuclei Embryos were collected, dechorionated and mounted on a MatTek dish under dark condition. For the imaging of MCP::GFP lines, the embryos were imaged on a custom-built Spinning Disc microscope with a Nikon Apo 40X/1.25 water-immersion objective. The pixel size is 409 nm and the image resolution is 1024 × 1024 pixels. At each time point a stack of 35 images separated by 3 µm was acquired. The temporal resolution was 40 s. For the imaging of MCP::mCh lines, the embryos were imaged on a Zeiss LSM710 microscope with a C-Apochromat 40x/1.2 NA water-immersion objective. A laser wavelength of 561 nm was used to excite MCP::mCh and an LED light source was used to illuminate the embryo to induce conformational change of CRY2::mCh::Bcd. Pixel size is 346 nm and a rectangular section with image resolution of 750 × 220 pixels was acquired in the middle region of the embryos . At eachNascent hb transcripts were detected from live-imaging data using both ImageJ  and Matlfunction . This me∧(Adjusted input – Ct(IP)) was used to normalize ChIP-qPCR data. Further, the relative % input for illuminated embryos was calculated as compared to the embryos in dark and summarized in a bar chart. In the case of the HbP2 +1 kb, data were compared to HbP2 results. Three independent replicates have been performed for each primer sets and the significance was calculated with a standard t-test.Late n.c. 14 embryos aged in the dark or exposed to light (at 4 mW) for 45 min were dechorionated in household bleach. Embryos were crosslinked for 15 min in a solution containing 2 ml of PBS, 6 ml of Heptane and 180 µl of 20% paraformaldehyde. Embryos were transferred to a 1.5 ml tube and the crosslinking was quenched with the addition of 125 mM glycine in PBS 15 min after the start of fixation. ChIP samples were essentially prepared as described in Embryos subjected to temporally patterned illumination were allowed to develop until the end of embryogenesis. The embryos were then dechorionated and incubated into a mixture of Hoyer’s medium and Lactic acid in a 1:1 ratio at 65°C between an imaging slide and a cover slip. For an exhaustive description of the method used see SCR_002285). For Hb, the intensity profile was normalized to the peak value of the posterior domain. To determine the boundary positions of Kr and Kni expression domains, we plotted the intensity profiles along the AP axis and defined the boundary at the position with intensity equals to half of the peak value.Confocal Z-stack images were Z-projected (maximum intensity) in Fiji for further analysis. Images were rotated to orient embryos anterior left and dorsal up and rescaled to same embryo length and width. Intensity profiles along the antero-posterior axis were measured in Fiji  and between the gap genes and morphogenetic inputs  and Tailless (Tll)). At times corresponding to scenarios ①-⑥ in Bcd,j are set to zero. The shifts in TBcd,j are assumed to be instantaneous. Scoring to the criteria outlined in the text is done manually. The parameter value variation between a subset, isubsetp, and the average of all n = 21 parameter setsiall,, p was scored for each parameter using The model analyzed is from Embryos were dechorionated as described earlier and mounted in 1% agarose on a Fluorinated ethylenepropylene (FEP) capillary . Embryos were then imaged on a custom-built Light-Sheet Microscope with a Nikon Apo 1 WD 25X/1.10 water-dipping objective. Embryos expressing CRY2::mCh::Bcd and the membrane marker Gap43::mCh were recorded using a 561 nm excitation laser. The pixel size was 510 nm and the image resolution was 1024 × 1024 pixels. At each time point a stack of 100 images separated by 2.5 µm was acquired. The temporal resolution was 30 s. This illumination setup mimics dark condition as no morphological defects were observed. To stimulate CRY2 conformational change in just one half of the embryo, a region on one lateral side of the embryo was defined from the most apical section up to 70 µm down. A stack of 100 sections separated by 0.7 µm were illuminated. The illumination was carried out from the beginning of the n.c. 14 (5 min after the establishment of the cellularization furrow) for 20 min with a 488 nm laser. Following illumination, embryos were imaged solely with the 561 nm excitation laser. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Drosophila embryo" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Didier Stainier as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Jun Ma (Reviewer #3).Thank you for submitting your article "Decoding temporal interpretation of the morphogen Bicoid in the early The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.As you can see below, all three reviewers recommended your study and were particularly excited about your ability to manipulate temporal parameters of the Bicoid gradient.The most significant recommendation was to perform some theoretical/computational analysis of the gap gene network, to examine your results, or call for the revision of existing models.Reviewer #1:For the last 30 years, the Bicoid gradient has been the most studied example of a morphogenetic gradient. It would thus seem difficult to find new important details that have been missed by these studies that went deep into molecular mechanisms and models of gene regulation. Yet, this paper appears to provide just that: not a huge amount of conceptual novelty, but a powerful way to manipulate temporal parameters of the gradient using a very clever method to render (some of) the activity of the protein light dependent.The authors achieved this by fusing the Bicoid protein in the context of its own rescue construct to a light sensitive moiety. Interestingly, this appears to only affect the transcriptional activity of the protein and not its DNA binding or its RNA regulatory capacity: the activation domain might be more sensitive, which is surprising but the data show a full rescue without light and no rescue with light. This allowed the authors to stop activation by Bicoid at various times during development. They conclude that the temporal pattern of Bcd activation is important, which appears to be in slight contradiction with the work of Thomas Gregor who also quantified very carefully the effect of Bicoid but could not manipulate it so precisely. It appears that genes that require higher Bcd concentration require Bcd for longer duration, while exposed to low Bcd (whose targets have high affinity Bicoid binding sites) can be specified with short early exposure.Drosophila AP patterning that I've seen in years. I'm not sure they solve a lot of the issues they raise, but the experiments and data are pretty interesting. I think understanding temporal contributions of transcription to cell fate are interesting, and it is great that the authors used this system to achieve this and their results are convincing: this is a powerful tool and they might be able to obtain new concepts with it.Other than that, it's the most novel contribution to the study of Reviewer #2:Drosophila blastoderm patterning. The authors find that Bcd is required from 1h post-fertilisation (cell cycle 10) and that specification of anterior regions requires longer durations of Bcd activity than posterior regions. The authors used the approach to generate a map of the temporal requirements for Bcd activity.Huang et al. use an optogenetic system to investigate the temporal requirement for Bcd activity in An optogenetically controllable Bcd is an elegant and powerful reagent which provides a new perspective to this well established system. As the textbook example of a morphogen, this study will be of interest not only to the Bicoid field but to the wider developmental biology community. Overall the experiments are convincing and well documented and the manuscript is clear and accessible. Defining differential time requirements for different target genes and demonstrating a role of Bcd prior to cell cycle 13 are important results that will lead to a re-evaluation of currently favored models of Bcd dependent patterning.The authors characterize the CRY2::mCh::Bcd in The authors suggest that illuminated embryos "resemble" the bcd knockout phenotype . In The mechanism by which the cryptochrome cassette blocks Bcd activity is unclear. The authors note that spatial distribution of Bcd and DNA binding activity is unaffected and the Caudal gradient is also unaltered. These experiments would be strengthened by controls in which Bcd and Cad gradients are disrupted, to gauge the sensitivity of the assays. The genotypes of these embryos should also be clearly stated in the figure legend.Experiments that directly test how quickly Bcd dependent transcription is lost in illuminated conditions and recovered following cessation of illumination would strengthen the interpretation of The data in Reviewer #3:Drosophila morphogen Bicoid (Bcd) for embryonic development. Their general conclusion is that cell fates dependent on higher Bcd concentration require the Bcd input for a longer duration of time. The concepts are interesting and most of the experiments are well controlled. The findings reported here are useful to the field. There are a few aspects that can be improved.Huang et al. describe an optogenetic manipulation approach for investigating the temporal requirement of the While the observed effects of light exposure on embryonic development and gene expression are convincing, the underlying mechanisms remain relatively unclear. This is because precisely how the CRY2::mCh::Bcd fusion protein becomes inactivated at the molecular or biochemical level has not been established. The authors describe experiments suggesting that neither the ability of Bcd in translation inhibition (monitored by Cad protein gradient profile) nor its DNA binding (monitored by ChIP) was affected. It is possible that, as suggested by the authors, the N-terminal fusion of CYR2 to Bcd may cause a light-induced conformational change that inhibits the transcriptional activity of Bcd (since its N-terminus does contain a regulatory motif modulating its transcription activity). But I am not fully convinced that the ChIP approach can sensitively and unambiguously rule out an effect on DNA binding. The fact that the expression domains of hb and Kr exhibit an anterior shift  is consiIt is generally acknowledged that weaker bcd alleles tend to affect preferentially the formation of the most anterior structures, suggesting that such structures are more sensitive to perturbations of the Bcd input. It would be useful to discuss how this study may (or may not) provide mechanistic insights into those earlier findings.It is useful to provide a brief discussion on the estimated time scales of both illumination-induced inactivation and "reactivation" in experimental setting described in the current study. The most significant recommendation was to perform some theoretical/computational analysis of the gap gene network, to examine your results, or call for the revision of existing models.We would like to thank the reviewers for their comments about our work, and the constructive questions and suggestions. We have performed additional experiments and theoretical analysis. We hope that our manuscript is now suitable for acceptance.The referee reports brought out five main points of discussion. We give a detailed discussion to each of these points below. After that, we highlight point-by-point responses to specific referee comments.The points of discussion of this letter are:1) Testing temporal perturbation using a gap gene network model2) The mechanism underlying light-induced Bcd-dependent transcriptional inhibition3) The kinetics of light-responsive loss and recovery of transcription activity4) Measurement precision of gene expression profiles4.1) Quantification of Cad gradients in altered Bcd dosages4.2) Further support for “5% anterior shift” in embryos subjected to early illumination5) Incorporating current findings with previous studies on Bcd-dependent patterning5.1) Reconciling temporal Bcd interpretation with previous quantitative workbcd alleles and short temporal perturbations5.2) Insights from phenotypic resemblance between hypomorphic 1) Testing temporal perturbation using a gap gene network modelDrosophila embryo development, with models identifying critical components in the gap gene network required for forming robust gene expression boundaries . These models use coupled differential equations to incorporate interactions between gap genes  and with maternal inputs, along with diffusion and protein degradation.Modelling has been a powerful tool in understanding early To test how such models perform under temporal perturbation of the Bcd input, we used the model outlined in Bieler et al., which is an advanced gap gene network model that incorporates the three-dimensional embryo shape, regulatory inputs from Bcd, Cad, Tailless and Huckebein, and the distinct regulatory functions of Hunchback monomers and dimers. The general form of this model is similar to other published models , and therefore we do not expect significant variation in our conclusions from using other, similar, models.We were able to effectively introduce into the model Bcd temporal deprivation by setting the interaction strength with Bcd to zero at different times in the simulation, see Materials and methods and new Results of these numerical simulations are provided in the main paper and in 2) The mechanism underlying light-induced Bcd-dependent transcriptional inhibitionAt the early phase of the project when we first observed the light-induced lethality in embryos expressing maternally loaded CRY2::mCh::Bcd protein in a wild-type bcd background, we considered several possible mechanisms underlying such phenomenon.  Light inbcd mutant embryos (excessive membrane ingression in the anterior end of the embryos) although the linking factors between Bcd morphogen to the regulation of cytoskeletal components remains unclear . We found that light also induces such cellularization phenotype in CRY2::mCh::Bcd embryos  Kinetics of light-responsive loss and recovery of transcription activityhb P2 promoter. The limitation of this reporter system lies in that we cannot visualize the transcription under dark condition in CRY2::mCh::Bcd embryos as the GFP excitation wavelength (488 nm) overlaps with CRY2 light-sensitive range (405–488 nm). We have recently developed and characterized a new reporter system where the MCP is tagged with mCherry instead. The updated findings are  tagged with GFP to detect nascent transcripts driven by the ings are  MCP::mChings are  The tranvelength 88 nm ove4) Precision in measurement of gene expression profilesDrosophila embryos given well-defined stages . We have made similar measurements on control embryos (OreR) and the variation of most gap gene boundary positions is within the range of 1~2% embryonic length (Gap gene profiles display extreme reproducibility in c length . Therefo4.1) Quantification of Cad gradients in altered Bcd dosagesTo verify our observation that the Cad gradient is not altered by the optogenetic perturbation of Bcd transcriptional activity, we tested the sensitivity of our assay by measuring the Cad gradient in embryos with one, two or four maternal copies of bcd. As shown in the new 4.2) Further support for “5% anterior shift” in embryos subjected to early illuminationWe have noticed that embryos illuminated before n.c.12 show a 5% anteriorly shifted Hb posterior boundary in the anterior domain as well as Kr anterior boundary, although these embryos are highly viable. A recent ChIP-seq study has shown that Bcd molecules serve a role of opening up chromatin states at a subset of its targets in early stages to gain accessibility for transcription . Our data suggests that early Bcd-dependent transcription events may have a chromosomal remodeling effect for the target sites to interpret a normal Bcd concentration in later stages. If embryos are deprived of its early Bcd-dependent transcription, target sites ‘see’ a lower Bcd gradient due to the compromised chromatin accessibility. To further support this observation, we have quantified the position of the cephalic furrow which shifts its position along the AP axis with altered bcd dosage. In consistency with our postulate, we observed a significant anterior shift of cephalic furrow position in embryos illumination before n.c.12 compared to dark condition  Incorporating current findings with previous studies on Bcd-dependent patterning5.1) Reconciling temporal Bcd interpretation with previous quantitative workno longer relevant for patterning precision ; (2) The embryonic patterning operates in a precisionist mode in that the information carried by gap genes at one defined time point is not only sufficient but also fully utilized by cells for decoding positional information, as probed by pair-rule genes .Studies from Gregor’s lab have been focusing on using information theory to understand the precision and reproducibility in Bcd-mediated embryonic patterning. Among many important findings, the most relevant to the current study are (1) While early target gene boundaries are set by the absolute Bcd concentration, the cross-regulatory gap gene network kicks in later to adjust the boundary positions and therefore Bcd is Our findings do not dispute the important role of the gap gene network in refining segmentation boundaries. In fact, embryos subjected to short period of illumination in late n.c.14 show only slight gap gene boundary shifts as a result of self-organized gap gene cross-regulation. However, we argue against the claim that Bcd in late n.c. 14 becomes irrelevant for patterning for the reason that, as we have shown, Bcd-dependent transcription is indispensable at this time for sustaining the proper expression level of gap genes, especially the most anteriorly expressed ones . The proper expression level of the anterior gap genes is in turn essential for precise anterior-most cell fate commitment.Our results are in agreement with a potentially optimized information flow from gap genes to downstream pair-rule gene patterns. We anticipate that the gap gene expression patterns under different illumination conditions can predict the pair-rule gene patterns using the same decoding algorithms as established by Petkova et al., 2016. However, what is not yet considered in these studies is the information flow from a snapshot patterning to the actual embryonic cell fate – just like Bcd, all the gap genes as well as pair-rule genes are transcription factors of which the duration of activity matters to cell fate determination. We have shown that indeed cell fates are determined not only by segmentation gene expression at a given time, but also its integration over time.5.2) Insights from phenotypic resemblance between hypomorphic bcd alleles and short temporal perturbations+ reduction can be distinguished, that is, the preantennal head region, the antennal and gnathocephalic head segments, and the thorax. While the weakest bcd alleles only affect the first region, defects sequentially extend to the second and third regions with increased allelic strength . Interestingly, the same distinction can be made by our temporally perturbed Bcd activity, with prolonged transcriptional inhibition giving rise to defects in sequentially extended regions.Three anterior embryonic regions with different sensitivity to bcdbcd alleles affect either the DNA binding affinity  or the activation potency  of the Bcd protein. The resemblance between hypomorphic bcd alleles and short temporal perturbations demonstrates that, constitutively low bcd activity is functionally equivalent to normal bcd activity with reduced active duration, both of which are sufficient to guide proper cell differentiation trajectories in the mid region of the embryo where target gene binding affinities are high. In comparison, in the more anterior region where target genes have low affinities, both wild-type level bcd activity and long duration are necessary for anterior induction .We have included within the Discussion a more detailed comparison between our optogenetic perturbations and the previously reported different Reviewer #1:For the last 30 years, the Bicoid gradient has been the most studied example of a morphogenetic gradient. It would thus seem difficult to find new important details that have been missed by these studies that went deep into molecular mechanisms and models of gene regulation. Yet, this paper appears to provide just that: not a huge amount of conceptual novelty, but a powerful way to manipulate temporal parameters of the gradient using a very clever method to render (some of) the activity of the protein light dependent.The authors achieved this by fusing the Bicoid protein in the context of its own rescue construct to a light sensitive moiety. Interestingly, this appears to only affect the transcriptional activity of the protein and not its DNA binding or its RNA regulatory capacity: the activation domain might be more sensitive, which is surprising but the data show a full rescue without light and no rescue with light. This allowed the authors to stop activation by Bicoid at various times during development. They conclude that the temporal pattern of Bcd activation is important, which appears to be in slight contradiction with the work of Thomas Gregor who also quantified very carefully the effect of Bicoid but could not manipulate it so precisely. It appears that genes that require higher Bcd concentration require Bcd for longer duration, while exposed to low Bcd (whose targets have high affinity Bicoid binding sites) can be specified with short early exposure.Please see section 5.1above, where we discuss our results in relation to other labs, particularly those of Thomas Gregor.Reviewer #2:[…] The authors characterize the CRY2::mCh::Bcd in We discuss possible mechanisms in the above section 2. We have also incorporated the main points into the Discussion, subsection “Mechanism underlying light-induced inhibition of Bcd transcription activity”.The authors suggest that illuminated embryos "resemble" the bcd knockout phenotype . In Figure 1—figure supplement 2 the authors show that the phenotypes are not identical. This should be highlighted earlier in the text and the phenotypic comparison given more interpretation, to help readers not expert in the system.bcd null and illuminated bcdCRY2 embryos in detail and explained the cause of such phenotypic differences.In the second paragraph of the subsection “Precise temporal control of Bcd-dependent transcription via optogenetic manipulation” we compare the phenotypes of The mechanism by which the cryptochrome cassette blocks Bcd activity is unclear. The authors note that spatial distribution of Bcd and DNA binding activity is unaffected and the Caudal gradient is also unaltered. These experiments would be strengthened by controls in which Bcd and Cad gradients are disrupted, to gauge the sensitivity of the assays. The genotypes of these embryos should also be clearly stated in the figure legend.We have tested the sensitivity of the assays by measuring Cad gradients in embryos with altered Bcd dosages , and found significant shifts in Cad boundary positions new . The meaExperiments that directly test how quickly Bcd dependent transcription is lost in illuminated conditions and recovered following cessation of illumination would strengthen the interpretation of We agree that such experiments are helpful. To this end, we have tagged the MCP protein with mCherry, enabling us to observe the system without triggering CRY2 conformational changes (which was an issue with the MCP::GFP protein). Full details are provided above in section 3. In the manuscript new supplemental figures are included – In new The data in in silico results and a new We agree that testing our results against current models of gap gene systems is beneficial. We have performed such an in silicoexperiment using the model of Bieler et al., 2011. A complete discussion is given above in section 1. We have included in the Results of the manuscript (subsection “Refining parameter estimation in models of gap gene network”) a discussion of the Related to this, the authors might want to extend their Discussion to consider the involvement of the dynamics of the Bcd controlled transcriptional network  in the differential sensitivity to durations of Bcd activity. This would allow a more direct comparison with the vertebrate neural tube where the dynamics of the Shh responsive transcriptional network are implicated.We discuss the similarities with the vertebrate neural tube in the Discussion . We fully agree that confronting our results with that of the Shh transcriptional network would be insightful. However, it would require extensive work and refinement of the current gap gene models.Reviewer #3:Huang et al. describe an optogenetic manipulation approach for investigating the temporal requirement of the Drosophila morphogen Bicoid (Bcd) for embryonic development. Their general conclusion is that cell fates dependent on higher Bcd concentration require the Bcd input for a longer duration of time. The concepts are interesting and most of the experiments are well controlled. The findings reported here are useful to the field. There are a few aspects that can be improved.While the observed effects of light exposure on embryonic development and gene expression are convincing, the underlying mechanisms remain relatively unclear. This is because precisely how the CRY2::mCh::Bcd fusion protein becomes inactivated at the molecular or biochemical level has not been established. The authors describe experiments suggesting that neither the ability of Bcd in translation inhibition (monitored by Cad protein gradient profile) nor its DNA binding (monitored by ChIP) was affected. It is possible that, as suggested by the authors, the N-terminal fusion of CYR2 to Bcd may cause a light-induced conformational change that inhibits the transcriptional activity of Bcd (since its N-terminus does contain a regulatory motif modulating its transcription activity). But I am not fully convinced that the ChIP approach can sensitively and unambiguously rule out an effect on DNA binding. The fact that the expression domains of hb and Kr exhibit an anterior shift  is consiWe agree that ChIP-qPCR experiment does not have the resolution to rule out any unspecific DNA binding caused by CRY2 conformational change. However, several experimental observations support that light-induced conformational change does not alter DNA binding of CRY2::mCh::Bcd molecules. We provide a detailed discussion of possible mechanisms and evidence for each one in section 2above. We have also extended the Discussion (subsection “Mechanism underlying light-induced inhibition of Bcd transcription activity”) to outline the mechanism underlying Bcd-dependent transcriptional inhibition in our experiments.Moreover, embryos illuminated before n.c. 12 should have wild-type Bcd activity present during n.c. 13-14; however, we still observed anterior shifts of Hb and Kr in these embryos. Since we have confirmed that the Bcd profile is not altered by illumination, we attribute the anterior shifts to the pre-patterning role of Bcd in early stages (see above section 4.2).Lastly, while local illumination by confocal microscopy results in a reduced persistence of transcription in n.c. 13, a thorough illumination by LED source can immediately and completely abolish transcription in n.c. 13  provide mechanistic insights into those earlier findings.We discuss these points in detail above in section 5.2. In the paper, we have extended the Discussion  to incorporate key points related to this comparison.It is useful to provide a brief discussion on the estimated time scales of both illumination-induced inactivation and "reactivation" in experimental setting described in the current study.We agree, and with our MCP::mCherry construct we are now able to image the developing embryo without simultaneously triggering CRY2 conformational changes (which was a problem with MCP::GFP). Full details are provided above in section 2. In the manuscript new supplementary figures are included –


Most of Earth’s glaciers are retreating, but some tidewater glaciers are advancing despite increasing temperatures and contrary to their neighbors. This can be explained by the coupling of ice and sediment dynamics: a shoal forms at the glacier terminus, reducing ice discharge and causing advance towards an unstable configuration followed by abrupt retreat, in a process known as the tidewater glacier cycle. Here we use a numerical model calibrated with observations to show that interactions between ice flow, glacial erosion, and sediment transport drive these cycles, which occur independent of climate variations. Water availability controls cycle period and amplitude, and enhanced melt from future warming could trigger advance even in glaciers that are steady or retreating, complicating interpretations of glacier response to climate change. The resulting shifts in sediment and meltwater delivery from changes in glacier configuration may impact interpretations of marine sediments, fjord geochemistry, and marine ecosystems. The reason some of the Earth’s tidewater glaciers are advancing despite increasing temperatures is not entirely clear. Here, using a numerical model that simulates both ice and sediment dynamics, the authors show that internal dynamics drive glacier variability independent of climate. This trend shows little spatial consistency, suggesting a dynamical rather than climate mechanism is responsible. Tidewater glaciers differ from their terrestrial terminating cousins because mass loss occurs not only by surface melt, but also by calving and ablation at the oceanic boundary. The mass budget of such glaciers is therefore sensitively dependent on conditions at the marine terminus such as water temperature, motion, and depth. One example of this dependence, first observed at Columbia Glacier3, is that the presence of a terminal shoal can lead to a considerable terminus advance relative to a position in absence of sedimentation. Such glaciers also tend to be unstable: ice flux from calving increases with terminus depth, and on a retrograde slope (such as the upstream side of a moraine) this positive feedback can lead to catastrophic retreat5. The coupling of nonlinear ice dynamics and sediment transport leads to a continuum of dynamical behavior known as the tidewater glacier cycle (TGC), which can be described in four archetypal phases6.Despite a globally consistent trend of glacier mass loss7) or subaerial . A glacier enters the retreating phase when the glacier can no longer maintain sufficient thickness to remain grounded on the shoal, and the associated reduction in basal drag leads to retreat into progressively deeper water, triggering the instability described above  and sediment transport are used. Such models are also capable of simulating the retreat phase, although the degree of hysteresis between the time scales of advance and retreat is not explicitly captured. Models were inconsistent about whether an external perturbation is required to initiate retreat. Models designed to simulate the TGC in temperate glaciers have invoked either glaciofluvial erosion13 or sediment deformation12 as a mechanism for transporting the terminal shoal. More physically complete models have been applied to the problem of coupled ice flow and sediment deformation, the primary mechanism of sediment transport for the relatively dry marine ice sheets15. Interestingly, such simulations were shown capable of producing unforced distinctly asymmetrical oscillations in ice extent with obvious analogies to the TGC, albeit over hundred thousand rather than 100 year time scales.Zero-dimensional modeling efforts have shown that sediment deposited at the glacier front can initiate advance through the calving reduction mechanism defined above15, and we include a physically consistent representation of both erosion and depositional processes using mass conservation13. We find that that this coupled model, when calibrated with observations, can produce all phases of the TGC without externally driven changes in climate and with a period and amplitude in general agreement with observations from the geologic record. These temporal and spatial scales are strongly influenced by the availability of meltwater, which drives the rate of sediment transport and subsequently controling the rate of glacier advance. This dependence is sufficiently strong that a change in meltwater availability due to climate warming may (perhaps counter-intuitively) trigger advance in tidewater glaciers that are currently stable or retreating. Although necessarily a simplification of the complete physical processes governing tidewater glacier dynamics, our results provide a basis for assessing how such natural variability in sedimentation and meltwater regimes could impact fjord ecosystems, interpretation of marine sedimentary records, and predictions of ice volume change.Here we test the hypothesis that TGCs can be explained solely due to the coupling of ice dynamics, glacial erosion, and fluvial sediment transport by running a series of numerical experiments with a new model that explicitly simulates both ice and sediment dynamics. Our approach builds upon previous work in two primary ways. First, we consider sediment transport due to glaciofluvial rather than deformational processes16. Fluvial erosion rates were calibrated with repeat radar measurements from Taku Glacier17. Fluvial deposition rates were derived from theoretical settling velocities of sediment samples taken from boreholes at Taku Glacier’s terminus. The conclusions presented here are robust to parameter choices . Sediment is carried to the terminus by meltwater, forming a shoal. The shoal acts as a plug, reducing ice flux through the calving front and yielding a positive mass balance that prompts thickening and advance onto the shoal. The sediment composing the shoal is recycled and continuously transported downstream by glaciofluvial processes, growing larger as more is produced and delivered from upstream. The shoal eventually breaks the surface (t = 240 years) and calving ceases. The sediment that reaches the terminus at this stage is transported subaerially, depositing at the downstream end of a growing outwash plain. The glacier in this configuration is effectively land-terminating, albeit with a bed that continues to be reshaped by fluvial transport. As accumulation and ablation approach balance (t = 286 years), advance slows. However, advection of the shoal is not subject to the same length limitations imposed on ice by its tendency to melt. Sediment continues to migrate from the upstream side of the shoal past the terminus even after ice advance has ceased. It is this process, in which the rate of advance of the shoal outpaces the advance of the ice, that leaves the glacier unsustainably grounded in progressively deepening water, eventually initiating retreat. A void at the upstream end of the shoal opens (t = 306 years), rapidly increasing basal motion from 30 to 350 m year−1 as the ice and bed decouple. Despite the longitudinal stresses imposed by the thin and rapidly deteriorating downstream, ice still grounded upon the shoal, more ice continues to reach the grounding line, triggering the irreversible positive feedback between thinning and ice flux4. Catastrophic retreat ensues over 20 years, leaving the glacier in the retracted phase and ready to begin the cycle anew. We emphasize that the model produces this cycle of advance and retreat in the absence of any externally imposed climate change: the TGC can be understood as a limit cycle rather than a sequence of alternations between two unstable steady states driven by an external climate trigger, as has been previously hypothesized13.The evolution of the modeled glacier exhibits each phase of the tidewater glacier Fig. . The gla17 or Brady9 Glaciers. However, the shoal at Columbia Glacier when advanced did not breach the water surface. Reaching the endmember case of a subaerial moraine is not necessary for the mechanism proposed above: if the shoal is advected faster than the glacier can advance while still subaqueous, then the unstable retreat is still initiated. Indeed, in the case of increased fluvial transport (either through enhanced meltwater or more easily transported sediment) the period and amplitude of cycles is reduced for precisely this reason. In addition, more robust diffusion may keep the shoal from breaching the surface . We find that the available meltwater cannot supply enough sediment to the terminus to overcome the shoal’s background diffusion: hillslope processes move sediment away from the shoal faster than deposition can supply it over the time scales considered here. Consequently, the shoal stays mostly stationary, as does the terminus . The meltwater regime under which fluvial sediment transport overcomes diffusion marks a bifurcation point at which sediment-driven advance begins  beginning mid-advance at t = 766 years  advance and the development of a TGC with a period of 1.4 ka and amplitude of 11 km. Thus glaciers in relatively warm polar regions such as Southeast Greenland or the northern Antarctic Peninsula may (counter-intuitively) advance in response to climate warming, albeit with a periodicity longer than in warmer climates. Such a scenario may also describe Svalbard’s glaciers, but the resulting dynamics would be superimposed upon the polythermal surge mechanism exhibited there22. Analysis of the convolved signal is difficult because of an incomplete understanding of either contributing process.Applying the same perturbation to our cold climate experiment, fluvial transport, and glacier advance both remain negligible. However, a more marked effect occurs when applied to an intermediate climate (meltwater availability between cold and temperate climates). Prior to warming, the glacier advances very little Fig. . However23. However, sediments may also provide critical micro-nutrients further from the glacier front24. TGCs destabilize the sediment delivery mechanism: during the extended phase, sediment is delivered subaerially. Conversely, during the retreated phase, sediment is delivered near the sea bed.Our results show that a relatively simple ice-sediment feedback mechanism can explain internally generated cyclicity in temperate tidewater glaciers over centuries to millennia, with ramifications for the coastal landscape and ecosystem. Changes in location and configuration of the proglacial moraine alters the manner in which sediment is delivered to the sea. In high melt regions like coastal Alaska and Greenland, sedimentation can suppress benthic productivity and biodiversity, due to drowning by marine snow and suppressed photosynthesis from turbidity25. Such circulation brings deep benthic and planktonic organisms to the surface where they become available to macrofauna26. Furthermore, invigorated circulation can also accelerate melting at the ice–ocean interface27.TGCs also introduce periodicity in freshwater flux partitioning and fjord circulation. In the canonical advanced phase, all glacier mass enters the ocean as liquid runoff because the terminus is grounded near or above sea level and freshwater enters the ocean via subaerial streams. The introduction of freshwater at the surface promotes stable stratification. We note that this is still the case for ice fronts that still calve but are grounded in shallow water as was the case for Columbia Glacier before its retreat. On the contrary, during the retreated phase freshwater is injected at depth leading to vigorous buoyancy plumes, which promote fjord circulation28. Icebergs are advected by wind, and their freshwater mixes more slowly and over a broader area than does runoff, adjusting fluxes of glacier-sourced nutrients into and out of fjords30. The chronic instability of sediment and freshwater fluxes may provide an important ecosystem service as waxing and waning of habitat suitability drives migration.When retreated, tidewater glaciers produce icebergs, which act as critical habitat for pinnipeds31. This mechanism may complicate regional studies of ice loss, which are important for predicting sea level rise and freshwater exports. However, shifts in climate have the potential to modify these cycles: an increase in melt enhances fluvial erosion and leads to faster, lower amplitude cycles, perhaps unto the point that they cannot be resolved. To the contrary, in cold regions, which lack the meltwater to produce TGCs, a warming climate may yield advance.The reduction in calving flux induced by sediment-driven shallowing of the terminus allows for advance even in a warming climate, which may explain the continued advance of Hubbard, Taku, Yahtse, and other glaciers, despite the use of these phenomena as evidence repudiating climate changeOur numerical model solves coupled equations representing ice and sediment transport. To compute ice flow, we use an approximation to the Stokes’ equations that accounts for both shear and membrane stresses, as well as a basal sliding that varies with changes in water pressure. Conceptually, ice is deposited by snowfall (which is a function of surface elevation), transported downstream by ice flow, and leaves the system either by calving  or by melt. Meltwater generated in this way is routed to the glacier bed and becomes available to move sediment, which is generated by the erosion from sliding at the glacier sole. We account for sediment transport due to fluvial entrainment and deposition, as well as gravitational diffusion. Sedimentation provides a feedback to glacial dynamics by adjusting the bed geometry, which alters the glacier’s stress configuration and potentially causes ice to either ground or float. In an effort to maintain simplicity, we neglect many interesting but conceptually non-essential physical processes including but not limited to tectonic uplift, isostasy, oceanic heat transfer, and sediment deformation.−1, far outpacing what could be expected based on sediment deformation17. This result is also supported theoretically32 and by large scale observations indicating that temperate glacial environments are the most fluvially erosive on earth16. Indeed, transport of sediment through pure deformation is not likely to account for the shoal advance rates observed in meltwater-dominated tidewater glaciers33. Depositional features directly observed in extant morainal shoals also suggest vigorous glaciofluvial transport34.Repeat radioechosounding at Taku Glacier has shown net erosion rates as high as 4 m year35). We simplify the momentum conservation equations by using the Blatter–Pattyn approximation37, which assumes hydrostatic pressure and negligible vertical resistive stresses. Such a model is suitable for simulating both creeping flow and sliding. Separation of the ice from the bed occurs when hydrostatic ice pressure drops below water pressure. We do not use a specific calving law, instead imposing a strong basal melt term to floating ice, such that half its thickness is lost annually. In addition, we neglect lateral drag for floating ice. This crudely simulates the presence of near terminus effects such as ice mélange when the front is pressed against an obstacle such as a terminal moraine, and has no effect on upstream dynamics otherwise5. With respect to existing calving formulations, this represents an adaptation of the “calving on flotation” parameterization, with differences occurring only when the glacier comes afloat but is still pressed against a downstream obstacle. This approach is empirically supported by the fact that the model produces the correct time scales of both advance and retreat when compared with the observed tidewater glaciers. Furthermore, the qualitative behavior of the model is insensitive to how quickly melting occurs, with the model still exhibiting periodicity in the case where no melt occurs and a floating tongue is allowed to develop. We neglect changes in water density due to freshwater flux.We use an ice flow model that simultaneously solves the equations of momentum and mass conservation  is produced and bedrock elevation B modified by glacial erosion mechanisms such as, quarrying, plucking, and abrasion, but we do not differentiate these. Instead, we assume that glacial erosion scales with the work done at the bed, which for our sliding law choice implies that it is non-linearly proportional to sliding velocity40:We define the equations for the production and transport of sediment and water over the entire domain, including both glacierized and unglacierized areas. Sediment , ρ is the density of ice and L the latent heat of fusion. We note that seasonality is not included in our computation of meltwater: only net annual ablation is included in runoff calculations, which would tend to underestimate the overall amount of water available. We convert the flux Qw to a vertically averaged water speed heff. Conservation of mass then implies thatρr and ρs are bedrock and sediment densities, and k the diffusivity of sediment due to hill-slope processes, 42, which leads to the expressionc is the fluvial erosion efficiency, which we chose such that net erosion 200 m upstream from the terminus is ~2 m year−1 and ~5 m year−1 at 1 km, based on repeated radar measurements17. The deposition rate is proportional to the fluvial sediment flux Qs normalized by the water flux−1, the settling velocity of fine silt43, which approximates the median grain size at the base of boreholes drilled by the authors near the terminus of Taku Glacier. We close the model with a second mass conservation equation accounting for mobile sediment, with the additional assumption that this quantity changes quickly relative to glacier time scales and the time derivative can be neglected, yieldingWe assume that sediment is fluvially transported. Water is provided by surface melt (which we assume immediately reaches the bed) and (to a much lesser degree) basal and englacial melt produced by sliding and deformation. These quantities are integrated along the hydraulic gradient to determine the total water flux F through the subglacial hydrologic system is a problem that is operationally unsolved. Approximating it requires explicit models of subglacial hydrology, which we did not implement because they are both computationally expensive and possessed of many unconstrained and unobservable parameters. We instead sidestep this issue by making the simple assumption that water flows in the direction of the ice surface gradient, with a parameterized flow depth providing what amount to balance velocities. If we also assume a geometry that is axially symmetric and transversely uniform (with respect to the downstream direction) then we may easily width integrate the model, reducing computational time by collapsing the model’s spatial extent to a single flowline. Such an assumption is certainly not valid over short length and time scales, with real glaciers exhibiting a great deal of flow-transverse heterogeneity in flux and efficient channels potentially transporting most of the water (and perhaps sediment) over a relatively small spatial footprint. However, we argue that our simulations are sufficiently long and the subglacial drainage configuration sufficiently variable, that averages of water and sediment flux over the glacier width capture the features salient to the purposes of capturing the interactions of ice and sediment at a large scale. A more theoretical justification of this assumption is outside the scope of this paper; instead, we appeal to heuristics by noting that the front of Taku Glacier exhibits a glacial outwash plane that extends across its entire terminus, despite extant major channels only exiting the glacier front in a few discrete locations.Specifying the flow direction FQ with the one-dimensional operator W(x) is the glacier width. The additional term Wmax = 14 km and Wmin = 3 km are the maximum and minimum domain widths, θ = 5 km is a length parameter, and κ = 1 is a shape function. We perform this lateral integration for the mass conservation equations of both ice and sediment. We do not account for lateral drag in the momentum conservation equations.In any case, this width-averaging has the effect of replacing the two-dimensional divergence operator ∇·37 in width-integrated form using a linear Galerkin finite element method in the horizontal dimension. We split the domain into ne = 1000 elements, for an average element size of ~Δx = 75 m. We discretize the vertical dimension using an ansatz spectral element method, in which we assume that the vertical velocity profile is adequately approximated by the linear combination of a zero-order and fourth-order polynomial35.We numerically solve the first order approximation of the momentum conservation equations44. We discretize the mass conservation equation in time using the Crank–Nicholson method, which provides second-order accuracy and good numerical stability. We used a time step of Δt = 30 days for all simulations. This timestep guaranteed convergence at each time step for all experiments. Further refinement did not substantially change the numerical solutions. All finite element discretizations were performed with the FEniCS library45.We solve the depth-averaged form of the mass conservation equation to evolve the ice geometry through time. As such equations are nominally hyperbolic, we use a Streamline Upwind Petrov–Galerkin (SUPG) finite element method to stabilize the transport equation46. This variational inequality solver allows for the specification of a lower bound on ice thickness to prevent the non-physical scenario of a negative ice thickness. In practice, we specified a lower ice thickness bound of 1 m to prevent numerical singularity in the momentum equations. Newton’s method requires a Jacobian matrix, which we computed analytically using the automatic symbolic differentiation capabilities of the FEniCS library.The non-linear mass and momentum conservation equations were solved simultaneously using the SNES VI Newton solver from the PETSc numerical solver libraryWe simultaneously solve the equations of mass conservation for deposited sediment, transported sediment, and meltwater transport. The former is discretized using Galerkin finite elements and the latter two using the SUPG finite element method. Time discretization is accomplished with the Crank-Nicholson method, and numerical solution of these non-linear equations is performed with the PETSc SNES VI solver, once again utilizing the FEniCS library’s capacity for symbolic differentiation. We specify a lower bound of zero on sediment thickness.Bmax = 2200 m at a sharp ridgeline, to sea level 30 km downstream. Here, the topography forms a fjord, which attains a maximum elevation of Bmin = 300 m another 45 km out to sea. Sinusoidal bumps with a wavelength of L/2 were superimposed, where L = 45 km is a characteristic length scale. The topography was given by the functionp = 0.3 is a shape factor and As = 100 m is the amplitude of topographic undulations.The initial bedrock geometry varies between f = 2 is a shape function. The temperate climate experiment prescribes a maximum accumulation rate of 2.5 m year−1 ice equivalent at 2200 m, and −8 m year−1 at sea level, representative of conditions in Southeast Alaska . The equilibrium line altitude is ~1000 m, and we impose no external climate variability. The cold experiment uses a −1 and −1. The intermediate climate has −1 and −1, respectively.In all experiments, climate is imposed through an elevation-dependent specific mass balance given by the functionhs uniformly zero). Our criterion for steadiness is that ice volume changes by <10−2% at each time step. After this initial state is computed, we allow the model to run through a single cycle of advance and retreat, and take the resulting sediment configuration as the initial condition for the model runs analyzed in the main body of the paper. We do this to eliminate transient signals associated with abruptly turning sedimentation on. In cases that do not exhibit periodicity over 10 kyr of model time, we initialize experiments from the 10 kyr state.We begin each model run from a steady state in the absence of sedimentation (i.e. Eqs. The Python script used to perform these simulations is distributed with this manuscript as Supplementary InformationSupplementary Movie 1Supplementary SoftwarePeer Review File