Zebrafish have an impressive capacity to regenerate neurons in the central nervous system. However, regeneration of the principal neuron of the evolutionary conserved cerebellum, the Purkinje cell (PC), is believed to be limited to developmental stages based on invasive lesions. In contrast, non-invasive cell type-specific ablation by induced apoptosis closely represents a process of neurodegeneration. We demonstrate that the ablated larval PC population entirely recovers in number, quickly reestablishes electrophysiological properties, and properly integrates into circuits to regulate cerebellum-controlled behavior. PC progenitors are present in larvae and adults, and PC ablation in adult cerebelli results in an impressive PC regeneration of different PC subtypes able to restore behavioral impairments. Interestingly, caudal PCs are more resistant to ablation and regenerate more efficiently, suggesting a rostro-caudal pattern of de- and regeneration properties. These findings demonstrate that the zebrafish cerebellum is able to regenerate functional PCs during all stages of the animal’s life. Regeneration of neurons in the central nervous system of mammals has been demonstrated to occur, but it is rare and limited to a few brain areas . In contpancreas-associated transcription factor 1a (ptf1a)  . In zebr (ptf1a) . Controv (ptf1a) . Togethe (ptf1a) .Based on the extensive adult neurogenesis and regenerative capacity, it is widely believed that all neurons within the zebrafish central nervous system are able to regenerate . Yet, inPCs constitute principal information processing neurons of the cerebellum and provide direct output to neurons of the vestibular nuclei . Hence, In zebrafish, during embryonic stages, the entire cerebellar primordium including PCs regenerates after surgical removal, due to repatterning processes in the hindbrain . Also, aYet, larval PC regeneration upon local wounding has been based on the appearance of new PCs, but this could represent ongoing growth of the PC population rather than a regenerative response. In addition, PCs considered to have regenerated cannot be distinguished from non-injured neighbors, therefore the proper physiological integration of regenerated larval PCs into existing circuitry could not be revealed, therefore also in larvae functional PC regeneration is still in question .Conversely, injuring the adult cerebellum may mask the ability to regenerate PCs. Extensive surgical ablations remove important progenitors and the cellular and extracellular environment likely required for PC regeneration. Local traumatic injuries instead may be counteracted by plasticity of neighboring PCs. In addition, acute traumatic cerebellum injuries affect numerous cell types beyond PCs in a defined area, and if invasive, induce wound healing that could influence regeneration .Instead, degenerative diseases of PCs affect the entire population of a single-cell type by non-invasive cell death mechanisms . Likely,We have recently introduced transgenic PC-ATTAC zebrafish, in which PCs can be selectively ablated by apoptosis through non-invasive administration of Tamoxifen . This moCarriers of the stable transgenic zebrafish strain PC-ATTAC express a membrane-targeted red fluorescent protein (FyntagRFP-T) as a reporter together with a Tamoxifen-inducible Caspase 8 selectively in postmitotic cerebellar Purkinje neurons . BetweenAbout 5–7 dpt first red fluorescent PCs reappeared, and already after about 11 dpt nearly half of the PC population compared to controls was replenished . This deThe partial PC regeneration observed within a few days could either represent a regenerative response, the continuation of PC layer development or a combination of both. Only in the latter case the PC population would regain its full size compared to controls. Therefore, PC numbers were quantified from 1 to 6 months post-treatment (mpt) . CompareIn addition, no apparent anatomical differences of the cerebellar cortex at 1 and 6 mpt could be observed by immunohistochemistry against the PC-specific antigen ZebrinII on sagittal sections from PC-ablated and control specimens . In addiTo obtain further insight into the temporal dynamics of PC replacement, proliferating cells were labeled by Bromodeoxyuridine (BrdU) treatment for 24 hr at different time points post-ablation followed by their immunohistochemical detection and quantification. During the first 7 days post-ablation no significant increase in proliferating cell numbers could be observed in PC-ablated larvae compared to EtOH-treated controls . The samgfap:EGFP) strain with green fluorescent radial glia cells into the PC-ATTAC background. Yet, after PC ablation none of the reappearing PCs expressed both fluorescent reporters  can be used for monitoring these VZ-derived progenitor cells (ptf1a:EGFP) larvae in the PC-ATTAC background were observed following PC ablation guration . These sguration . First Pguration .ca8:GFP)bz12 background with ablation rates above 90% were investigated vu12 background, in which ECs are marked by EGFP expression  are considered unable to replace lost PCs, like the mammalian cerebellum . The effWe therefore, induced apoptotic cell death in heterozygous PC-ATTAC adults at 5 months of age  by threeptf1a:EGFP) adult fish showed that a source of progenitor cells for PCs is still present at advanced adult ages (ptf1a:EGFP) × Tg(PC-ATTAC) zebrafish, at least a few differentiated PCs (RFP positive) showed remaining GFP fluorescence derived from ptf1a:GFP progenitors providing evidence of ptf1a-expressing cells as progenitors of PCs also in the adult cerebellum (Fluorescent stereomicroscopy recordings from living fish confirmed the results of PC quantification from fixed tissue. At 2 weeks post-treatment (wpt) control PC-ATTAC fish displayed a strong and continuous red fluorescence from the dorsal surface of their CCe, while in PC-ablated specimens, fluorescence was faint at best, or completely absent . Strikinult ages . Furtherrebellum .PC quantification on sagittal sections by immunohistochemistry against TagRFP-T and the PC-specific epitope ZebrinII  confirmeThese findings demonstrate that degeneration of the entire PC population in the adult zebrafish cerebellum does not represent an irreversible loss of these principal cerebellar neurons as previously thought. Instead, the adult zebrafish cerebellum maintains an impressive capability to regenerate significant numbers of PCs counteracting degeneration.With an average of 8% PC survivors, the caudal area of the adult CCe was more resistant to induced apoptosis than the rostral area, in which PC survival was sparse. When PC regeneration in sagittal sections of the CCe was evaluated based on morphological landmarks, medial and caudal regions of the PC layer were more efficient in replenishing lost PCs compared to rostral areas, irrespective of subdividing the CCe into two or three areas . This paRecently, four different PC subtypes have been distinguished based on soma size correlating with a different dendrite morphology and physiology, but not being segregated with respect to their location in the PC layer . By measFunctional PC regeneration implies the restoration of behavior caused by adult PC loss known to result in a compromised exploration . In the Acute invasive injuries have demonstrated the ability of the teleostean cerebellum including zebrafish to regenerate damaged neuronal structures by proliferation, migration and differentiation of neuronal progenitors . With reAcute injuries, which may represent only minor cases of cerebellar damage, have a massive impact on the surrounding neural tissue and are either locally restricted, as in the case of stab wounds, or lead to the destruction of the entire cerebellar architecture, as in the case for hemisphere removal or blunt force damage . More coptf1a. Interestingly, this replacement of PCs did not occur by a rapid wave of significantly increased progenitor cell proliferation, but instead by a continuation of cerebellum growth accompanied by a slow but steady replenishment of PCs in number, until a PC population indistinguishable in size from non-PC-ablated control fish was reached.We have readdressed this open question of PC regeneration with the help of the PC-ATTAC model, in which PCs can be ablated specifically by inducing apoptosis and reappearing PCs can be monitored by fluorescent protein expression . Using tAn explanation for the slow but steady regeneration and stability of the circuitry can be derived from our physiological studies. Attached cell patch-clamp recordings from PCs in non-ablated control specimens confirmed that zebrafish PCs mature physiologically quite rapidly within 2 days, and the entire PC population displays stable electrophysiological patterns already at 8 dpf, 5 days after the first PCs are born . In PC-aWe showed that during the acute phase of PC apoptosis this loss of inhibitory input resulted in ECs, the direct PC efferences, in a significantly increased average firing frequency of 47% and highest burst frequency of 82% at 3 dpt. With the reappearance of PCs at 4 dpt, a reduction in EC firing frequency back to normal values at 10 dpt was observed. This suggests that reappearing PCs quickly reintegrate into the PC circuitry. Thus, larval PCs do not only regenerate in cell number, but likely establish proper synaptic contacts. Alternatively, ECs adapt by plasticity to the lack of PC input along the same time-course.Interestingly, the pattern of physiological maturation appeared to be the same between wild-type and regenerating PCs. An initially increased complex to simple spike ratio was reduced during PC maturation from 5 to 7 dpf, similar to firing patterns recorded from regenerating PCs from 5 to 7 dpt. This alteration in complex to simple spike ratio has been suggested to be derived from multiple climbing fiber innervation of PCs that is subsequently reduced by pruning until the strongest climbing fiber input remains . Once prA fast recovery of mature and stable physiological patterns in regenerating PCs predicts a similarly quick recovery of PC function in governing behavior. In zebrafish larvae, impaired PC function has been shown to result in eye movement defects by means of saccade performance during the OKR , to leadStudies on acute cerebellum injuries have established that PCs lose this ability to functionally regenerate during juvenile and young adult stages at about 3 months post-fertilization . HoweverWe have therefore reinvestigated the potential of PC regeneration in the adult zebrafish cerebellum in the context of PC-specific cell ablation clearly beyond young adult stages starting at 5 months post-fertilization. This approach more closely reflects a degeneration of the PC population, as it occurs in the many known human ataxias. Surprisingly, we observed a strong and extensive regenerative response reestablishing about 28% of the entire PC population within 4 months and 30% within 1 year. Moreover, PCs also reestablished direct efferent projections to the vestibular nuclei in the ventral hindbrain. Compared to larvae, both the process of PC ablation and PC death as well as the regeneration required extended time periods. This is likely due to both a deceleration of neurogenesis and a reduced progenitor population  as well ptf1a-expressing progenitor cells to differentiated PCs in adults compared to the larval cerebellum, a lack of need for further regeneration, prolonged PC differentiation processes in a compact and mature cerebellar cortex or incipient aging influencing or counteracting PC generation.Behavioral tests revealed that PC regeneration in adults was accompanied by recovery of PC-mediated compromised exploratory behavior. Therefore, PCs do not only reappear, but also reestablish characteristic functions of a wild-type PC population. Furthermore, these findings suggest that about one third of the size of the adult wild-type PC population is sufficient to reestablish cerebellum-controlled behavior maybe eliminating a need for further PC regeneration. Clearly, the adult zebrafish cerebellum is capable of significant functional PC regeneration under conditions of cell type-specific PC loss. That not the entire adult PC population is reestablished may be due to the apparent lower ratio of A recently established zebrafish model with compromised adult PC functions displayed defects in both exploratory and locomotor behavior . InteresIn addition to regional differences, patterned PC death in mammalian cerebellar diseases is also dependent on PC subtypes . The exibrass pigmentation mutant zebrafish were used for matings as indicated for each experiment. No selection criteria were used to allocate zebrafish of both sexes to any experimental group. Embryos and larvae were maintained in zebrafish 30% Danieau rearing medium 2, and 5 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] pH 7.2) at 28°C. Larvae older than 7 days and adults were maintained in fish tanks according to standard protocols at 28°C, under light–dark conditions (simulating day–night cycle), and constant running water exchange. Stable transgenic lines used in this work include the Tg(ca8:-FynTagRFP-T2A-Casp8-ERT2)bz11 line abbreviated (PC-ATTAC) (5ca8:EGFP)bz12, Tg(olig2:EGFP)vu12, Tg(ptf1a:EGFP), and Tg(gfap:EGFP)mi2001. All experimental protocols for animal research were approved by governmental authorities of Lower Saxony, LAVES, (AZ33.19-42502-04-20/3593). All efforts were made to use only the minimum number of experimental animals necessary to obtain reliable scientific data.All animals were raised and kept in the zebrafish facility in accordance with established practices adhering to the guidelines of the local government . AB wildC-ATTAC)  approvedFor PC ablation experiments in larvae (4–6 days post-fertilization), 4-OHT  at a concentration of 8 µM was added to the rearing medium for 18–20 hr. Since EtOH was used as solvent for 4-OHT, 0.4% EtOH was applied to the rearing medium of a control group for the same time period. On the next day, the medium was exchanged three times and the ablation rate of PCs was analyzed under a confocal laser scanning microscope (LSM). Ordinary ablation rates higher than 85–90% at day 2 post-treatment (dpt) compared to the control group were considered successful to use these larvae for further experiments.To deplete PCs in adults, 5 months old zebrafish were incubated overnight in fish facility water supplemented with 4 µM Endoxifen , an active metabolite of 4-OHT, which proved more efficient in adult PC ablations. Three consecutive treatments were performed each overnight at 28°C in the dark with resting and feeding periods during the day. Since dimethyl sulfoxide (DMSO) is used as solvent for Endoxifen, control groups were treated with 0.13% DMSO. Afterwards, the chemicals were washed out at least three times with fresh fish facility water and specimens were returned to the fish facility.Click-IT Alexa Fluor 647 Imaging kit, Thermo Fisher, C10340) at 22 dpt during 24 hr at 100 µM (stock solution 2.5 mg/ml diluted in DMSO) in a 6-well plate.For proliferation studies in larvae, 10 mM BrdU diluted in Danieau’s solution was added to the fish water after 4-OHT treatment in a 24-well plate (1 ml/well) for 24 hr, or up to 5 consecutive days in case of cumulative BrdU analysis. For BrdU/EdU double pulse chase experiment, incubation of BrdU was performed at 18 dpt for 24 hr at 10 mM, and incubation of EdU (t-test for two groups comparison (unpaired t-test two tailed) was applied.After the treatment, larvae were euthanized and the brains were isolated and processed for immunohistochemistry. The number of proliferating cells in each group (4-OHT/EtOH solvent control) within the PC layer of the cerebellum, and therefore excluding the caudal upper rhombic lip, was counted using the cell counter plugin of the image analysis software FIJI (see below). Student’s Brains of both, larvae and adults, were isolated and fixed by immersion in 4% paraformaldehyde (PFA) overnight at 4°C. For isolation of larval brains, fish were euthanized with an overdose of Tricaine and prefixed with PFA for 20–30 min, to allow for complete removal of the skin and brain isolation (using a minute pin 00 as dissection tool). After overnight fixation of the brain, PFA was washed out with an excess of phosphate-buffered saline (PBS), and the tissue was processed to a methanol/PBS series  for gradual dehydration and final storage in 100% methanol at −20°C.4, Alexa Fluor azide, 1× Click iT buffer additive; following the manufacturer’s instructions), incubated for 30 min at RT in the dark, and finally washed with 3% BSA in PBS.For immunostaining, larval brains were rehydrated in a methanol–PBS series  and washed several times in PBS. Larval brains were processed for immunostaining as whole mount. After washing with PBS, antigen retrieval with 10 mM citrate buffer was achieved by heating samples in a water bath until boiling. After cooling to room temperature the brains were rinsed five times in PBS-T (PBS with 1% Triton X-100) for 5 min each, incubated in 100% precooled acetone for 15 min at −20°C to improve tissue permeabilization, followed by additional five PBS-T washing steps. Nonspecific protein-binding sites were blocked in 5% normal goat serum in PBS-DT-I: PBS, 1% bovine serum albumin (BSA), 1% DMSO, 1% Triton X-100 for 1 hr at room temperature followed by primary antibody incubation in PBS-DT-II  overnight at 4°C and additional 3 hr at room temperature. The primary antibodies: Rabbit anti-tagRFP , mouse anti-ZebrinII , chicken anti-GFP , and rat anti-BrdU . Before anti-BrdU antibody application an additional incubation step with 2 N HCl was performed for 30 min at 37°C to denature the DNA for better anti-BrdU antibody accessibility. Subsequently brains were washed five times in PBS-T several times for 5 min each at room temperature with constant agitation and incubated with the secondary antibody diluted in PBS-DT-II overnight at 4°C. Secondary antibodies: Alexa goat anti-rabbit 568 , Alexa goat anti-mouse 488 , donkey anti-chicken-FITC , and goat anti-rat 488 . For DAPI  stained brains were incubated for 30 min at RT followed by several PBS-T washes. Finally, brains were thoroughly rinsed in PBS-T and PBS before imaging. In the case of EdU staining, EdU was detected after the incubation with primary antibodies  during the immunostaining procedure. The brains were washed twice with 3% BSA in PBS, washed with 0.5% Triton X-100 in PBS, washed twice with 3% BSA in PBS, added Click-IT reaction cocktail . Brains were pre-embedded in 1% low-melting agarose in PBS, and then embedded in 3% agarose. Vibratome settings: speed: 1 mm/s, vibration frequency: 75 Hz. The immunostaining was performed as described above, except for the antigen retrieval and acetone incubation. All incubations steps using adult brain sections were performed on floating sections, and eventually transferred to glass slides for confocal microscopy imaging.Zebrafish embryos were injected at the one cell stage with plasmid DNA (25 ng/µl supplemented with 25 ng/µl mRNA encoding Tol2 transposase) .zebrafish artificial cerebro spinal fluid  containing 10 µM d-Tubocurare . After complete loss of motor functions, the medium was removed and the larva was embedded in 2% low-melting agarose diluted in z-ACSF. Skin and skull tissue were then carefully removed with a sharp thin glass needle without damaging the underlying brain tissue to expose the cerebellum bz11 and Tg(–7.5ca8:GFP)bz12. Double-positive larvae were screened at 4 dpf and PCs were ablated by standard procedures. Double transgenic larvae incubated in 0.4% ethanol for 20 hr served as controls. The ablation rate was monitored by imaging larvae from 0 to 2 dpt each day under a confocal LSM and cell number analysis was performed. If the ablation rate was stable among all larvae and at 2 dpt over 90% compared to controls of the same age, the experiment was considered successful and patch-clamp recordings and imaging was performed from 3 to 21 dpt (10–28 dpf).For regeneration studies of PCs, double transgenic fish carrying the PC-ATTAC and the –7.olig2:EGFP)vu12 vu12  were croElectrophysiological data were recorded with the Patchmaster Software from HEKA  and afterwards analyzed offline with Igor Pro 6.37  with a program code written by Marcus Semtner . The counting of complex spikes was performed manually, whereas simple spike counting was automated. By this algorithm-based analysis all simple spikes were marked by the program, complete identification of all simple spikes was confirmed by subsequent visual inspection. Only traces which were stable for a minimum of 75 s and showed consistent spike patterns with comparable amplitudes were considered successful and taken for the final data analysis. The datasets from all recordings were collected in Excel 2019  and then further analyzed and graphically represented with Prism9 . Statistical relevance was analyzed by two-way analysis of variance (ANOVA) with Šídák’s multiple comparisons test.t-test for parametric or normal distributed data.The setup for this visuo-motor behavior test consisted of a movie projector playing a stimulating movie with vertical black and white stripes moving horizontally in constant speed projected over 180° across a circular screen as previously reported . Single The analysis of free-swimming using larvae was performed using a custom-built zebrafish box , illuminated with infrared and regular bright field light. Specimens were automatically tracked with the Ethovision XT 12 software . The larvae placed in a 15-cm Petri dish were transferred to the behavior test room (at 28°C) for acclimatization at least 2 hr before the experiment. Subsequently, larvae were placed in a 12-well plate  and let alone for an adaptation period of 5 min. The tracking was performed over a time period of 6 min. In order to quantify thigmotaxis, the arena was subdivided into middle and edge zones by the analytical software. Additional parameters measured were swim speed and total distance traveled. The larvae were returned to their respective fish tanks, and sacrificed at the end of the experiment. For more consistency of the results, the test was always carried out between 2:30 pm and 5:30 pm, with alternating of each group for swimming test analysis. The statistical tests applied were: ANOVA test for multiple group comparison .The NTT was performed with a custom-built zebrafish box  illuminated with infrared and regular bright field light, and automatic tracking with the Ethovision XT 12 software . The fish were placed into behavior test room (at 28°C) approximately 1 hr prior to testing for acclimatization. For the NTT, the swimming of the fish was recorded for 6 min without a pre-adaptation period to the novel tank. The fish tank used for the test had a volume of 1.5 l . After finishing the tracking, adult fish were returned to their respective home tanks, and sacrificed at the end of the experiment. For the tracking, the arena was subdivided into upper and bottom zones by the analytical software. Due to some variability of the NTT behavioral assay, individual control fish that showed clear exploration behavior of at least 5% occupancy in the upper half of the tank were chosen for further analysis. Mann–Whitney two-tailed test for no parametric data was applied.For imaging, larvae were anesthetized with 0.015–0.02% Tricaine  dissolved in 1% low-melting agarose/Danieau solution. Images of the larval and adult zebrafish cerebellum were acquired using an SP8 laser scanning confocal microscope  with a ×40 objective. Images were processed with the imaging and analysis Leica LasX software.https://imagej.net) and Ethovision . The quantification of PCs in juvenile and adult cerebelli was carried out after vibratome sectioning and immunostaining with anti-tagRFP and anti-ZebrinII antibodies. Semi-automated counting of cells was performed with the cell counter plugin of the FIJI software, in which every cell is initially identified manually followed by their automated detection with a z-stack of images to avoid, that cells are counted twice. The total amount of PCs per brain corresponds to the sum of all the sections. In order to minimize the error of double counting the same PCs from consecutive sections, cutting thick (70 µm) vibratome sections was considered the most appropriate sectioning method.Additional analysis of data was carried out with following software: FIJI .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact. Using different zebrafish lines in combination with morphological and functional analysis, the authors provide compelling evidence that ventricular progenitors retains the life-long ability to regenerate PCs. At larval stages the newly regenerated PCs form fully functional circuits that lead to normal behavior. In adult, PC regeneration is less efficient but sufficient to support exploratory behavior. This fundamental study resolves the controversial issue of whether adult PC regeneration is possible and demonstrates that newly formed PCs at larval and adult stages can form functional circuits that supports normal behavior. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Marianne Bronner as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Luis Rodrigo Hernandez-Miranda (Reviewer #3).Thank you for submitting your article "Lifelong regeneration of cerebellar Purkinje neurons after induced cell ablation in zebrafish" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.The reviewers agree that this is quite exciting work; however, they all coincide in two flaws that need to be addressed to convincingly support the conclusions of the study.The first is related to cell proliferation. This is not exhaustively analysed and therefore it is unclear whether it does contribute to Purkinje neuron regeneration at all the stages analysed.The second issue relates to the identity of the adult precursor cells, which should be established.The reviewers raise a few other criticisms that deserve attention if possible. The manuscript will also benefit from a more careful structure and labelling of the figures, which is not always accurate.Reviewer #2 (Recommendations for the authors):The manuscript would profit from a better emphasis on important conclusions. One specific example is some of the titles in the Results section which are rather descriptive and miss the opportunity to give the reader the immediate conclusion of the coming section (for example Progenitor cells of regenerating PCs). In general, this manuscript has a lot of exciting, important findings, which feel sometimes hidden in a lot of descriptive phrasing.Comments and questions:1. Is functional recovery due to new PCs being generated or could remaining cells and circuits rewire?Would inhibition of progenitor cell proliferation  assess whether functional recovery is inhibited in that scenario and therefore indeed due to regeneration of PCs?Alternatively, would a second tamoxifen treatment, which should ablate newly regenerated PCs, lead to a lack of functional recovery?2. Regeneration of PV neurons does not reach control numbers in the adult cerebellum, what is the interpretation of that result?Are the Ptf1a numbers in the adult cerebellum lower in relation to the ones in young animals, such that the ratio of progenitors to neurons is less than in larvae. Are there potentially too few progenitors to lead to full regeneration? Can this be quantified?3. A GFP transgenic reporter (ptf1a:gfp) was used to determine that these cells give rise to Purkinje cells and that gfap positive radial-glia-like cells are not the source.Can it be excluded that gfap-positive cells generate ptf1a-positive progenitors which in turn would give rise to PCs? In the pictures in figure 2, it seems that gfap- and ptf1a reporter-positive cells are located in very similar regions, could there even be double-positive cells? It would be important to determine their respective localization and potential co-localization.Reviewer #3 (Recommendations for the authors):+; Supplementary Figure 1A-D) during the regeneration of Purkinje cells. Which area was chosen for these quantifications?. When I look at Supplementary Figure 1A, I would quantify 34 BrdU+ cells in the PC-ablated condition vs 15 BrdU+ cells in the control condition. Please delineate the area that was chosen for the quantifications.1) The authors describe no change in proliferative cells  cells is seen in the ablated condition, as one of two possibilities can occur during the regeneration of Purkinje cells: (a) precursors divide faster to compensate for the loss of Purkinje cells or (b) precursors keep cycling over prolonged times, that is over more days than in controls.I would recommend the authors carry out a small experiment to assess the cycle length of precursor cells in the ablated larval condition, that is by combining pulses of BrdU and EdU. This could explain why no difference in proliferating (BrdU2) The authors define ventricular zone Ptf1a+ cells as the precursors for the regenerated larval Purkinje cells. This, in my view, is not surprising as ventricular zone Ptf1a+ cells are precursors of Purkinje cells in most animals that have a cerebellum. Here, I have a few suggestions for the authors which can help the reader of this work to better understand this work:a) Could you please indicate, in the introduction, the temporal window of Purkinje cell production in Zebrafish?b) Could you please describe, in the introduction, whether ventricular zone Ptf1a+ cells are the natural precursors of Purkinje cells in Zebrafish?c) Could you please mention whether inhibitory interneurons emerge from ventricular zone Ptf1a+ cells, as is the case in mammals? This can also be discussed in the Discussion section.3) Whereas the authors show that Ptf1a+ cells are the precursors for the regenerated Purkinje cells in the larval stage, the suggestion that these cells are the source of regenerated Purkinje cells in the adult stage must be tested in this work. In my view, the elucidation of this can be one of the most exciting aspects of this work.Therefore, I would recommend the authors carry out an experiment similar to that described in Figures 2A-D. That is using the Tg and Tg in the adult ablated PC-ATTACTM Zebrafish background, and quantified the number of double positive RFP/GFP cells in the regenerating Tg(gfap:EGFP);PC-ATTACTM and Tg(ptf1a:EGFP);PC-ATTACTM animals. I would suggest focusing at least on the first 4 months post-regeneration. I know that this is a 4-month experiment, but I think this experiment will significantly add to this work.4) Please restructure the manuscript. It is very confusing the description of Figures in the text versus the actual labeling of the Figures. It took me a lot of time to understand that the provided Supplementary Figure 1 contains the panels described for Figure 1 —figure supplement 1A-D; Figure 2—figure supplement 1E; Figure 2—figure supplement 1F; etc. Please separate the Figures and label them clearly. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.The reviewers agree that this is quite exciting work; however, they all coincide in two flaws that need to be addressed to convincingly support the conclusions of the study.ELife that due to a parental leave of both lead authors until October 2022, working on the revision of the manuscript was delayed. We have carefully read all concerns and have addressed them point by point also delineating the changes that were made to the carefully revised manuscript.We are grateful for the smooth and balanced handling of our manuscript. We appreciate very much the understanding of The first is related to cell proliferation. This is not exhaustively analysed and therefore it is unclear whether it does contribute to Purkinje neuron regeneration at all the stages analysed.We have followed this advice and have added the suggested analysis of BrdU positive cells at a later stage of PC regeneration, which we answer in our response to the first question raised by reviewer 1. In addition, we have performed the dual BrdU/EdU-labeling experiment to address further upregulation of proliferation and a potential prolongation of cell cycle exit as suggested in the first comment of reviewer 3. We would like to refer the editor to our answers to these points and we hope that we could have clarified this concern.The second issue relates to the identity of the adult precursor cells, which should be established.ptf1a:GFP expressing cells are progenitors of PCs. For this we have established double transgenic ptf1a:GFP/PC-ATTAC adult zebrafish in which we were able to identify red fluorescent PCs expressing with remnants of GFP expression. We explain these findings in our answer to question 3 of reviewer 3 and reveal these new data in a revised new Supplementary Figure 13.We have also attempted to solve this question, and we now show that indeed in the adult cerebellum ptf1a:GFP cells as progenitors of regenerating PCs in adults requires the establishment and validation of additional sophisticated transgenic strains, which in our opinion represents a whole scientific project on its own that extends beyond the current focus of the manuscript.To however trace The reviewers raise a few other criticisms that deserve attention if possible. The manuscript will also benefit from a more careful structure and labelling of the figures, which is not always accurate.We have read through all these points of concern and have carefully addressed each criticism. Please find our detailed answers below which we hope resolve of points of dispute.Reviewer #2 (Recommendations for the authors):The manuscript would profit from a better emphasis on important conclusions. One specific example is some of the titles in the Results section which are rather descriptive and miss the opportunity to give the reader the immediate conclusion of the coming section (for example Progenitor cells of regenerating PCs). In general, this manuscript has a lot of exciting, important findings, which feel sometimes hidden in a lot of descriptive phrasing.We thank the reviewer for these supportive comments. We have followed the advice by reformulating the headlines of the individual paragraphs of the result section into conclusions and we have significantly revised the entire manuscript accordingly.Comments and questions:1. Is functional recovery due to new PCs being generated or could remaining cells and circuits rewire?Based on the PC ablation and functional recovery in adults, we estimate that about 1/3 of the PC population is required to establish sufficient cerebellar circuitry for functional recovery in larvae. With ablation rates close to or above 90% of the PC population, the few remaining cells will not be able to reestablish a functional cerebellar cortex in larvae. In particular, since the cerebellar PC population is known to be subdivided in different functional compartments such variation in PC connectivity could not be covered by the few remaining PCs. Therefore, it is highly unlikely that rewiring of ablation-resistant PCs is sufficient to recover cerebellar functions, while the many regenerating PCs do not reestablish proper circuitry. Moreover, our electrophysiological data showing that all replenished PCs establish expected patterns of neurotransmission argue against such a scenario.Would inhibition of progenitor cell proliferation  assess whether functional recovery is inhibited in that scenario and therefore indeed due to regeneration of PCs?Alternatively, would a second tamoxifen treatment, which should ablate newly regenerated PCs, lead to a lack of functional recovery?This is an interesting experimental suggestion, but it is technically not feasible. Global inhibition of cell proliferation will also affect PC afferent and efferent structures of first and higher orders making it impossible to discriminate between an impaired functional recovery caused by insufficient numbers of PCs or defects in differentiation along the circuitry.ptf1a-enhancer. But this regulatory element drives expression as well in progenitors of eurydendroid cells, the first order efferents of PCs, and inhibitory interneurons as first order afferents of PCs not allowing one to discriminate between manipulating PC, EC and inhibitory interneuron progenitor proliferation.For a PC progenitor specific inhibition of proliferation using genetics the best regulatory element available would be the Alternatively, would a second tamoxifen treatment, which should ablate newly regenerated PCs, lead to a lack of functional recovery?Authors: We have followed this advice and performed this experiment. 10 days after the first Tamoxifen-induced PC ablation, when roughly 50% of the PC population was reestablished (49.1 %), a second Tamoxifen-treatment was performed reducing again the PC population to 6.6%. Within the following 10 days the PC population recovered to 64% compared to numbers of PCs in control larvae. This shows that PCs can repeatedly regenerate and also replace the newly regenerated PCs for another time.To address the functional recovery of these PCs we performed electrophysiological recordings. Similar to the first ablation, the average frequency and highest frequency of PCs decreased significantly, while the ratio between complex to simple spikes increased during the acute degeneration phase (3 days post second treatment). With the second 10 days regeneration period the average frequency and complex to simple spike ratio recovered to values indistinguishable to PCs in non-ablated control larvae. This indicates that also after the second ablation repeatedly regenerating PCs recover to normal firing patterns.Moreover, if an ablation resistant PC population existed and regenerating PCs remained non-functional, this ablation resistant fraction of PCs would have reestablished normal PC firing patterns by plasticity mechanisms during the first regeneration period. Consequently, the second ablation should not affect the physiological signature of these potential ablation resistant PCs anymore. Yet, our physiological measurements revealed clear physiological changes and patterns of recovery reminiscent to the first PC ablation and regeneration recovery. This is strong evidence against the existence of a small but ablation resistant PC population that reestablishes the function of the entire PC population merely by plasticity. We are therefore convinced that the repeated functional recovery of the large majority of the ablated PC population is driven by the incorporation and proper rewiring of newly added regenerating PCs.We thank the reviewer for this constructive suggestion which we believe made our data about PC regeneration stronger and further convincing. We have therefore added these findings to the manuscript and display the corresponding data in a new Supplementary Figure .2. Regeneration of PV neurons does not reach control numbers in the adult cerebellum, what is the interpretation of that result?Are the Ptf1a numbers in the adult cerebellum lower in relation to the ones in young animals, such that the ratio of progenitors to neurons is less than in larvae. Are there potentially too few progenitors to lead to full regeneration? Can this be quantified?ptf1a:GFP progenitors in adults are apparently lower than in larvae and juveniles as these undergo cerebellar development with a peak of PC generation. We have added this possible explanation to the discussion. In addition, in adults PC generation and differentiation occurs at a much slower pace. But even in adults one year of age ptf1a:GFP positive progenitor cells can be identified and seem to add PCs to the PC population. Hence there is no indication that the PC progenitor population is exhausted.The numbers of Functional recovery in adults is achieved when about 30% of the PC population has been re-established. At this level of recovery, the pressure for further regeneration is lifted, which may contribute to slowing down PC regeneration. In addition, PC differentiation in adults requires much longer time periods likely due to reduced differentiation signals, longer migration pathways and a more compact tissue that progenitor cells have to cope with. Furthermore, ongoing PC regeneration may be further influenced or counteracted by beginning aging-associated processes at 1.5-2 years in the nervous system. To provide explanations for the incomplete regeneration of the adult PC population, we have added these arguments to the discussion which reads now:ptf1a-expressing progenitor cells to differentiated PCs in adults compared to the larval cerebellum, a lack of need for further regeneration, prolonged PC differentiation processes in a compact and mature cerebellar cortex or incipient aging influencing or counteracting PC generation.”“Furthermore, these findings suggest that about one third of the size of the adult wildtype PC population is sufficient to reestablish cerebellum-controlled behavior maybe eliminating a need for further PC regeneration. Clearly, the adult zebrafish cerebellum is capable of significant functional PC regeneration under conditions of cell type specific PC loss. That not the entire adult PC population is reestablished may be due to the apparent lower ratio of 3. A GFP transgenic reporter (ptf1a:gfp) was used to determine that these cells give rise to Purkinje cells and that gfap positive radial-glia-like cells are not the source.Can it be excluded that gfap-positive cells generate ptf1a-positive progenitors which in turn would give rise to PCs? In the pictures in figure 2, it seems that gfap- and ptf1a reporter-positive cells are located in very similar regions, could there even be double-positive cells? It would be important to determine their respective localization and potential co-localization.gfap-positive cells are progenitor cells of ptf1a-expressing cells and we have stated that in our manuscript (line 182-183). We did not find GFP-expression of transgenic gfap:GFP carriers to colocalize with red fluorescent PCs of the PC-ATTAC strain. While this argues against gfap-positive cells to give rise to PCs, we cannot exclude that GFP expression is not sufficiently stable to still provide sufficient green fluorescence in differentiating PCs.The reviewer is right that we cannot exclude that gfap-positive radial glia cells are progenitor cells of ptf1a:GFP positive PC progenitors, we undertook additional experiments which are displayed in a new Supplementary Figure 4 and we refer to them in the manuscript. Because gfap:GFP and ptf1a:GFP transgenic carriers express the same fluorescent protein (GFP), we could not make use of double transgenic larvae. We therefore used an anti-Brain lipid-binding protein antibody (Blbp) for immunohistochemistry, which detects almost all gfap-positive radial glia cells in the developing zebrafish cerebellum (90%). Anti-Blbp immunohistochemistry in ptf1a:GFP zebrafish larvae revealed only a small fraction of about 10% of ptf1a:GFP positive cells to coexpress Blbp and an even smaller fraction of olig2:GFP positive cells to coexpress Blbp, while none of the red fluorescent PC progenitors were found to coexpress Blbp. These findings argue that a small fraction of cells in the cerebellum expressing radial glia proteins in ptf1a:GFP cells likely contribute to the small fraction of olig2:GFP expressing oligodendrocytes of the cerebellum rather than to specific neuronal populations such as PCs.To address whether ptf1a:GFP but not gfap:GFP positive cells to act as progenitors of PCs, these data are still not unambiguously conclusive as a potential short half-life of Blbp would interfere with the identification of radial glia protein expression in the majority of ptf1a:GFP cells. The conclusive identification of radial glia gene expression in ptf1a:GFP and PC-ATTAC cells will require permanent fate mapping approaches such as Cre-recombinase mediated permanent reporter gene expression in gfap:GFP derivatives and require genetic tools which are currently not available.While these data further argue for Reviewer #3 (Recommendations for the authors):+; Supplementary Figure 1A-D) during the regeneration of Purkinje cells. Which area was chosen for these quantifications?. When I look at Supplementary Figure 1A, I would quantify 34 BrdU+ cells in the PC-ablated condition vs 15 BrdU+ cells in the control condition. Please delineate the area that was chosen for the quantifications.1) The authors describe no change in proliferative cells  cells is seen in the ablated condition, as one of two possibilities can occur during the regeneration of Purkinje cells: (a) precursors divide faster to compensate for the loss of Purkinje cells or (b) precursors keep cycling over prolonged times, that is over more days than in controls.I would recommend the authors carry out a small experiment to assess the cycle length of precursor cells in the ablated larval condition, that is by combining pulses of BrdU and EdU. This could explain why no difference in proliferating  and have been added to the last paragraph of describing the results presented in Figure 1.We have followed this helpful advice of this reviewer and have implemented into this study the questions of reviewer 1 to address cell proliferation in the PC regenerating cerebellum at around 3 weeks after PC ablation. A first pulse of BrdU-labeling was applied 18 days after the ablation followed by a pulse of EdU-labeling four days later. Subsequently, four days after the EdU labeling BrdU2) The authors define ventricular zone Ptf1a+ cells as the precursors for the regenerated larval Purkinje cells. This, in my view, is not surprising as ventricular zone Ptf1a+ cells are precursors of Purkinje cells in most animals that have a cerebellum. Here, I have a few suggestions for the authors which can help the reader of this work to better understand this work:a) Could you please indicate, in the introduction, the temporal window of Purkinje cell production in Zebrafish?First Purkinje cells expressing PC specific genes such as carbonic anhydrase 8 or ZebrinII appear in the zebrafish cerebellum about 3 days postfertilization (dpf). And this first wave of PC generation lasts until 6dpf when a sufficiently rudimentary PC population has been established and a first plateau in PC generation has been reached . Subsequently, fewer PCs are added slowly over time to achieve a slow continuous increase in PC numbers aligned with larval growth. We have now added this information to the second paragraph of the introduction.b) Could you please describe, in the introduction, whether ventricular zone Ptf1a+ cells are the natural precursors of Purkinje cells in Zebrafish?c) Could you please mention whether inhibitory interneurons emerge from ventricular zone Ptf1a+ cells, as is the case in mammals? This can also be discussed in the Discussion section.We agree that such information is helpful to the reader who is not familiar with zebrafish cerebellar development. We have therefore added the following information to the introduction:pancreas associated transcription factor 1a (ptf1a) . In zebrafish, a first wave of postmitotic PCs appears from 3 to 6 days postfertilization (dpf), afterwards fewer PCs are added slowly over time to achieve a slow continuous increase in PC number aligned with larval growth . Controversial reports exist about whether in the adult zebrafish cerebellum new Purkinje cells are still generated . Together, these studies have laid a solid foundation for regeneration surveys of cerebellar neurons and PCs in particular .““Furthermore, clarification of the developmental origin and differentiation program of zebrafish cerebellar neurons, their physiology and functional contribution to locomotor control, motor learning and socio-emotional behavior has been revealed. For example, like in mouse, Purkinje cells, together with inhibitory interneurons and eurydendroid cells – deep nuclei neuron equivalents – in zebrafish have been shown to arise from progenitor cells derived from the cerebellar ventricular zone expressing the ptf1a-expressing progenitor cells in the late larval and adult zebrafish cerebellum as well as the limited space in the discussion, we prefer not to elude too much about the differentiation potential of these progenitors into cerebellar interneurons as we have not investigated this in detail in the present study.Given the current lack of genetic tools to unambiguously trace 3) Whereas the authors show that Ptf1a+ cells are the precursors for the regenerated Purkinje cells in the larval stage, the suggestion that these cells are the source of regenerated Purkinje cells in the adult stage must be tested in this work. In my view, the elucidation of this can be one of the most exciting aspects of this work.Therefore, I would recommend the authors carry out an experiment similar to that described in Figures 2A-D. That is using the Tg and Tg in the adult ablated PC-ATTACTM Zebrafish background, and quantified the number of double positive RFP/GFP cells in the regenerating Tg(gfap:EGFP);PC-ATTACTM and Tg(ptf1a:EGFP);PC-ATTACTM animals. I would suggest focusing at least on the first 4 months post-regeneration. I know that this is a 4-month experiment, but I think this experiment will significantly add to this work.ptf1a:GFP/PC-ATTAC double transgenics and we were able to reveal that also in adults of about 6 months of age double fluorescent cells exist in the PC layer therefore showing for the first time that ptf1a:GFP progenitors cells continue to give rise to PCs also during adult stages. These findings have been added now to the new Supplementary Figure .We agree with the reviewer that this is an interesting question to address. We have therefore investigated adult zebrafish ptf1a during the course of differentiation. Therefore, GFP expression will be lost in these progenitors over time, and it remains questionable whether GFP-stability will allow for observing double fluorescent regenerated Purkinje cells. Only in the case of a positive outcome, this experiment will be interpretable. In case, none GFP-positive regenerated PC-ATTAC Purkinje cells in the ptf1a-GFP background could be observed, it cannot be excluded that ptf1a+-cells do not represent the progenitor cells of regenerating Purkinje cells, as these progenitors could have lost GFP fluorescence prior to activating tagRFP-T expression. In order to address whether ptf1a+-expressing cells are the progenitor cells of regenerating Purkinje cells in the adult cerebellum a permanent labeling method for ptf1a+ cells e. g. by a Cre-recombinase-mediated approach would be needed. Such a study requires generation and validation of stable transgenic zebrafish strains, and we hope the reviewer agrees with our opinion that these fate mapping studies represent a full project of their own, which is beyond the scope of the current manuscript.To address this same question for regenerating PCs is technically far more challenging than assumed by the reviewer and has to await the advent of additional genetic tools. As shown in our studies the differentiation of regenerating progenitor cells into Purkinje cells occurs much more slowly compared to larval stages. Progenitors of Purkinje cells downregulate their expression of 4) Please restructure the manuscript. It is very confusing the description of Figures in the text versus the actual labeling of the Figures. It took me a lot of time to understand that the provided Supplementary Figure 1 contains the panels described for Figure 1 —figure supplement 1A-D; Figure 2—figure supplement 1E; Figure 2—figure supplement 1F; etc. Please separate the Figures and label them clearly.eLife and our initially submitted manuscript was returned to us demanding that we stick to these figure labeling rules. Yet, in the revised manuscript we have more clearly separated the supplementary material and hope that this will facilitate to relate supplementary material to main figures.We thank the reviewer for this advice, and we agree that the naming of supplementary material is cumbersome. But this labeling of supplementary figures adheres to the format required by


Recent highly pathogenic avian influenza A(H5N1) outbreaks in two Korean cat shelters highlight the need to enhance surveillance for cross-species viral transmission into animal populations kept by humans for non-agricultural or non-conventional livestock farming purposes from a One Health perspective. In this Comment, the authors describe recent outbreaks of highly pathogenic avian influenza in cat shelters in Seoul, South Korea. They discuss potential routes of transmission and describe implications for surveillance of spillover infections in animals in non-agricultural settings. The current COVID-19 pandemic has clearly illustrated how cross-species viral transmission can negatively impact both human and animal health, with the potential to trigger future pandemics, thereby highlighting the importance of having effective and efficient surveillance for cross-species viral transmission in place. Nonetheless, the challenge always remains in identifying the interfaces for cross-species viral transmission and distributing surveillance efforts optimally amongst these interfaces.Cross-species viral transmission refers to the infection of a new host species by a virus, commonly referred to as ‘spillover'4. However, surveillance in animal populations raised for non-conventional production purposes (e.g. minks and foxes for fur production) and those in non-agricultural settings  remains scarce globally, despite increasing attention to surveillance on mink and fox farms5 in the context of recent SARS-CoV-2 and highly pathogenic avian influenza (HPAI) A(H5N1) outbreaks in these settings9.The research and public attention on cross-species viral transmission has primarily focused on enhancing our knowledge about a pool of potential zoonotic viruses in wildlife, monitoring the prevalence of known zoonotic viruses within wildlife or livestock, and assessing the risk of their transmission to humans, along with syndromic surveillance targeted at unexplained clinical cases in livestock and humans. Above all, these surveillance activities are concentrated at the interfaces where the economic and health impacts of cross-species transmission are considered significantth and 31st, 2023, highly pathogenic avian influenza A(H5N1) infection, which has the potential to cause fatal disease in humans10, was confirmed in two cat shelters in Seoul, South Korea13. Briefly, at the cat shelter where the infection was first confirmed (Shelter 1), 38 out of the 40 cats died within a month, with the time between two consecutive deaths being ≤2 days13. This surge in deaths prompted the owner of Shelter 1 and its veterinarian to suspect a cause other than common respiratory infections in cats. Samples from two of the cats that had died from suspected respiratory infections were sent to a private diagnostic laboratory and confirmed to contain HPAI A(H5N1) virus by the Laboratory of Virology at the College of Veterinary Medicine, Seoul National University13. Then, the infection was confirmed again by the Animal and Plant Quarantine Agency (APQA)11 after it was formally reported to the agency according to the Act on the Prevention of Animal Contagious Diseases: the Act mandates reporting animals, including cats, that have died of unidentified diseases or are suspected or confirmed to have been infected by contagious animal diseases types I, II, and III as defined by the Act14. Several days later, a veterinarian reported respiratory symptoms in a cat from another shelter in Seoul (Shelter 2), and HPAI A(H5N1) infection was subsequently confirmed by the APQA12. As of October 2023, influenza A(H5N1) infection has been confirmed in five cats from Shelter 1 and four cats from Shelter 2, all of which were reported to have died16. The information on the number of cats with negative test results has not been released yet.On July 2517. Not surprisingly, the detection of the virus in cats in both these cat shelters within a short timespan attracted widespread attention. A subsequent epidemiological investigation by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) detected influenza A(H5N1) virus in cat food collected in Shelter 2, which contained duck meat, but not in cat food containing chicken meat. The MAFRA investigation concluded that raw duck and chicken meat had not been properly processed by the company that produced the cat food found to be contaminated18. In a swift response, the MAFRA ordered the recall of all cat food production batches containing duck or chicken meat that were deemed at risk. Approximately 13,200 units of the two recalled products had already been sold to 286 customers, but no signs of infection have been reported amongst their cats which were monitored twice at a 14-day interval15. Considering also that all cats in the two shelters were known to have stayed indoors and HPAI infection had been confirmed on the country’s poultry farms including duck farms until April 202316, it is plausible that influenza A(H5N1) viral incursion originated from contaminated duck meat contained in cat food, particularly in Shelter 2. However, as of this writing, no definitive epidemiological or genomic evidence has been released by the MAFRA, and therefore, it is unknown whether cats in Shelter 1 were fed the same or similar products and whether cat-to-cat transmission occurred following initial exposure in the two shelters and, if it did, what its relative contribution was compared to potential food-to-cat transmission.Avian influenza surveillance in South Korea is primarily focused on the detection of infections on poultry farms and in wild birds. Surveillance in mammals started early this year. However, it only focused on specific wild mammalian species and did not include domestic catsSo far, the management of the influenza A(H5N1) outbreaks in the cat shelters can be considered an example of good practice of surveillance and epidemiological investigations related to cross-species viral transmission. It has successfully detected influenza A(H5N1) infection in cats, identified a potential source of infection in at least one of the two shelters, and resulted in prompt transmission control measures. However, the question arises whether the MAFRA and APQA would be able to respond as successfully to cross-species viral transmission occurring in other epidemiological contexts. It has to be emphasised that the detection of influenza A(H5N1) outbreaks in the two cat shelters was initiated by the sudden deaths of multiple cats in Shelter 1 noticed by its owner and veterinarian. Subsequent steps taken by the veterinarians and virologists then led to the notification of the authorities, thereby initiating more detailed epidemiological investigations. This underscores the potential challenge of detecting cross-species influenza A(H5N1) infection and identifying a potential source of contagion if contaminated cat food was solely distributed to privately-owned cats rather than shelter cats, or if any element of the aforementioned chain of events was absent. There are several possible reasons for this:1, although it is currently unknown whether any cat-to-cat transmission had occurred in the two shelters.Firstly, under a given level of viral pressure, stochastic viral extinction may be more likely to occur when only a few cats in individual households are exposed to the virus as opposed to a larger group of cats. This is due to the increased contact rates between cats in a shelter as well as the increased likelihood of the virus successfully adapting to cats and subsequently transmitting between them, especially considering immunological barriers within individual cats19 and is difficult to distinguish during the early infection stages. The fact that the reporting from Shelter 1 was made only after a substantial number of cats had died further emphasises the challenge veterinarians would face with limited prior experiences and knowledge about the risk and clinical manifestation of HPAI infection in cats. The recent identification of influenza A(H5N1) infections in privately-owned cats in Poland demonstrates that detecting the infection in such a scenario is not entirely implausible20. However, it also indicates that undetected exposure of privately-owned cats to the virus could be frequent, consistent with serological evidence of avian influenza A infections in cats from different countries including household and shelter cats in South Korea22. Finally, if the virus is spread to cats through other routes (e.g. hunting and ingestion of infected birds by stray or privately-owned cats with outdoor access), the ability to establish plausible and specific epidemiological links would be challenged by the difficulty of conducting timely and targeted sampling as well as comprehensive history taking. While inadequately processed uncooked poultry meat leftovers in Shelter 2 were successfully collected and tested, it remains uncertain if the same could be achieved in other scenarios. This is exemplified by the recent influenza A(H5N1) infections of cats from individual households in Poland, where purchased food could only be tested for suspected cases, but not for confirmed cases20.Secondly, the presence of infection of cats in different households would be more likely to be missed by animal health authorities. This is due to the challenge that veterinarians would face in suspecting influenza A(H5N1) infection when individual cats are presented to different veterinary clinics. Although HPAI infection in cats is notifiable, it is much less common than other respiratory infections in cats20, suggest the need to enhance the effectiveness of infectious disease surveillance and prevention in any settings where a group of potentially susceptible animals are kept in close contact. This is particularly important for groups of animals that typically are not covered by regulatory efforts aimed at maintaining or improving animal health and welfare standards. These animal groups could not only provide environments for cross-species viral transmission, promoting zoonotic risk, but could also serve as sentinels for detecting such events, thereby offering opportunities to assess and then mitigate such risk. Several strategies can be implemented:The influenza A(H5N1) outbreaks in the cat shelters in South Korea, along with an increasing number of reports of influenza A(H5N1) infection in mammalsFirstly, it is important to require the aforementioned settings to record adverse animal health events at individual  and group levels , along with environmental factors  and husbandry conditions (e.g. food and water source), in systematic ways. In the event of cross-species viral transmission, this information will help to better understand the source of infection and thus inform the implementation of preventive and control measures. Such a recording practice appears to have been lacking in these particular settings, very much in contrast to conventional livestock farming where the recording is routinely practised.Secondly, regularly training staff in the aforementioned settings to report any unusual clinical signs or trends is crucial for timely and sensitive early detection, coupled with the above recording practice. For this, animal health authorities should define clear reporting criteria, ensure their staff’s familiarity with these criteria, and work to minimise the risk of underreporting due to potential stigmatisation. Additionally, animals in these settings need to be regularly examined by veterinarians to aid the timely recognition of unusual health events, which are often neglected due to economic constraints. Importantly, animal health authorities could play a role by designating veterinary staff to provide regular trainings to the staff about the recording practice and reporting criteria and to examine the animals.23. Finally, there is a need for continued discussion on how to implement measures to prevent and control cross-species viral transmission. It is crucial to involve all relevant stakeholders in this discussion, particularly the staff of the aforementioned animal settings, as their understanding of the implications of animal keeping for animal and human health might be less than amongst those involved in conventional livestock farming.Thirdly, the identification of inadequately processed uncooked poultry meat as a potential common source of influenza A(H5N1) infection in cats in both South Korea and Poland provides strong justification for prohibiting or limiting feeding of uncooked meat to animals kept for non-conventional production purposes and those in non-agricultural settings, in order to minimise the risk of cross-species transmission of influenza A(H5N1) virus and other viruses. Given the widespread availability of heat-treated commercial pet foods and the option to cook raw meat before feeding, this recommendation might be feasible for most animal species in these settings. Furthermore, if necessary, a ban on feeding uncooked meat to certain animal species could be implemented where cross-species transmission has been already reported or the risk is considered high , similar to the bans on swill feeding in many countries to prevent the introduction of African swine fever virus into domestic pig farmsIn conclusion, the recent influenza A(H5N1) outbreaks in South Korean cat shelters illustrate the risk of cross-species viral transmission in animals kept for non-agricultural or non-conventional livestock farming purposes, considered to present potential threats to human health. These events emphasise the need to strengthen surveillance of cross-species viral transmission events within these specific animal populations. However, it is also important to acknowledge that such efforts would require supplementary resources for national animal disease surveillance systems. While the scale of these additional resources may be relatively modest compared to those already invested in conventional livestock health surveillance, it could still pose a significant economic burden, especially in regions where the animal disease surveillance system is already poorly resourced. Therefore, it will be crucial to set realistic priorities by conducting risk assessments on the interfaces at which cross-species transmission has already been detected or expected to occur, in order to allocate limited surveillance resources based on the outcome of these risk assessments. By adopting such approaches, we can better prepare to protect against future zoonotic threats, thereby safeguarding both human and animal populations globally.


In Drosophila, the timer genes are expressed broadly across much of the blastoderm, which segments simultaneously, but their expression is delayed in a small ‘tail’ region, just anterior to the hindgut, which segments during germband extension. Specification of the tail and the hindgut depends on the terminal gap gene tailless, but beyond this the regulation of the timer genes is poorly understood. We used a combination of multiplexed imaging, mutant analysis, and gene network modelling to resolve the regulation of the timer genes, identifying 11 new regulatory interactions and clarifying the mechanism of posterior terminal patterning. We propose that a dynamic Tailless expression gradient modulates the intrinsic dynamics of a timer gene cross-regulatory module, delineating the tail region and delaying its developmental maturation.In insect embryos, anteroposterior patterning is coordinated by the sequential expression of the ‘timer’ genes  Gryllus bimaculatus or the beetle Tribolium castaneum, to more or less simultaneous, blastoderm-based patterning in the fruit fly Drosophila melanogaster  expression, which broadly regulates segmentation gene expression in time and space . We idendal cad; , Dichaethaete D; , and oddred opa; , all of erm band . In addiosophila .However, we currently do not understand how the timer genes themselves are spatiotemporally regulated within the embryo. What accounts for their local sequential activation in segmenting tissues, and why are these dynamics so deeply conserved across species? How is their expression globally regulated along the AP axis, and why is this regulation so evolutionarily flexible?Drosophila embryo, exploiting the fact that segmentation in this model species is not quite so simultaneous as it is often described. Although most of the Drosophila blastoderm is patterned simultaneously before gastrulation, the most posterior part of the segmental ectoderm is not patterned until germband extension  refers to the anterior boundary of parasegment n.Morphological segments are offset from the initial metameric subdivisions of the embryo, the parasegments, by about 2/3 of a segment repeat . The nthen stripe is en1 because it marks the anterior of parasegment 1, and the 14th en stripe is en14. The first wingless (wg) stripe, expressed just anterior to en1, is wg0, and the 14th wg stripe, expressed just anterior to en14, is wg13.Segment-polarity stripes are conventionally numbered according to the parasegment they are located within . Thus, tDrosophila embryo/larva  and, to the best of our knowledge, a 16th parasegment boundary has not been described. To investigate this issue, we used multiplexed HCR ISH to re-examine the expression of the parasegment boundary markers wingless , segment-polarity domains are initially patterned by stripes of pair-rule gene expression . In the eve expression at the posterior of the embryo is well-known, although it has been described as a remnant of eve15 .To the best of our knowledge, the slp15 domain has not been described previously. Persistent of eve15  or the 7e stripe  rather tDrosophila embryo after gastrulation  in these regions during blastoderm stages and early germband extension  To aid with fine-scale staging of embryos, we have divided stage 5, which lasts ∼40 min at 25° C, into five timeclasses based on gene expression and morphology (see Appendix 1).Given that cad, D, and opa transcripts are expressed sequentially over stages 4–6; first cad, then D, then opa , reaches appreciable levels at stage 4.4 (nuclear cycle 13), rapidly reaches a very high peak at stage 5.2, then declines sharply, with residual expression clearing by stage 6, replaced ventrally by persistent expression in the neuroectoderm  or absent (opa) through most of stage 5. At stage 5.4, a D tail domain emerges within the lateral part of the cad tail domain, rapidly strengthening and extending dorsoventrally. D protein becomes prominent in the tail domain at stage 6 (opa expression expands into the tail region from late stage 5 (described below).In the tail, a similar he trunk . cad is  stage 6 , again rcad tail domain is mostly anterior to the wg posterior domain, with an overlap of a single cell row (cad/wg merge). At stage 5.4, cad is actively transcribed in a domain 3–4 cells wide, but this shrinks to 2–3 cells wide by stage 6, with transcription ceasing at the anterior edge , and also overlaps the cad domain by about one cell row . This suggests that opa transcription gradually invades the cad tail domain from the anterior edge, with cad transcription then ceasing in these cells as Opa levels increase (cad-intron merge). Supporting this interpretation, we confirmed that a posterior expansion of Opa expression is evident in published live-imaging data  and cad zygotic mutants (cadm+z-) in addition to cad null mutants (cadm-z-) in order to disentangle maternal and zygotic effects , but the residual expression indicated that both the clearance of D expression from the trunk and the appearance of the D tail domain may have been delayed. The posterior wg domain, the posterior border of the cad tail domain, and the posterior border of the opa domain were all modestly anteriorly shifted relative to wild-type  largely rescued the cadm-z- phenotype, except that the D tail domain was lost prematurely, during germband extension  rescued the D tail domain fully and partially rescued the wg posterior domain  and stage 5 (nuclear cycle 14); we noticed that this border also retracts by about 3–4 nuclear diameters over the course of stage 5  mutants  mutants has also been described previously , we next asked how these regions are distinguished from each other. Forkhead (Fkh) is a zygotic transcription factor that is expressed in the posterior of the embryo from stage 4.4 (nuclear cycle 13) downstream of Tor  as in wild-type embryos  show a similar morphogenetic delay , and we model how Fkh, Wg, Cad, D, and Opa are expressed in response. Each of these seven factors is modelled as a logical variable, some of which  may take one of three levels of expression (off/weak/strong), while the others  may take only two (off/on). The AP axis is modelled as four discrete regions, 1–4 , which differ in their hard-coded Hkb and Tll inputs over time.  Each simulation consists of four time points, t0–t3 . At t0, Cad is on in all regions, and the other output factors are off. Expression at subsequent timepoints is computed from expression at t(n − 1), according to factor-specific logical rules . Mutants are simulated by keeping the relevant factor(s) turned off for all timepoints.The modelling framework we have chosen is very simple . Briefly, Hkb and Tll are assumed to be extrinsic inputs to the system  and posterior midgut (Fkh only) fates (Fkh (both regions), and differentially regulating Wg (repressed by Hkb in region 4). In region 1 (trunk), where Tll and Hkb are not expressed, gene expression is shaped by the intrinsic dynamics of the timer gene network: as D is activated and the level of Opa builds up, first Cad and then D are repressed. Finally, in region 2 (tail), these dynamics are modulated by transient expression of Tll, which delays the activation of D and Opa, and thereby prolongs the expression of Cad. Crucially, this Tll expression is weaker than in region 3, and so does not activate Fkh and (therefore) Wg.Regions 1–4 generate different gene expression as a result of their different inputs from y) fates , specifiFkh, Cad, D, and Opa  Wg are not expressed, while in regions 1 and 2 the activation of D is reduced. Simulated mutants of the ‘outputs’  and Opa  have per embryos , we can Tll and Hkb  into region 3 (hindgut). The tll- mutant transforms region 2 into region 1 but produces novel expression dynamics in region 3: D expression is transiently repressed (as in wild-type region 2) but Opa is not, producing a posteriorly shifted, transient Cad stripe and precluding any late expression of D. Finally, in the tll-opa- mutant, the repression from Opa on D and Cad seen in the tll- mutant is removed, and region 3 is fully transformed into region 2.In contrast, simulated mutants involving the ‘inputs’ Fkh and (therefore) Wg is spuriously delayed in the hkb- simulation  cross-activation of tll by Hkb , while D represses cad and Opa represses cad and D  in the sequence). opa is not cross-regulated, however, making it an ‘input-only’ component of the three gene network (at least in the blastoderm context).This work provides evidence for a set of cross-regulatory interactions between cad, D and opa in the tail region, indirectly allowing cad expression to be maintained. The localised inputs from the posterior terminal system are overlaid on global temporal regulation provided by the nuclear:cytoplasmic ratio  that had not been cloned at the time most of the original work was completed. All told, we have identified 11 new regulatory interactions involved in Drosophila AP patterning, put forward the first formalised model (to our knowledge) for the patterning of the tail, and provided a solid foundation for future quantitative analyses of this system.In this work, we have investigated blastoderm gene expression downstream of the posterior terminal system, revisiting a patterning network that was most intensely studied in the late 1980s and early 1990s . The modtll alleles can be arranged into a coherent phenotypic series in which the most posterior structures within the Tll-dependent region are the most sensitive to tll perturbation and the most anterior structures are the least  unless Tll expression was stronger, helping explain the transition from tail fate to hindgut fate as Tll levels increase. Furthermore, the retraction of the Tll domain over time explains the posterior shifting dynamics we found for the timer genes within the tail region, which contrasts with the anterior shifting dynamics previously described for the pair-rule and gap genes  Although further research is necessary to determine the mechanism by which Hkb cross-regulates tll, the phenotype implies that the size of the tll domain is not an unmediated response to terminal signalling. , with the anal pads located in PS15/A10  15 parasegment boundaries  However, PSB16 appears extremely dorsoventrally restricted and may have little functional significance in the organism. As the number of abdominal segments varies across insects , and tor RNAi embryos fail to express cad and wg in the posterior of the embryo, resulting in AP truncation  and simultaneously segmenting embryos like Drosophila? One key difference is likely to be the role of a posterior Wnt signalling centre: there is evidence from many different sequentially segmenting species that Wnt signalling is important for activating cad expression and maintaining the segment addition zone , we can see how appropriate segment addition zone dynamics might naturally emerge , cad3 (gift from H. Skaer), cad2 (Bloomington #7091), Dr72 (gift from S. Russell), opa8 (Bloomington #5340), torXR1 (gift from T. Johnson), hkbA321R1 (Bloomington #2059), Df(3R)Exel6217 , and fkh6 (gift from K. Roeper). Mutant lines obtained from the Bloomington Drosophila Stock Centre were verified by cuticle preparations as described in tll-opa- double mutant was generated by the Cambridge Fly Facility by recombining Df(3R)Exel6217 and opa8. Mutants were balanced over marked balancer chromosomes expressing lacZ during early embryogenesis: CyO, hb-lacZ (Bloomington #6650) for the second chromosome and TM6C, twi-lacZ Sb1Tb1 (Bloomington #7251) or TM3, hb-lacZ Sb1 (gift from S. Russell) for the third.Stock maintenance and embryo fixation  was performed as described in cad- germline clones were generated using the heatshock induced FLP/FRT system as described in cad2FRT40A/CyO virgin females (Bloomington #7091) were each crossed with 10 hsFLP w; ovoD1 FRT40A/CyO males . Adults were flipped to new vials every 2 days, resulting in a total of ∼100 vials. When crawling L3 larvae were visible, vials were heatshocked at 37°C in a waterbath for 1 hr, allowed to recover at 25°C for 24 hr, then heatshocked again at 37°C for 1 hr. Approximately 600 non-CyO virgin females (some presumably with cad2/cad2 ovaries) were collected from the heatshocked vials and crossed with ∼300 cad3/CyO, hb-lacZ males. Resulting embryos without lacZ expression lacked both maternal and zygotic cad (cadm-z-), while embryos with lacZ expression were paternal rescues (cadm-z+). Zygotic cad mutants (cadm+z-) were offspring from cad3/CyO, hb-lacZ parents that lacked lacZ expression; note that this genotype is also heterozygous for maternal cad.opa coding sequence was obtained from the Drosophila Genomics Resource Center. Gateway attB primers were designed to express 386 amino acids from the N-terminus of Opa (amino acids 3–389), spanning the zinc finger region in the centre of the protein. The forward primer included a Shine-Dalgarno sequence; the reverse primer was designed to be in-frame with the C-terminal fusion of the Gateway expression vector pET-DEST42 (Thermo Fisher Scientific). A two-stage PCR procedure was used to obtain a final amplicon carrying the attB-sequences at each end of the N-terminal opa sequence.Clone FI01113 containing Primers for the first amplification wereAAAAAGCAGGCTTCGAAGGAGATAGAACCATGAACGCCTTCATTGAGCpaDM-F: AGAAAGCTGGGTTGTCGTAGCCGTGGGATGpaA-R: Overlapping primers for the second amplification to complete the attB regions wereGGGGACAAGTTTGTACAAAAAAGCAGGCTattB1adap-F: GGGGACCACTTTGTACAAGAAAGCTGGGTattB2adap-R: The attB-opa amplicon was obtained by PCR with Phusion proofreading polymerase (Thermo Fisher Scientific) using primers opaDM-F and opaA-R. This first amplicon was diluted 1000-fold, then Phusion PCR was repeated with primers attB1adap-F and attB2adap-R. This attB-opa amplicon was recombined into Gateway donor vector pDONR (Thermo Fisher Scientific) using the BP Clonase II kit (Thermo Fisher Scientific). Plasmid DNA from a sequence-verified clone was then recombined into pET-DEST42 using the LR Clonase II kit (Thermo Fisher Scientific).Escherichia coli (Thermo Fisher Scientific). Opa protein was expressed in two ways, firstly by IPTG induction of exponentially growing cells (0.75 mM IPTG for 2.75 hr), secondly by overnight culture in TB Overnight Express (Novagen). The Opa fusion protein in pET-DEST42 had a C-terminal 6-His tag. Protein was purified from bacterial pellets, each from 100 ml of cells induced in IPTG or TB Overnight Express. Purification was carried out using Ni-NTA Agarose (QIAGEN), under 8 M urea denaturing conditions according to the manufacturer’s protocol. Purified protein was dialysed against water, then concentrated using an Amicon Ultra-Ultracel 5 kDa centrifugal filter (Millipore). Antibodies were raised in two guinea pigs by Eurogentec. Aliquots are available from EC on request.For expression of the fusion protein, plasmid DNA was transformed into One Shot BL21 Star (DE3) chemically competent Prior to staining, fixed embryos stored in methanol were put through a rehydration series of 5 min each at 75, 50, and 25% methanol in PBS + 0.1% Tween-20, then washed three times with PBS + 0.1% Tween-20.HCR in situ hybridisation was performed using probes and hairpins produced by Molecular Instruments, following the protocol for whole-mount fruit fly embryos included in For antibody staining following HCR, embryos were incubated for 30 min in blocking solution , at room temperature with rocking. Embryos were then incubated overnight in preabsorbed primary antibody diluted in blocking solution, at 4°C with rocking. Embryos were washed four times for 15 min in 5× SSC + 0.1% Triton X-100, at room temperature with rocking, then incubated for 30 min in blocking solution, at room temperature with rocking. Embryos were then incubated for 2 hr with fluorescently labelled secondary antibody diluted in blocking solution at room temperature with rocking. Embryos were washed four times for 15 min then one time for 30 min with 5× SSC + 0.1% Triton X-100 at room temperature with rocking. Antibody staining without prior HCR was performed as above with the exception that PBS was used instead of 5× SSC. Primary antibodies were guinea pig anti-Opa (this work) at 1:5000, rabbit anti-Dichaete  at 1:10,Following HCR and/or antibody staining, embryos were incubated for 30 min with 1 ng/μL DAPI (Thermo Fisher Scientific) in 5× SSC + 0.1% Tween-20, at room temperature with rocking, then washed three times for 30 min in 5× SSC + 0.1% Tween-20, at room temperature with rocking. Prior to mounting, embryos were stored in 1.5 mL tubes in SlowFade Gold Antifade Mountant (Thermo Fisher Scientific).Embryos were mounted in SlowFade Gold Antifade Mountant (Thermo Fisher) on glass microscope slides (Thermo Scientific) with #1.5 coverslips (Corning). #1.5 coverslips were used as bridges to prevent embryos from being squashed. Clear nail varnish was used to seal the edges of the slide.Microscopy was performed on an Olympus FV3000 confocal microscope at the Department of Zoology Imaging Facility (University of Cambridge). Acquired images were 12-bit, with a 1024 × 768 scan format and a 2 μs/pixel dwell time. Whole embryo images were acquired using an Olympus UPlanSApo 30 ×1.05 NA silicon immersion oil objective, a physical pixel size of 0.47 μm × 0.47 μm, and a z-stack step size of 1.5 μm. The close-ups in lacZ probe or a probe to a gene covered by a deficiency was additionally labelled with one of these same fluorophores, so that homozygous mutant embryos could be easily identified.) All imaging channels were acquired sequentially to minimise cross-talk. The laser lines and collection windows were: 405 laser and 443–472 nm window for DAPI; 488 laser and 500–536 nm window for Alexa Fluor 488; 561 laser and 566–584 nm window for Alexa Fluor 546 or Alexa Fluor 555; 594 laser and 610–631 nm window for Alexa Fluor 594; 640 laser and 663–713 nm window for Alexa Fluor 647. Alexa Fluor 514 (514 laser and 519–540 nm window) was used in place of Alexa Fluor 488 for a round of HCR experiments carried out when the 488 laser was awaiting repair. When necessary, a transmitted light channel was also collected to allow for embryo staging based on the progress of cellularisation.In each experiment, embryos had been stained for up to four transcripts and/or proteins of interest plus nuclei, generally using Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, and DAPI.  using the libraries NumPy  or generated for custom colours using a macro provided by Nicolás De Francesco (https://github.com/ndefrancesco).Embryo staging was based on Bownes stages , with sues NumPy , SciPy  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Drosophila embryo" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Claude Desplan as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Shelby A Blythe (Reviewer #1).Thank you for submitting your article "A timer gene network is spatially regulated by the terminal system in the The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Please clarify the presentation of Figure 8. Two reviewers found it hard to follow (see detailed comments below).2) Please move the "by gene" quantitative comparisons into the main figure, perhaps at the expense of the images of embryos. This change could be limited to the figures that compare genotypes, and all images can still be included as supplements.3) The observation about the size of Caudal germline clones should be removed or reinterpreted. The observation is quite tangential to the point of the paper and likely unrelated to anything having to do with Caudal.4) The manuscript should better highlight the broader implications. The data are really nice but the way it is currently written (particularly the Results section) somehow downplays the advance to the extent that it sounds more incremental and niche than it actually is.Reviewer #1 (Recommendations for the authors):This work is, as always, done to the highest degree of rigor and scholarship, and will be invaluable for the field both in terms of its direct focus , as well as a general reference for gene expression patterns of a broad set of patterning genes including some, like fkh, that have overall received less attention over the years.I am concerned that the paper, in its current form, is overly comprehensive in its presentation of details that it will be inaccessible to all but the most seasoned experts in the field of fly embryo patterning. While I appreciate the comprehensive treatment of each experimental result, I often lost the thread of the writing and had to search for summary paragraphs to make sure that my reading was in line with what the authors meant to convey. One example of this is in the nuanced presentation of the minimal computational model at the end of the Results section. I found myself wanting more of a summary or narrative to this section, rather than a point-by-point description of the model's output. Similar points could be raised about most of the other figures. Because this is a stylistic point, I understand if the authors disagree, but hopefully, some effort at streamlining the Results section could be attempted.Whether or not such streamlining of the text occurs, I did have some suggestions for improving the presentation of the figures. I found it difficult to follow in the main figures how the plots of gene expression patterns changed from timepoint to timepoint because the plots were always "by embryo" and not "by gene". For instance, in Figures 4 – 6. The critical comparison that we need to make in these two figures is 'by gene over time, between genotypes'. I see that the appropriate 'by gene' plots are provided in the supplemental data. While it will be ok for those reading the paper *online* to be able to flip between all these supplements, those who read a.pdf version in whatever form will be challenged to find this data . In this case, it might be better to swap some of the beautiful embryo images for the 'by gene' plots in the supplement in order to substantiate the important points in the main text and move the nice images to the supplement.If such a drastic change is not wanted, I would at the very least request that in figure 6, the 'matrix' of embryos have equivalent stages across rows between genotypes. For instance row 2 of panels A and B are slightly different stages, but my tendency was to assume we are to compare across these rows.Reviewer #2 (Recommendations for the authors):1) The shorter, wider phenotype in the germline clone embryos that is attributed to the loss of Cad is something that is usually observed with germline clone embryos generated by the FLP/DFS system. I do not think it is specifically related to the cad mutation. If the data in Fig4 supplement 6 are to remain in the manuscript, the authors should include an analysis of germline clone embryos carrying an unrelated mutation, as I suspect they will have the same shape change.2) The ectopic activation of Dichaete in the neuroectoderm in cad mutants, and therefore inhibition of neuroectodermal activation of D by Cad is not discussed in relation to the model, which simply shows Cad activating D. Can the authors justify this? What changes in neuroectodermal expression are predicted by the model if Cad has a role in suppressing D activation?3) If I understand the model correctly, inputs such as Tll can be strong, weak, or off. As the authors mention in the Discussion that there is a phenotypic series of tll alleles, could some of these be used to test whether the model can capture the changes in the network expression patterns when the Tll input is weak instead of strong?4) In Fig 8 it would help the reader to include, for some key mutants and time points, the same visual representation of the expression patterns based on the observed staining patterns. This would help the reader make a quick and simple comparison - currently, the authors direct the reader to the Source Data 1 but the data are provided in table form. Essential revisions:1) Please clarify the presentation of Figure 8. Two reviewers found it hard to follow (see detailed comments below).We were asked to clarify the presentation of the modelling results from Figure 8. We have moved the genotype-by-genotype description of the model output from the Results section to a new Appendix, and replaced it with a new, narrative summary of the key findings.2) Please move the "by gene" quantitative comparisons into the main figure, perhaps at the expense of the images of embryos. This change could be limited to the figures that compare genotypes, and all images can still be included as supplements.We were asked to move the "by gene" quantitative comparisons from the supplementary figures into the main figures that compare gene expression across genotypes . We have incorporated the relevant plots as suggested and agree that it is useful to have this information presented in the main figures.3) The observation about the size of Caudal germline clones should be removed or reinterpreted. The observation is quite tangential to the point of the paper and likely unrelated to anything having to do with Caudal.caudal germline clones. We have rewritten the relevant paragraph to make clear that we do not know whether the morphological changes are related to the loss of Cad expression or the use of the FLP-DFS system. While we agree the finding is of little relevance to the rest of our study, we have retained the observation in the manuscript as we think it is useful information for anyone exploring our embryo dataset or indeed using the FLP-DFS system themselves.We were asked to remove or reinterpret the observation about the size of the 4) The manuscript should better highlight the broader implications. The data are really nice but the way it is currently written (particularly the Results section) somehow downplays the advance to the extent that it sounds more incremental and niche than it actually is.We were asked to revise the text  to better highlight the broader implications of our work. We have completely rewritten the Abstract, partially rewritten the Introduction, and completely rewritten the modelling results subsection of the Results (as mentioned in point 1). We have also streamlined the experimental results subsections by editing the text, adding sub-subheadings to signpost the content of each subsection, and more strongly highlighting the “In summary” paragraphs at the end of each subsection. We should stress that the paper is intentionally written so that a casual reader can get by with only the summary paragraphs. However, the rest of the material is necessary if readers want to be walked through the expression data in the main figures, so we prefer to retain it in the main text.Reviewer #2 (Recommendations for the authors):2) The ectopic activation of Dichaete in the neuroectoderm in cad mutants, and therefore inhibition of neuroectodermal activation of D by Cad is not discussed in relation to the model, which simply shows Cad activating D. Can the authors justify this? What changes in neuroectodermal expression are predicted by the model if Cad has a role in suppressing D activation?D neuroectodermal domain. The neuroectodermal domain is activated at the end of our period of interest and its regulation presumably depends on factors not included in our network, such as dorsoventral patterning genes; it is therefore beyond the scope of our simple model. However, in principle, we see no difficulty with Cad both activating D’s early blastoderm expression and repressing D’s later neuroectodermal expression, for example through different enhancers and/or indirect effects.We have now clarified in the Results text that we do not attempt to model the 3) If I understand the model correctly, inputs such as Tll can be strong, weak, or off. As the authors mention in the Discussion that there is a phenotypic series of tll alleles, could some of these be used to test whether the model can capture the changes in the network expression patterns when the Tll input is weak instead of strong?tll dosage mutants, to further interrogate the quantitative effects of Tll.  However, it would be preferable to analyse quantitative effects such as these in the context of a fully quantitative model, rather than the minimalist qualitative model we have used for this study. For the avoidance of doubt, we should also stress that by modelling Tll as a qualitative variable that can be strong, weak, or off, we are not claiming that Tll literally switches between discrete “strong” and “weak” regulatory effects at a specific concentration threshold, but rather that we need at least 3 distinct levels of Tll in our model to reproduce the qualitative patterning phenomena we wish to explain.It would be very interesting in future to work to compare gene expression across these different alleles, and also 4) In Fig 8 it would help the reader to include, for some key mutants and time points, the same visual representation of the expression patterns based on the observed staining patterns. This would help the reader make a quick and simple comparison - currently, the authors direct the reader to the Source Data 1 but the data are provided in table form.hkb- mutant); as a consequence, a visual comparison would have all but duplicated the existing figure. We hope that the rewritten section of the Results now describes the performance of the model much more clearly, obviating the need for a visual comparison.We had considered this option when originally deciding how to present Figure 8, but we decided against it for two reasons. Firstly, rigorously converting real embryo data to the minimal “4 region” representation we used in our model would have required so many  decisions about AP axis positions and intensity thresholds that it felt more straightforward and transparent to simply provide the cross-references to the real data in table form. In this table (now Appendix 4—table 1), we describe noteworthy qualitative aspects of the simulations, and provide figure panel references to the corresponding expression data within the paper. Secondly and more importantly, the qualitative recapitulation of real patterning dynamics by the simulations was actually so accurate that there is only really one discrepancy between the real and simulated data (a spurious delay in the activation of Fkh in the simulated