There are three main reasons for this: (1) Scarce availability of the nuclide. Despite a relatively large number of globally existing cyclotrons capable of producing 211At, few cyclotron facilities produce the nuclide on a regular basis. (2) Lack of a chemical infrastructure, that is, isolation of 211At from irradiated targets and the subsequent synthesis of an astatinated product. At present, the research groups that work with 211At depend on custom systems for recovering 211At from the irradiated targets. Setting up and implementing such custom units require long lead times to provide a proper working system. (3) The chemistry of 211At. Compared with radiometals there are no well-established and generally accepted synthesis methods for forming sufficiently stable bonds between 211At and the tumor-specific vector to allow for systemic applications. Herein we present an overview of the infrastructure of producing 211At radiopharmaceuticals, from target to radiolabeled product including chemical strategies to overcome hurdles for advancement into clinical trials with 211At.Despite the consensus around the clinical potential of the α-emitting radionuclide astatine-211 ( The main rationale has been treatment of remaining minimal residual disease after primary treatments such as surgery, external radiotherapy, and/or chemotherapy.in vivo, only one nuclide remains, that is, astatine-211 (211At).There are ∼400 (5-100%) α emitting radionuclides when including isotopes. However, among those only a few fulfill the criteria for nuclear medicine applications, that is, suitable half-life, absence of long-lived and/or toxic daughters, and feasible production of clinically relevant amounts. Narrowing the list to those with a half-life >1 h and no serial decay, which potentially will contribute to toxicity 227Th), actinium-225 (225Ac), and radium-223 (223Ra).5–7 Including also nuclides with shorter half-lives, bismuth-213 (213Bi) and the lead-212/bismuth-212 (212Pb/212Bi) system can be added as potential choices.9 All these nuclides have been used in clinical settings but Ra[223Ra]-dichloride  is the only clinically approved α-emitting radionuclide.10A few more radionuclides can be considered disregarding serial decay. These are thorium-227 . Despite the fairly straightforward production of 211At and a number of existing possible manufacturing sites, there are few facilities currently producing the nuclide. From this it can be concluded that the problem of 211At availability is not exclusively reflected by the possibility of radionuclide production. 211At needs to be converted into a chemically useful form after cyclotron production and it needs to be coupled to a carrier molecule. A chemical infrastructure must hence also be in place within a reasonable close proximity to the cyclotron production site.However, when comparing the α-emitting radionuclides proposed for nuclear medicine applications in detail, 211At-radiopharmaceuticals in clinical settings. So far there has been two minor clinical phase I trials completed with 211At-antibody radiopharmaceuticals, mainly financed by academic funding.12 The results from these studies indicate therapy potential with no or very mild toxicity related to the treatment. To further prove the clinical potential of 211At, including progress into phase II/III studies, substantial funding is required in which both academy and industry need to share the economic risk. However, normally the industry needs evidence of the clinical potential before taking financial risk. So, in addition to the described practical hurdles of 211At production and availability, the funding of such research additionally suffers from this chicken and egg dilemma.To improve availability and motivate an increased production of the nuclide it is also essential to prove the potential of 211At is not reflected by difficulties in any of the radiopharmaceutical production steps. The production, isolation, and chemistry are well documented and feasible. However, a complete radiopharmaceutical infrastructure is less widely spread.It is important to note that the scarce use of 211At including a chemical infrastructure to provide activities needed for patient treatments, using existing chemistry.In this study we will, despite the hurdles, show a working outline to produce 13 The astatine isotope with the longest half-life is astatine-210 (210At) with a half-life of 8.1 h. 210At, however, decays by polonium-210 , making it unsuitable for medical purposes. Because of the lack of stable or long-lived isotopes, many chemical and physical properties of astatine are still not known but derived from its neighboring halogens.15Astatine, initially denominated “eka-iodine,” was first discovered in the United States in 1940 where it was produced at University of California, Berkeley through α-particle bombardment of natural bismuth.211At, 227Th, 225Ac, 223Ra, 213Bi, 212Pb/212Bi) in detail, 211At emerges as perhaps the most promising alternative  or by electron capture (EC) to polonium-211 (211Po). After the EC, 211Po promptly decays by α-emission to stable lead-207 (207Pb). This means that each decay will yield one α-particle. In addition, the EC branch to 211Po will yield characteristic polonium X-rays in the range of 70–90 keV. This enables simple quantification and the energy of the X-rays are in the range where it can be imaged with a γ camera.16When comparing the physical properties of the α-emitting radionuclides proposed for nuclear medicine applications  is another astatine isotope that decays in two branches either by α-emission to bismuth-205  or by positron emission (β+ decay) to polonium-209 . Due to high abundance of β+ it has been proposed as a theranostic pair to 211At.17 However, as 209At is produced by high-energy proton spallation of actinide targets,18 production of clinically relevant amounts can be foreseen to be problematic in a global perspective. In this sense, iodine-124 (124I) and 123I are more realistic theranostic pairs for 211At.Astatine-209  production, including targetry and cyclotron production of the nuclide followed by (2) the chemistry infrastructure, including isolation and work-up of 211At from the irradiated target and synthesis of the 211At-radiopharmaceutical. These steps are illustrated in The overall infrastructure for producing 211At, starting with a bismuth target: either indirectly through a 211Rn/211At generator in which 211Rn is produced by heavy ion irradiation of bismuth-209 (209Bi) through the nuclear reaction 209Bi211Rn,19 or directly through cyclotron production utilizing the nuclear reaction 209Bi211At.20 The advantage with the 211Rn/211At generator system is the longer half-life of 211Rn compared with 211At (14.6 h vs. 7.2 h). This will increase the distribution area of 211At from the production site with maintained astatine activity compared with the delivery of 211At alone. Although feasible, this route requires heavy ion high-energy beam irradiations and careful isolation and containment of the produced 211Rn, which is more technically demanding than the latter medium energy cyclotron production route. It is also possible to produce both 211At and 211Rn by high energy proton spallation of actinide targets but radiochemical yields from this production are to this date comparatively low.21There are two possible routes for producing 211At can be produced by heavy ion irradiation or spallation, the main production route has been and still is through the 209Bi211At reaction. The reason for this is the relative simplicity of production, the comparatively high yields, and significantly higher number of available facilities. A previous review by Zalutsky and Pruszynski related to these issues and the worldwide potential of producing 211At showed that there are a number of existing medium energy cyclotrons around the world with the capacity to produce 211At.22 However, many of the cyclotrons mentioned in that review are old and some of them have been or are under decommissioning. In addition, a few new medium energy cyclotrons have recently been installed or are under installation and cyclotron models that can produce 211At are today available for purchase “off the shelf” from, for example, IBA, Belgium and Sumitomo, Japan. Because of this, an updated, nonexhaustive, worldwide list of cyclotrons capable of producing astatine is provided in Although 209Bi211At reaction shows a maximum production yield using an α-beam of 31–35 MeV of the incident α particle. However, the energy should be kept at ∼28 MeV to avoid or minimize the competing reaction 209Bi210At. Coproduction of the 210At is unwanted because of its decay to the potentially toxic daughter 210Po.23The excitation function for the 211At. As bismuth is readily abundant, there will not be any lack of target material, which enables a theoretical indefinite production of 211At. This can be compared with several other α-particle emitters such as 225Ac, 213Bi, and 223Ra, where nuclear legacy material has been initially used for production and where future targetry and/or production of clinical amounts is either limited in availability or complicated by the handling of the starting material. 24–27There are several advantages by using natural bismuth as a target for producing 211At from bismuth, either thin targets for internal cyclotron irradiations or thick targets for extracted beam irradiations.28 For thick target preparation the bismuth can, for example, be electroplated or melted on the backing29 or used as granulates that are melted during irradiation.30 Thin targets are mainly prepared using physical vapor deposition of the bismuth on a backing material.31 In any case, targetry for the production of 211At is a relatively straightforward and cost-effective process. However, one difficulty encountered with using bismuth as target material is its fairly low melting point (271.4°C). This in combination with a low melting and boiling point (Bp) of astatine  means that an effective cooling of the targets and measures to enclose potentially evaporated astatine during the cyclotron irradiation are required. Therefore, the targets often have liquid cooling on the backside and front-side gas cooling.32 In addition, melting of bismuth and evaporation of 211At during thin target irradiation can be prevented by application of a fine layer of aluminum on top of the bismuth layer.33There are two main target set-ups for producing 211At is related to the helium ion source, the α-beam energy, the current of the beam, and the cooling of the target. These parameters need to be optimized, taking into account the described physical properties of bismuth and astatine.The efficacy in cyclotron production of 211At production reported come from Zalutsky et al. in Durham, NC.32 They reported a maximum produced amount of 211At of 6.6 GBq at end of bombardment. Providing an efficient chemistry this would allow clinical radiopharmaceutical production for several patients in a single production run, in addition to transport within an area corresponding to several hours of decay from the production site.As for today the most efficient and highest yields of 211At embedded in the bismuth layer of the target must be isolated and converted into a chemically useful form for further chemical processing, that is, labeling and production of astatinated molecules. The isolation from the activated target can be performed either by wet extraction or by dry distillation.34 Using the wet extraction method, the irradiated target is partially or fully dissolved in strong acid, for example, nitric acid.35 The 211At is then recovered using solvent extraction into an immiscible organic phase.36 Before the solvent extraction step, it can be necessary to change the aqueous phase by distillation of the dissolution acid. In general the astatine also has to be back extracted from the organic phase or the solvent changed to achieve a chemically useful form for further labeling.29 This sequential manual processing results in good yields but is time-consuming compared with the short half-life of the nuclide.37 An alternative method to solvent extraction after target dissolution is astatine adsorption on a solid support followed by elution.37After production 211At (Bp = 337°C) releasing it from the bismuth (Bp = 1564°C), which remain as a solid/liquid. The evaporated 211At can then be condensed in a cold trap or captured through gas scrubbing utilizing different solvents.38 Before the distillation it is advantageous to remove the activated bismuth layer from the aluminum backing of, for example, a thin target, as this allows the use of a small distillation unit that can fit in a hot cell or a glove box. Furthermore, reducing the volume of target material and preheating the distillation furnace will speed up the subsequent dry distillation, that is, the evaporation of 211At. In this way, dry distillation can be completed within 2 min.33 Furthermore, if capturing the 211At in a cold trap (as a dry residue) it can be eluted in a small volume of a preferred solvent. In this way the overall preparation time, distillation, and work-up, can be limited to <20 min from the start of the distillation.33In dry distillation the irradiated target is heated well above the Bp of 211At. It readily solvates high activities of the nuclide and the resulting activity in the solvent is stable for at least a couple of hours. The high vapor pressure of chloroform enables evaporation of the solvent without loss of 211At activity, leaving the astatine as a dry residue. Although the 211At speciation in chloroform is not known, recent results suggest that radiolysis products of chloroform such as chlorine species may be important for the apparent stability of 211At in this solvent.39 However, it should be noted that distribution ratios for 211At into chloroform from nitric acid are low, making this solvent unsuitable for astatine recovery using wet extraction.15After distillation care should be taken when selecting the eluting solvent and to the following activity concentration. Chloroform has been found to be a suitable solvent for 211At from the cold trap is to use sodium hydroxide or an aqueous solvent containing a reducing agent to define an astatide state.14 However, by introducing a reducing agent the subsequent halogen chemistry might be compromised. Yet another option is to use methanol. Contrary to chloroform, this may allow for direct labeling of protein conjugates but activity eluted in this solvent should be used immediately as 211At is not fully stable in methanol. Introducing an oxidizing agent, for example, a halogen-based oxidizing agent such as N-chlorosuccinimide, has been found to increase the stability of 211At in methanol.41Another option for eluting 211At and hence also higher yields, distillation configurations with a cold trap enable elution of the 211At in small volumes ready for immediate use in the subsequent chemistry.33In comparison, the dry-distillation method shows some advantages over manual wet-extraction methods. Besides generally faster isolation of 211At in terms of external shielding, lead-glassed glove box window, or similar, is less important because of the low abundance of high-energy gammas [211At (687 keV) and 211Po (569.7 and 897.8 keV)] besides 211Po X-rays (70–90 keV), giving low external dose.Independent of method, the isolation and work-up procedure should be performed as to minimize risk of internal contamination from the potentially volatile α-particle emitter, that is, in a sealed environment such as a negative pressure glove box or hot cell. Radiation protection for personnel from the decay of 211At compounds is based on previous inorganic and organic chemistry of astatine and for more detailed information on this topic we like to refer to a comprehensive review by Guerard et al.42This section provides an overview of the different chemical approaches to produce astatinated radiopharmaceuticals. Much of the chemical development of 211At has been found to form weak bonds with the sulfhydryl groups of cysteine.43 This apparent instability of the direct 211At-protein bond compared with iodine can be ascribed to the fact that astatine, besides its halogen character, also displays some metallic properties.44 The metalloid behavior of astatine has led to a few investigations in which its chelating possibilities were evaluated.46 Although demonstrating the metallic properties, no chelate has so far resulted in a sufficiently stable bond with astatine. The development of radiopharmaceuticals with astatine has therefore, instead of using a metal-like chelate, mainly been directed to the formation of covalent bonds such as the aryl–astatine bond. Several routes, based on iodine chemistry, for synthesizing aromatic astatine compounds, have been investigated. These chemical reactions include halogen exchange,47 diazonium salt reactions,48 aryliodonium salt reactions,49 and electrophilic substitution of metal-functionalized aromatic compounds.51 Recently reactions involving boronic esters and boronic acids as leaving groups for aromatic astatination of biomolecules have also been explored.52 Among the functionalized aromatic compounds, the most common strategy so far has been aryl–tin agents in which the 211At is introduced as an astatine–carbon bond in the aryl ring through an electrophilic destannylation reaction.54Astatine is the second heaviest element of the halogen group and it possesses many similar characteristics compared with its neighbor, iodine. In general, halogen properties can be used in astatine-labeling chemistry but there are also some obvious differences between astatine and iodine. For example, unlike iodine, astatine cannot be stably coupled to tyrosine residues of proteins. Instead of binding to tyrosine 55 The stability of the 211At-aryl carbon bond was recently evaluated by Teze et al. and the results showed that the apparent instability can be attributed to oxidative decomposition, which in vivo particularly occurs in lysosomes after cell internalization.56 This also relates to the nonresidualizing properties of halogens and may explain the low in vivo stability of small 211At-labeled molecules, which after internalization processes in the kidney are decomposed, resulting in systemic reabsorption of free 211At.57 To overcome the hurdle of exocytosis of 211At after internalization, a guanidinomethyl-containing reagent that increases intracellular retention of halogens  was developed by Zalutsky and colleagues.58 The guanidine residue of the astatinated version of this reagent is postulated to sterically prevent cleavage of the 211At carbon bond after cell internalization.59 Pyridine instead of aryl derivatives have also been used in protein halogenation in attempts to increase intracellular retention of 125I and 211At.61It should be noted that the bond strength of the astatine–carbon bond has been calculated to be weaker than the corresponding iodine–carbon bond.An overview of the different aromatic labeling strategies is given in 211At compounds showing favorable in vivo distribution, for example, [211At]-meta astato benzylguanidine,62211At-labeled poly (ADP-ribose) polymerase (PARP) inhibitor,63 and 211At-labeled sigma receptor ligands.65 IgG antibodies have been the main vector for targeted α therapy using 211At and although labeled by aromatic reagents, the 211At antibodies show relative stability in vivo, particularly for intracompartmental applications.12Despite the relative instability there are some recent reports on aromatic 211At-carbon bond have been shown between boron and astatine.66 Based on this chemistry, Wilbur et al. developed and investigated several boron cage reagents, nido- and closo-carboranes, for radiohalogenation of biomolecules.68 These reagents have also been implemented in protein labeling and were found to be more stable in vivo compared with the corresponding astatinated proteins labeled by aromatic reagents.70Higher bond energies compared with the 211At cannot be labeled to tyrosine residues of proteins and an intermediate bifunctional reagent utilizing one of the above-detailed astatination strategies is therefore required. It commonly includes an N-succinimidyl ester, isothiocyanate, or a maleimide moiety, which can be coupled to amines or sulfhydryl groups of proteins and peptides.71–73 The conjugation of these bifunctional reagents to proteins can, depending on the nature of the reagent, be performed in two routes: either two radiochemical steps, astatine labeling to the bifunctional reagent followed by conjugation of the astatinated reagent to the protein, or one radiochemical step where preconjugation to the protein is performed before the astatine reaction .76 In this system, the target is mounted in a target dissolution block in which parts of the target and produced 211At are dissolved by nitric acid. The nitric acid is then distilled and the activity redissolved in hydrochloric acid before being and loaded onto a resin column. The column is washed with an acidic wash solution followed by elution with an eluting solvent. The complete procedure from dissolution is controlled by software. Higher radiochemical yields of 211At isolated from the target and an overall simpler procedure is achieved with this method compared with previously reported manual wet extraction methods.An automatic wet extraction system for recovery of 37 The difference from the method reported by O'Hara is mainly the solid support, which in this case is based on tellurium. The irradiated target is dissolved by nitric acid and the nitric acid is converted to hydrochloric acid by addition of hydroxylamine hydrochloride. The solution is then loaded onto the tellurium column. The column is washed and the 211At is eluted with sodium hydroxide. High yields were obtained within a 90- to 100-min preparation time.A similar automated wet extraction method was evaluated by Li et al.The advantage with this system is that it is somewhat less reliant on the target configurations of the cyclotrons compared with the described dry-distillation method.211At-BC8-B10, before donor stem cell transplant. The study aims to determine side-effects and the best dose of 211At-BC8-B10. There are no results reported from this study yet (NCT03128034).At Fred Hutchinson Cancer Research Center , there is currently a clinical phase I/II study enrolling patients with acute myeloid leukemia and acute lymphoblastic leukemia for treatment with an anti-CD45 antibody, 211At-labeled antibodies: one as a postsurgery boost treatment for recurrent brain tumor and another with intraperitoneal treatment of ovarian cancer patients having relapsed after primary treatment.12 Both these intracavitary studies showed mostly mild (grade I–II) side-effects across all dose levels from the treatment. In both studies, the antibodies were labeled using aromatic bifunctional agents with Stannyl-based according to There are two completed phase I clinical studies using 211At-labeled chimeric anti-tenascin monoclonal antibody 81C6 was given locally in the resection cavity followed by salvage chemotherapy. Results from 18 treated patients showed no dose-limiting toxicity. A few patients experienced low-grade neurotoxicity but no grade 3 toxicity was related to the treatment. The study showed that such a local intracavitary therapy in this dose range is safe and may result in a prolonged survival for the patients.11In the brain cancer study, performed at Duke University, Durham, North CA, 71–347 MBq 211At-MX35(Fab)2 antibody fragment, targeting the sodium-dependent phosphate transport protein 2B (NaPi2b). Nine women who had relapsed after standard primary treatment were, after treatment by salvage chemotherapy in complete, or near complete, remission, subsequently treated with 211At-MX35(Fab)2 in 1–2 L of peritoneal dialysis fluid (icodextrin 7.5%). By blocking the sodium/iodide transporter with potassium perchlorate in sodium iodide symporter-expressing organs such as the thyroid and stomach, a significant reduction in uptake of free 211At in the thyroid could be recorded. After the initial nine patients, an additional three patients were treated after a change in the protocol regarding preparation of the radiopharmaceutical. The synthesis route was simplified through preconjugation of the antibody fragments compared with previous two-step labeling, as given in 211At activity decayed in the abdominal cavity. Dosimetric evaluation of organs at risk showed that this treatment can be delivered without causing any acute toxicities. Indeed, most toxicities recorded were low grade (I–II) and mainly related to the treatment procedure, with no observed hematological toxicity, thus no dose-limiting toxicity was reached.77In the ovarian cancer trial performed in Gothenburg, Sweden, patients were treated intraperitoneally with 211At therapy, long-term stochastic risks may arise and should be considered, particularly for an upfront adjuvant therapy.78 Weighing dose to various size microtumors79 with recorded patient dosimetry12 and calculated long-term stochastic risks,78 an activity concentration of 200 MBq/L of 211At-labeled antibody may be a justifiable dose in future clinical intraperitoneal trials.77Despite low risk of acute toxicity from 223Ra]RaCl2, which is used for palliative treatment of bone pain resulting from skeletal metastases in disseminated castrate-resistant prostate cancer.80 Another α-emitting radionuclide that currently is intensively investigated for treatment of cancer is 225Ac.82 Similar to 223Ra, 225Ac has frequently been clinically used on late-stage castrate-resistant prostate cancer.84 Yet another radionuclide that has recently attracted interest for targeted α-therapy is 227Th, the rationale being an in situ generator of 223Ra.85 At present, patients are enrolled in a phase I study for treatment of certain mesothelin-expressing cancers using 227Th (BAY2287411).Targeted α therapy is an emerging strategy for treatment of cancer, where the main rationale is treatment of small-scale disease, considering the high linear energy transfer and short path length of the α-particles. Therefore, α-particle therapy can be envisioned as an adjuvant treatment after standard primary treatments with, for example, surgery and chemotherapy. However, no such upfront targeted α-therapy setting for minimal residual cancer exists today. The only approved α-emitting radiopharmaceutical today is Xofigo, that is,  FDG or 68Ga-DOTA-octreotate. In this way the synthesis will facilitate reproducible and repeatable production. An automatic approach would also be a possible solution to the chemistry infrastructure problem presently limiting 211At research. A system that automatically converts 211At into a chemically useful form after cyclotron production75 and automatically produces the radiopharmaceutical74 would meet the safety requirements in handling 211At and facilitating larger clinical trials and multicenter studies. Standardization and reliable quality production of 211At and a chemical infrastructure is a requirement for future routine hospital production of 211At radiopharmaceuticals. One may envision several different approaches for implementing the logistics of the supply of 211At and the subsequent synthesis of the 211At radiopharmaceutical. Besides optimization and improvement of the cyclotron production, a delivery chain must also be established. Target distribution from cyclotrons to surrounding treatment centers within a circa one half-life radius followed by automatic on-site production would most likely allow for the best use of the produced activity, limiting radiolysis of the product. In densely populated areas, or where larger hospital and producing cyclotrons are located in close proximity, target work-up and pharmaceutical synthesis at the cyclotron facility may also be an alternative.A common way of facilitating chemical synthesis in nuclear medicine applications is automation utilizing radiopharmaceutical synthesizers, for example, in the production of [in vivo instability of 211At compounds, particularly important for general systemic applications with this nuclide. Although relatively high stability of astatinated compounds is often observed in vitro, deastatination may occur in vivo, predominately when small targeting vectors that are metabolized by the kidneys are used.89 The reabsorption of free 211At after sequestering of 211At molecules by the kidneys is believed to be related to intracellular oxidative decomposition after exocytosis and subsequent systemic recycling.90An additional issue that needs to be addressed is the apparent in vivo stability of 211At compounds to enable general nuclear medicine applications.92 Many of the hampering features of 211At relate to its halogen characteristics. Like iodine, astatine is nonresidualizing after cell internalization and instability is to some extent related to intracellular decomposition and exocytosis of free 211At. Efforts to improve retention of 211At intracellularly have been made by utilizing different modifications of the aryl ring.61 Despite the stability issue, relative stability of a few 211At compounds, proteins, or small molecules labeled by aromatic intermediates have been observed and may be explained by fast kidney clearance in relation to slow or noninternalization properties.93 In addition, 211At-labeled antibodies synthesized with existing well-established chemical methods show sufficient stability in intracavitary applications and allow for treatment in several different cavity modalities such as intraperitoneal, intrathecal, and in the blood compartment following, for example, leukemia or lymphoma.There are intense efforts made to improve the 211At, that is, the feasibility of producing 211At and the implementation of systems for target work-up and chemical synthesis. By providing a worldwide supply chain of 211At utilizing existing and new installations of cyclotrons and implementation of an efficient chemistry infrastructure, the main obstacles with this nuclide can be overcome and hence the demand concerning 211At radiopharmaceuticals for research and clinical trials could both be met and significantly enhanced.In this review we show that despite existing challenges, there have been major steps taken in research for nuclear medicine applications with


Preventive measures are key to reducing COVID-19 morbidity and mortality. Our primary aim was to assess factors associated with practice of COVID-19 preventive measures among senior high school students in the Bawku Municipality, Ghana. A cross-sectional study was conducted, and data on 624 students were analyzed using multivariable logistic regression. An estimated 31.5%  of the students wore a face mask often or always. Students who reported that COVID-19 can be transmitted via droplets from the nose or mouth  and students who reported that asymptomatic persons can transmit the virus  had higher odds of wearing a face mask. However, students who reported that COVID-19 was not deadly were associated with lower odds of wearing a face mask . Handwashing/hand sanitizing was practiced by 49.5%  of students. Students in the technical program compared to the science program  and those who reported that COVID-19 was not deadly  had lower odds of handwashing/hand sanitizing. An estimated 46.2%  of students practiced social distancing. Students who reported that COVID-19 can be transmitted via droplets from the nose or mouth were positively associated with social distancing . There is the need to intensify education about COVID-19 in senior high schools while enforcing the practice of COVID-19 preventive measures. Key among these strategies included testing, tracing, and treatment (“3 Ts”) approach; lockdowns in some parts of the country; and ensuring that all Ghanaians practice COVID-19 preventive measures.17 Despite these measures taken by the government of Ghana to control the COVID-19 pandemic, the WHO observed that Ghana is one of the countries with a substantial increase in the number of COVID-19 cases.18In Ghana, the first case of COVID-19 was reported on March 12, 2020.19 However, some students tested positive to COVID-19 after returning to school,20 which raises concern whether schools have implemented COVID-19 safety protocols. Despite the emphasis on students to practice COVID-19 preventive measures while in school, we do not know the factors associated with practicing COVID-19 preventive measures among students in Ghana. Our primary aim was to assess factors associated with the practice of COVID-19 preventive measures among senior high school students in Ghana.As part of measures to control the pandemic in Ghanaian schools, the government of Ghana in June 2020 introduced a phased reopening of schools, commencing with final year students in tertiary institutions, and junior and senior high schools to enable them prepare for their final examination, while ensuring that they observe COVID-19 safety protocols.This was a cross-sectional study conducted in the Bawku Municipality in the Upper East Region of Ghana. The Upper East Region is one of 16 regions of Ghana. Data were collected from final year students from four senior high schools in the Bawku Municipality. The municipality, schools, and students were conveniently sampled for the study. The inclusion criteria were students in any of the four selected schools who were aged 18 years and older. Data were collected by administering structured questionnaires to students in selected schools who met the inclusion criteria. Data were collected via self-administered questionnaires on August 3–7, 2020. The minimum sample size required for the study was estimated based on our assumption that 50% of students observed COVID-19 preventive measures. Using a 5% margin of error with a 95% CI, a minimum sample size of 384 was required. We however approached 631 students, but seven students were ineligible because they did not meet the inclusion criteria. The remaining 624 students participated and completed the questionnaires. The questionnaire consisted of questions on sociodemographics, knowledge about COVID-19 common symptoms, transmission, and preventive measures against COVID-19.14 and “0” for students who either do not wash/sanitize their hands or sometimes handwash/sanitize. Social distancing was defined as maintaining at least one meter distance between a student and others.14 It was dichotomized as “1” for students who practiced it always or often and “0” for students who did not practice it or practiced it sometimes.Our primary outcomes of interest included three key COVID-19 preventive measures. The following primary outcomes were measured: wearing of face mask, handwashing with soap and water or with alcohol-based hand sanitizer, and social distancing. Wearing of face mask was dichotomized as “1” for a student who always or often wears a face mask irrespective of the type of face mask and “0” for those who reported wearing the face mask sometimes or not at all. Handwashing with soap and water or hand sanitizing was also dichotomized as “1” for a student who always or often washes his/her hands with soap and water or sanitizes the hands with alcohol-based hand sanitizerWe explored variables of interest that included knowledge of COVID-19 symptoms, knowledge of COVID-19 mode of transmission, knowledge on COVID-19 preventive measures, and whether an educational/training program was a source of information about COVID-19.With regard to knowledge of COVID-19 symptoms, we compared students who knew one or more symptoms of COVID-19  with students who did not know any of the symptoms.On mode of transmission of COVID-19, we compared students who reported that COVID-19 can be transmitted via droplets from the nose or mouth of an infected person with students who reported that this was not a transmission route. A comparison was also made on students who reported that COVID-19 can be transmitted by touching droplets on surface or objects from an infected person with students who reported that this was not a transmission route. We also compared students who reported that an infected asymptomatic person can transmit the virus with students who reported otherwise. Students who reported that COVID-19 can be transmitted among adolescents and young adults were also compared with students who reported that transmission was not possible.On knowledge about COVID-19 preventive measures, we compared students who reported that avoiding crowdedness in public places can prevent infection with COVID-19 with students who reported that it was not a preventive measure. Students who reported that covering the nose and mouth while coughing can prevent COVID-19 were also compared with students who reported that it was not a preventive measure. We also compared students who reported that avoiding the touching of the eyes, nose, and mouth can prevent infection with COVID-19 with students who reported otherwise. Students who reported that COVID-19 was not deadly and there was no need for preventive measures were compared with students who reported that the virus was deadly.Students who reported they had heard about COVID-19 via an educational or training program were compared with students who did not hear about it through an educational/training program.Covariates included age, gender , father’s level of education , mother’s level of education , has health insurance (yes or no), place of residence when school is not in session , and program of study . The program of study refers to the course the student is majoring in the school.Data analysis was conducted using SAS version 9.3 . Descriptive statistics was used to report the characteristics of the study population. We used three multivariable logistic regression models to assess the relationship between our predictor variables of interest and each of our primary outcomes while controlling simultaneously for all our covariates. We obtained adjusted odds ratio (aOR) estimates for the predictor variables in each of the models. Our study protocol was approved by the Christian Health Association of Ghana Institutional Review Board. We also obtained informed consent from students before data collection as well as observing COVID-19 safety protocols.The study population consisted of 624 students aged 18 years and older. The mean age of the study population was 19.9 years, with a SD of 1.48. A majority of the students were males (54.2%). Many of the students had a health insurance (78.5%), and most of them were in the technical program (38.0%), compared with other programs .An estimated 31.5%  of the students wore a face mask often or always; 49.5%  of the students practiced handwashing or sanitized their hands with alcohol-based sanitizers often/always. The prevalence of social distancing among students was 46.2% . The odds of wearing a face mask among students who reported that asymptomatic persons with COVID-19 could transmit the virus was twice the odds of wearing a face mask among students who reported that asymptomatic persons cannot transmit the virus . The odds of wearing a face mask among students who reported that COVID-19 was not deadly had a 40% lower odds of wearing a face mask than those who reported that COVID-19 was deadly  .The odds of handwashing or hand sanitizing among students in the technical program had a 60% lower odds of practicing handwashing or hand sanitizing than students in the science program . Students who reported that COVID-19 was not deadly had a 40% lower odds of practicing handwashing or hand sanitizing than those who reported that the virus was deadly  .Our analysis found that at least 50% of the students either did not wear a face mask, practice handwashing/hand sanitizing, or practice social distancing often/always. Students who reported that COVID-19 can be transmitted via droplets from the nose or mouth of an infected person and students who reported that asymptomatic persons can transmit the virus were positively associated with wearing a face mask. However, students who reported that COVID-19 was not deadly were less likely to wear a face mask than those who reported that the virus was deadly. Similarly, students who reported that COVID-19 was not deadly had a lower odds of practicing handwashing or hand sanitizing. It was also revealed that students in the technical program were also less likely to practice handwashing or hand sanitizing than those in the science program. The study also established that students who reported that COVID-19 can be transmitted via droplets from the nose or mouth were positively associated with the practice of social distancing.The low prevalence of practice of COVID-19 preventive measures in our study might be due to several possible reasons. Many students might not be well informed about the morbidity and mortality associated with COVID-19. Some students might also not be aware of the mode of transmission and the importance of practicing COVID-19 preventive measures. School authorities might also not be enforcing the practice of COVID-19 safety protocols. Schools might also not have adequate hand sanitizers for students or handwashing stations at vantage points on campus. The low prevalence of practice of COVID-19 preventive measures in our study is worrying as this can lead to local outbreaks in schools if a student gets infected.21 compared with students who did not know that transmission via droplets was possible. Our findings on the positive association between students who reported that asymptomatic persons can transmit the virus and wearing a face mask might be because students are aware of the recent evidence suggesting that COVID-19 can be transmitted from an asymptomatic person.22 We did also observe that students who reported that COVID-19 was not deadly were less likely to wear a face mask than those who reported that the virus was deadly. This finding reflects the lack of knowledge among some students about mortality associated with COVID-19. Although there is the need to educate students on the importance of wearing a face mask, students need to know that COVID-19 can be fatal.We found that students who reported that COVID-19 can be transmitted via droplets from the nose or mouth of an infected person were more likely to wear a face mask than those who reported otherwise. This finding is not surprising as students with knowledge that COVID-19 can be transmitted via droplets from the nose or mouth of an infected person might also be aware of the protective effects offered by face mask against droplets,24Our study found that students in the technical program were less likely to practice handwashing or hand sanitizing than those in the science program. This finding in our study might be because students in the science program have a better understanding on the importance of handwashing/hand sanitizing in disease prevention than their peers in the technical program. We did also find that students who reported that COVID-19 was not deadly had a lower odds of practicing handwashing or hand sanitizing than those who reported that the virus was deadly. This emphasizes the need for students to understand that COVID-19 can be fatal and the important role handwashing/hand sanitizing plays in COVID-19 prevention. We also found that students who reported that COVID-19 can be transmitted via droplets from the nose or mouth of an infected person were more likely to practice social distancing than those who reported that the virus cannot be transmitted via droplets from the nose or mouth of an infected person. Students who reported that COVID-19 transmission via droplets was possible practiced social distancing as they might be aware that social distancing reduces the virus transmission by droplets.Our study had some limitations. The cross-sectional design of our study does not allow for causal interpretation of our findings. Another limitation is that our primary outcomes were self-reported and were subject to recall bias. However, we expect recall bias to be similar between exposed and unexposed predictor variables. Our primary outcome, social distancing, was also not objectively measured, and was therefore subject to measurement error. Another limitation of our study was the inability to account for school differences. One other limitation is that the true population of students who practiced any of the COVID-19 preventive measures is likely overestimated in our study as students are obliged to observe COVID-19 safety protocols according to the directive by the government of Ghana. Finally, our study took place in selected schools in one of the 16 regions in Ghana; therefore, our findings might not be generalizable to the entire country. Nonetheless, our findings fill an important gap in the literature and provide relevant information on factors associated with the practice of COVID-19 preventive measures among students in Ghana.This is the first study in Ghana to explore factors associated with practice of COVID-19 preventive measures among senior high schools. Our findings provide guidance to schools on factors that might contribute to ensuring that students of senior high schools in Ghana practice COVID-19 preventive measures. It may also be relevant for COVID-19 health education to be introduced in senior high schools. Our findings may also be relevant to those of senior high schools in low- and middle-income countries contemplating on reopening schools within this pandemic.


Strongylocentrotus purpuratus, an emerging model for diverse pigment function. We took advantage of single cell RNA-seq (scRNAseq) technology and discovered that pigment cells in the embryo segregated into two distinct populations, a mitotic cluster and a post-mitotic cluster. Gcm is essential for expression of several genes important for pigment function, but is only transiently expressed in these cells. We discovered unique genes expressed by pigment cells and test their expression with double fluorescence in situ hybridization. These genes include new members of the fmo family that are expressed selectively in pigment cells of the embryonic and in the coelomic cells of the adult - both cell-types having immune functions. Overall, this study identifies nodes of molecular intersection ripe for change by selective evolutionary pressures.Cells bearing pigment have diverse roles and are often under strict evolutionary selection. Here, we explore the regulation of pigmented cells in the purple sea urchin  Fromia milleporella) to a black and white striped brittle star (Ophiactis savignyi) to the variegated sea urchin (Lytechinus variegatus), pigment is a pervasive feature of this phylum. Because of the molecular techniques now available for echinoderms, mechanisms controlling pigmentation in these animals are being revealed , which in turn activates several pigment cell genes, including: polyketide synthase1 (pks1), three flavin monooxygenases , a sulfotransferase (sult) and a dopachrome tautomerase (dopt)  and later during blastula stage they become restricted to the aboral NSM  cells is sufficient to induce a similar single cell migration to the ectoderm and the accumulation of pigment in these cells, but not the acquisition of the typical morphology of pigment cells  . Among te (dopt) . The enze (dopt) . Pks1 ise (dopt) . Genes eoral NSM . At the elopment . Some evare lost . Moreovent cells . Gcm is hallenge . Thus, wgcm in pigment cell development through single cell transcriptomic approaches, and define polytypic pigment cell subpopulations. Our analyses revealed two distinct groups of pigment cells, a mitotic population and a differentiated population. We further analyzed the diversity of fmo genes, essential players in the pigmentation pathway, and found a group of fmo genes specifically expressed in the embryonic pigment cells and in the coelomocytes of the adult immune system. We also performed a single cell mRNA analysis of gcm morphants and revealed that pigment cell specification is dependent on gcm. Our study leverages our understanding of the Gcm transcription factor, an essential factor in the development of pigmentation in the larvae . Other state here instead of cell type for the compelling reason that distinctions in these cell groupings by this technology does not always mean a distinction in cell morphology, physiology, function, or cellular location, key characteristics of the use of cell type  to the gcm-enriched cluster of a time-course single cell analysis of sea urchin embryos encompassing eight developmental time points, from eight-cell stage to late gastrulae . Of these cell clusters expressing gcm, we found enrichment for other pigment cell terminal differentiation genes in three clusters in late gastrulae  and two clusters in larvae  and analyzed the average logFC for pks1 in each cluster. We found that pks1 was highly expressed in clusters 2 and 13 with respect to cluster 12, where pks1 levels were low. To determine if the pks1 low levels are stage dependent we normalized the data and compared cluster 12 to cluster 13 at 48hpf and 72hpf separately and found that at both stages pks1 levels in cluster 12 were lower than in clusters 2 and 13 . Another cluster that included mesodermal markers (12) expressed gcm in gastrulae, and low pks1 expression, suggesting that this is a population of pigment cell mesodermal precursors.We then analyzed the expression of 2 and 13 . Similarcdk1, pcna, DNA polymerases, DNA ligases, condensins, and centromere proteins  and pks1  by double in situ hybridization. Among these genes, ABCG11 is an ATP-binding cassette transporter homolog of the white half-transporter that transports pigment precursors and intermediates into the Drosophila eye  is a Cholecystokinin Receptor Type A, a G-protein coupled receptor that binds to cholecystokinin peptide hormones to modulate feeding and dopamine-induced behavior , and we detected a spotty expression in this region by FISH (arrowhead in gluR6). GluR6 was not detectable by single cell sequencing at 48hpf, and double FISH analysis showed only faint signals in gcm+ cells (violin plots in GluR6 was detectable at 72hpf though at low levels in cluster 2 (differentiated pigment cells) and cluster 16, a cluster of neurons. Double FISH with pks1 at 72hpf showed that gluR6 was expressed in pks1+ cells in or underneath the ectoderm  are never expressed in the mesodermal cluster (cluster 12), but rather in the mitotic (cluster 13) and post-mitotic (cluster 2) pigment cell clusters.MIF (macrophage inhibitory factor) is a member of a gene family of cytokines that regulate innate immunity . Of thesh stages . A knownh stages . Single 1+ cells . Opioid behavior . scRNA-spks1, we investigated the expression of a class of enzymes that at least one representative modifies the polyketide pigment scaffold, the Flavin-dependent monooxygenases (Fmo). Fmos are present throughout phylogeny, and although their conserved protein domain is typical of proteins with oxidizing activity, it is still unclear the exact role in the sea urchin pigment biosynthetic pathway/s. To determine the expression of fmos in echinoderms, we sought all Fmo domains in the S. purpuratus genome , and we found that not all of the identified fmos were specifically expressed in pigmented cells. We performed a phylogenetic analysis to look for protein subfamilies, and found 4 Fmo clades: one is in the same clade with vertebrate Fmo proteins (clade 2), two are not closely related to Fmo proteins in other organisms (clades 1 and 3), and one sub-family is in the same clade with fly orthologs  were all expressed in the mitotic and differentiated pigment cell clusters, but not in the mesodermal cluster 12 , knocking out the gene function of fmo3 resulted in a change in pigmentation of the adult, distinct from the albino phenotype of the pks1 gene knockout  as an additional tool to determine the temporal expression of representative genes from the 4 clades of fmos. Clades 2, 3 and 4 fmos were expressed at low levels during embryonic development, with less than 1000 transcripts per embryo , while the other clades of fmos are poorly or not detectable at all in embryos.We next determined the expression of r embryo . Only fms/embryo . We invee embryo . Our expfmos were not expressed during development, we analyzed their temporal expression in adult pigmented structures (spines and tube feet) and in the adult immune system (coelomocytes) by performing quantitative PCR in these tissues . Taken together, these results suggested that of the four fmo families, only clade 1 was found specifically in pigment cells of the embryo and in the adult red spherule cells, two cell-types with known immune functions. Within this clade, fmo2-2 and fmo2 are expressed also in embryonic cells that do not contain pigments, pointing these to a distinct oxidizing function. Other fmos are expressed in the spines  where pks1 is expressed at low levels, suggesting a role for non-clade 1 fmos independent of pigment biosynthesis and from gcm and pks1. Overall, we conclude that the many pigmented cell populations in the larvae and adults of S. purpuratus have distinct gene profiles.Since our results showed that many  tissues . We firs tissues . We foun lacking . Tube fedetected . Coelomoher fmos . These cher fmos . In the gcm expression is associated with the expression of pigment cell genes, we tested whether pigment cell gene expression was dependent on gcm. Gastrulae in which gcm is knocked down have a unique phenotype with no pigmentation and low levels of pks1, sult1c2 and fmo3 , determined but not differentiated yet. This dynamic gcm expression would have been more difficult to establish without single cell analysis, since it was not detectable so far using other techniques.nt cells . There welopment . Here, bme cells , it is pgcm loss of function is the lack of pigment cell precursors migration during gastrula and lack of pigment synthesis . In particular, gluR6 is expressed in a cluster of neurons located in the blastocoel basal to the ciliary band that resemble the localization of the lateral ganglion cells . It has been suggested that in adult sea urchin a population of pigment stem cells is set aside in the pre-metamorphosis larva to serve as a reservoir for the adult cells . This isfmo) enzymes that contribute to the pigment biosynthesis. In adult spines and tube feet under control conditions  pks1 is not transcribed, or is so at very low levels. Two scenarios are possible to explain this observation: either cells could have a level of enzyme (protein) sufficient to synthesize some pigment or in the absence of degranulation of pigment in response to infections or injuries, the synthesis of pigment is halted. In the case of cells with predicted similar functions, for example coelomic-pigmented cells (the red spherule cells), versus larval-pigmented cells, the composition of pigment synthesizing enzymes and other gene expression is instead similar. Cells with immune functions  always express the same group of fmos, together with the enzyme pks1 and the transcriptional factor gcm. Both cells produce the pigment echinochrome A, a molecule with antimicrobial properties  groups together with the fly orthologs. Flies have two fmos, one with housekeeping functions and another involved in development  in embryos, nor in the coelomocytes, as it is only expressed in spines. Sea urchin spines are covered by a thin epithelium  off the California coast and kept in artificial seawater at 16°C. Gametes were obtained by shaking adult sea urchins or by intracoelomic injection of 0.5M KCl. Eggs were fertilized in the presence of 1 mM 3-amino-triazol (3-AT) . Embryos were cultured at 16°C in filtered seawater from the Marine Biological Laboratory (MBL).Adult AB_2572409). Labeled probes were transcribed from linearized DNA using digoxigenin-11-UTP , or labeled with DNP  following kit instructions. Probes were synthesized using primers in SCR_017861).For fluorescent whole mount in situ hybridization (FISH), we followed the protocol outlined in SCR_013527) in 1 mg/ml Bovine Serum Albumin (BSA) and 4% sheep serum in PBST. Samples were then washed three times with PBST and incubated for 2 hr at room temperature with the secondary anti-mouse antibody conjugated to Alexa 488  diluted 1/500 in 1 mg/ml BSA in PBST. 5-ethynyl-2-deoxyuridine (EdU) pulse labeling of the pigment cells was performed with the Click-IT EdU imaging kit . Embryos and larvae were soaked in 10 μM EdU in FSW for 30 min (considering a 20 min cell cycle), washed five times with FSW and fixed in 4% PFA/FSW. Following EdU detection with fluorescent azide (according to the manufacturer's instructions), samples were stained with the Sp1 antibody as described above. 20 to 40 larvae were stained for imaging. Samples were mounted for imaging with an Olympus FV3000 Confocal Microscope (RRID:SCR_017015) equipped with high sensitivity GaAsP detectors managed by cellSens software (RRID:SCR_016238) from the Brown University Leduc Bioimaging Core Facility (RRID:SCR_017861). Raw files were analyzed and figures were made using the software Image J (RRID:SCR_003070).Larvae and adult tissues were fixed in 4% paraformaldehyde (PFA) in filtered seawater (FSW) for 15 min at room temperature, washed multiple times in phosphate-buffered saline with 0.1% Tween-20 (PBST), and incubated overnight at 4◦C with the Sp1 antibody 1:50  was used at a concentration of 0.5 mM. MOs were injected with 20% glycerol and 10,000 MW fluorescent dextran (injection solution). Eggs were dejelled by washing in pH4.0 seawater. For each experiment, around 600 zygotes were injected with ∼2–4 pl of oligonucleotide injection solution by constant pressure injection in with 1 mM 3-AT (Sigma). For each condition, only 300 of these injected embryos with the expected phenotypes were dissociated at 48hpf and used for single cell RNA seq. The gcm morphant has a unique phenotype with a straight gut and no pigmentation. As a negative control, fertilized eggs were injected with a MASO sequence irrelevant to S. purpuratus . It is used to compare to the gcm morphants. We chose to use a gcm morpholino to knockdown gcm instead of CRISPR/Cas9 as used previously (Translation-blocking antisense morpholino (MO) against Gcm  previously used in our lab  were useSCR_000667)  . ProteinRNA from 100 embryos was isolated with the RNeasy Micro kit , while RNA from adult tissues was isolated from Trizol . cDNA synthesis was performed using Maxima kit . qPCR was performed using ABI 7900 real time instrument with Maxima SYBR master mix  and normalized to ubiquitin transcripts. Experiments were run in three biological replicates, and every biological replicate was run on the qPCR machine with three technical replicates. In the graphs, the mean of the three technical replicates is shown, and error bars represent biological replicates. List of primers used for qPCR are in Embryos were collected and washed twice with calcium-free seawater, and then suspended in hyalin-extraction media (HEM) for 15 min . For enrSCR_016387) (2 × 150 bp paired-end runs). The non-injected 48hpf and 72hpf samples were sequenced in separate lanes (350M reads per lane). The morpholino injected samples were sequenced together in the same lane . Single cell unique molecular identifier (UMI) counting was performed using Cell Ranger Single Cell Software Suite 3.0.2 from 10X Genomics . The custom transcriptome reference was generated from assembly Spur_4.2 using Cell Ranger. Cell Ranger matrices were further analyzed using the R package Seurat v 3.1.4  (https://www.ncbi.nlm.nih.gov/geo/] (GSE155427).Single cell RNA sequencing: Single cell encapsulation was performed using the Chromium Single Cell Chip B kit on the 10x Genomics Chromium Controller. Single cell cDNA and libraries were prepared using the Chromium Single Cell 3’ Reagent kit v3 Chemistry. Libraries were sequenced by Genewiz on the Illumina Hiseq (RRID:_007322) . Cells w_007322) . The seq In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This manuscript uses single-cell sequencing to characterize the development of pigment cells in the purple sea urchin. In addition to producing various chemically interesting pigments, these cells play a role in the animal's innate immune system . This work presents a new view of the process of pigment cell development in the purple sea urchin, clarifies the relationships between larval pigment cells and different adult pigmented cell types and some immune cells, and will serve as a useful resource for the field.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Marianne Bronner as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Jonathan Patrick Rast (Reviewer #2).Thank you for submitting your article "Regulation of dynamic pigment cell states at single-cell resolution" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:Strongylocentrotus purpuratus. In addition to producing various chemically interesting pigments, these cells play a role in the animal's innate immune system . Thus, understanding how these cells are generated and characterizing the biochemical pathways used to synthesize their diverse pigments should be of broad interest.This manuscript by Perillo et al. uses single-cell sequencing to characterize the development of pigment cells in the purple sea urchin, fmo) is involved in pigment synthesis, the authors examined fmo genes present in the S. purpuratus genome, and found one group (of the four families they describe) with enriched expression in embryonic pigment cells and adult coelomocytes (immune cells). Finally, based on previous work showing that the transcription factor glial cells missing (gcm) was required for pigmentation, the authors perform single-cell analysis of gcm knockdown embryos and conclude that this gene is required for pigment cell specification. Overall, this work presents a new view of the process of pigment cell development in the purple sea urchin as well as the relationship of larval pigment cells to different adult pigment cell types and to red spherule immune cells. Pending the clarification of the issues outlined below and substantial editing of the text, it will serve as a useful resource for those interested in this system.Single-cell analysis at distinct developmental time points (gastrulae and larvae) identified two pigment cell clusters in larval ectoderm and one cluster of mesodermal pigment cell precursors in gastrulae. One of the larval pigment cell clusters expresses cell cycle-associated genes and the authors show that cells expressing a pigment cell marker can incorporate EdU in gastrulae and larvae. New pigment cell markers identified via single-cell sequencing were validated by double fluorescent in situ hybridization. Because a flavin-dependent monooxygenase  The authors seem to be overstating the case that they have identified a population of pigment stem cells. It has been evident for some time that pigment cell numbers increase after they delaminate from the archenteron and before they differentiate  consistent with a round of cell division after delamination. What evidence is there from the single cell sequence data that these cells have stem cell qualities and are not just progenitors at the last division before differentiation. Their data show that pigment cells can divide, but their subsequent fates after taking up EdU have not been followed. In the absence of additional evidence, the authors should soften their language about stem cells and stem cell niches.2) Do the authors know whether the dividing Cluster 13 cells are granular at this point in development? A description of the morphology/behavior of the cluster 12 and cluster 13 cells would be interesting if the authors know anything about this. Cluster 12 is of interest since it is most unlike cluster 2 and 13. As GCM disappears at 72 hrs in these cells, the cluster remains in the tSNE plot. Do the authors know where these cells are at this time? Are these some type of blastocoelar cell?fmo gene families in S. purpuratus, the authors conclude that two of the protein families are not found in other organisms. Because the only animals represented are humans and S. purpuratus , it would be reasonable for the authors to broaden the sampling of organisms on their phylogenetic tree to include a more diverse set of animals.3) In their characterization of gcm , they should clarify why they instead used morpholino knockdowns for the scSeq experiments reported here.4) Because the authors have previously published the lack of pigment cells following CRISPR/Cas9 targeting of gcm MO embryos, this should be quantified. It would also help the reader if the rationale for this experiment were made clearer at the outset. In its current form the results are described in the middle of a paragraph about the phenotype of gcm knockdown embryos without any introduction or explanation.5) In Figure 6F it is not obvious that pigment cell numbers are unaffected by PKS Cas9. Based on the image shown, a naïve reader could conclude that there are fewer pigment cells and that they are larger. As they have done for 6) What is covered in Foster et al., 2020, in review? How is it non-overlapping with this paper? What is the significance of the early non-pigment cell GCM expression in Figure 2? It is difficult to see the data in detail here and this may be important since the finding seems to contradict earlier in situ data, FP transgene analysis and transcript quantification data. It may be that single-cell sequencing can discern sporadic transcription that is seen as background in previous studies but more information on estimated expression levels would be useful here. Is this seen for other well characterized transcription factors in the developmental scRNA seq data?gcm expression analysis with the gastrula and larva scSeq (starting at the second paragraph of the subsection “Gcm is Enriched in Three Clusters at Gastrula and Early Larva Stages”) was unclear. For example, the reader is told that following integration of the various time points, "one cluster is marked by the expression of gcm…", but this starting point is never shown. It may be useful for the reader if somewhere in the figure legends, the 7 detected cell states could be defined, since they are all numbered in the figures. The authors do a very nice job of presenting relevant numbers in other figures , but this section is very unclear on where the data are coming from: how many total cells? what percentage express gcm? etc…7) The rationale and description of integrating this 8) To help the authors clarify their presentation, the extensive editorial comments of reviewer #3 (included below) should be addressed in the revised manuscript. Essential revisions:1) The authors seem to be overstating the case that they have identified a population of pigment stem cells. It has been evident for some time that pigment cell numbers increase after they delaminate from the archenteron and before they differentiate (Gibson and Burke 1987) consistent with a round of cell division after delamination. What evidence is there from the single cell sequence data that these cells have stem cell qualities and are not just progenitors at the last division before differentiation. Their data show that pigment cells can divide, but their subsequent fates after taking up EdU have not been followed. In the absence of additional evidence, the authors should soften their language about stem cells and stem cell niches.We have tempered the language of pigment stem cells in the Abstract, Introduction, and Results, but we have retained this consideration in the Discussion section to ensure a broader dialog within the community2) Do the authors know whether the dividing Cluster 13 cells are granular at this point in development? A description of the morphology/behavior of the cluster 12 and cluster 13 cells would be interesting if the authors know anything about this. Cluster 12 is of interest since it is most unlike cluster 2 and 13. As GCM disappears at 72 hrs in these cells, the cluster remains in the tSNE plot. Do the authors know where these cells are at this time? Are these some type of blastocoelar cell?The cluster 13 cells are not blastocoelar cells because we did not find any blastocoelar markers . Cluster 12 has mesodermal markers and might represent undifferentiated mesoderm. From Sp1 staining they look migratory, extending filopodia as the non-dividing cells, but we cannot yet identify pigment levels in these cells.3) In their characterization of fmo gene families in S. purpuratus, the authors conclude that two of the protein families are not found in other organisms. Because the only animals represented are humans and S. purpuratus , it would be reasonable for the authors to broaden the sampling of organisms on their phylogenetic tree to include a more diverse set of animals.fmos including sequences from mouse, frog, fish, flies, chicken. In this new tree we found that Fmo5-6 belongs to clade 3 (which previously did not group with any other sequence), but all other associations were consistent. We now document that two different methods of tree construction (maximum likelihood and maximum parsimony) resulted in the same outcome, providing additional confidence in our interpretations.Good point – We made a new phylogenetic tree of 4) Because the authors have previously published the lack of pigment cells following CRISPR/Cas9 targeting of gcm , they should clarify why they instead used morpholino knockdowns for the scSeq experiments reported here.gcm. A paraphrased version of this explanation is now in the Materials and methods section.CRISPR/Cas9 randomly mutates the targeted genomic sequences. These are, after all, wildtype animals and not strains of homogenous animals that have a single type of mutation. We wanted to have data that could be clearly interpreted and reproducible and the morpholino will always bind the same sequence of the mRNA to stop its translation. Every injection of CRISPR/Cas9, however, will yield different mutations  that could have different effects in the gene expression and the phenotypes obtained. Moreover, with CRISPR/Cas9, the resulting truncated proteins are still being translated and could still be active depending on the mutations. Such variations may complicate the scRNA-sq results so we decided to use the morpholino as a more reliable method to downregulate 5) In Figure 6F it is not obvious that pigment cell numbers are unaffected by PKS Cas9. Based on the image shown, a naïve reader could conclude that there are fewer pigment cells and that they are larger. As they have done for gcm MO embryos, this should be quantified. It would also help the reader if the rationale for this experiment were made clearer at the outset. In its current form the results are described in the middle of a paragraph about the phenotype of gcm knockdown embryos without any introduction or explanation.The Pks1 – KO here is used to test homeostasis in the pigment cells and whether PKS may have any function, directly or indirectly, in regulating gene expression. This is topical in the Discussion. We have now also mentioned this briefly in the Results section as justification for doing the experiment, but retained its position in the paragraph. It is an experimental result that contrasts with the GCM experiment, so we believe the order in the paragraph is appropriate. We have now also quantitated these results as requested and these data are now shown in Figure 6F, G and H.6) What is covered in Foster et al., 2020, in review? How is it non-overlapping with this paper? What is the significance of the early non-pigment cell GCM expression in Figure 2? It is difficult to see the data in detail here and this may be important since the finding seems to contradict earlier in situ data, FP transgene analysis and transcript quantification data. It may be that single-cell sequencing can discern sporadic transcription that is seen as background in previous studies but more information on estimated expression levels would be useful here. Is this seen for other well characterized transcription factors in the developmental scRNA seq data?S. purpuratus development from 8 – cell stage to 48 hours. We use that dataset here to extract gcm mRNA expression, which was not performed in Foster et al., 2020. As seen for several genes, the mRNA profile is more broad and earlier than seen by in situ RNA or qPCR experiments. Foster et al., 2020, discusses this phenomenon extensively, and we refer to that discussion but only briefly highlight the finding here. The Foster et al., 2020 manuscript was recently accepted for publication (Development) so readers of this pigment paper will also have access to that single cell RNA-seq data.Foster et al., 2020, presents a single cell RNA-seq resource dataset to the community of 8-timepoints of 7) The rationale and description of integrating this gcm expression analysis with the gastrula and larva scSeq (starting at the second paragraph of the subsection “Gcm is Enriched in Three Clusters at Gastrula and Early Larva Stages”) was unclear. For example, the reader is told that following integration of the various time points, "one cluster is marked by the expression of gcm…", but this starting point is never shown. It may be useful for the reader if somewhere in the figure legends, the 7 detected cell states could be defined, since they are all numbered in the figures. The authors do a very nice job of presenting relevant numbers in other figures , but this section is very unclear on where the data are coming from: how many total cells? what percentage express gcm? etc…gcm+ cells, 1% of total cells. At morula stage 125 cells express gcm, 3.48%. Early blastula:13.9% Hatched: 9.5% Mesenchyme blastula: 7.6% Early gastrula: 6.5% and late gastrula 4.7%. This result is now presented in the figure legend of Figure 2.Gcm is first detected at 64-cell stage, when there are 9 8) To help the authors clarify their presentation, the extensive editorial comments of reviewer #3 (included below) should be addressed in the revised manuscript.Thank you for this effort. We have now done so and have addressed each issue below.


Zootoca vivipara) are weakly decreased by a daily restriction in free-standing water availability (less than 0.5°C contrast). The influence of air humidity during the day on thermal preferences depends on time of the day and sex of the lizard, and is generally weaker than those of of free-standing water (less than 1°C contrast). This shows that short-term dehydration can influence, albeit weakly, thermal preferences under some circumstances in this species. Environmental humidity conditions are important methodological factors to consider in the analysis of thermal preferences.Thermoregulation is critical for ectotherms as it allows them to maintain their body temperature close to an optimum for ecological performance. Thermoregulation includes a range of behaviors that aim at regulating body temperature within a range centered around the thermal preference. Thermal preference is typically measured in a thermal gradient in fully-hydrated and post-absorptive animals. Short-term effects of the hydric environment on thermal preferences in such set-ups have been rarely quantified in dry-skinned ectotherms, despite accumulating evidence that dehydration might trade-off with behavioral thermoregulation. Using experiments performed under controlled conditions in climatic chambers, we demonstrate that thermal preferences of a ground-dwelling, actively foraging lizard ( Thermoregulation is a critical determinant of the ecological performances of many organisms, and of the sensitivity and resilience of biodiversity to global changes –3. In maStudies of thermoregulation in ectotherms have identified numerous factors that generate variation in PBT both within and between species, including seasonal factors, inter-sexual differences, trophic interactions or local environmental conditions [e.g., lizards, Podarcis lizard species. In these species, the changes in thermoregulatory behavior are explained by an increased use of shelters, whose availability in the environment determines opportunities for a behavioral conflict between thermoregulation and hydroregulation [see also, In reptiles, dehydration is the consequence of an imbalance between water loss, mainly evaporative water loss through the skin and through respiration, and water input, which is accounted for by drinking, at least in snakes and lizards ,31,32. EZootoca vivipara, that typical inhabits cold and humid environments across Eurasia. In this species, chronic restriction in water availability leads to individual dehydration and is associated with a decrease in locomotor activity and thermal preferences, and changes in the selection of thermal and moist refuges during the daytime [Here, we tested for short-term effects of daily changes in free water availability and air humidity on the thermoregulatory behavior of a ground-dwelling lizard species,  daytime ,38–40. I daytime ,34,41. S daytime , then thZootoca vivipara) from semi-natural populations located at the CEREEP-Ecotron IleDeFrance  close to the southern distribution of the species range in lowland Northern France. We selected yearlings to standardize age and reduce differences in physiological state (due to reproduction) among individuals. In addition, earlier studies have shown that yearlings have similar average preferences than adults, except pregnant females . Studied individuals were captured between 22–27 May 2017  from captive populations maintained in fenced, outdoor enclosures (10 x 10 m). In this study site, this sampling period corresponds to the end of the mating season. All yearlings were transferred to a nearby laboratory upon capture, sexed by coloration, morphology and visual search for the presence of hemipenis in males. Lizards were measured for body size  and body mass at capture . Females were longer and weighed higher on average than males . During maintenance and not experimentation, lizards were individualized in terraria with sterilized peat soil, and kept under stable day-night light and temperature conditions in a temperature-controlled room (16h of night at 16°C and 8h of day at 23°C). Each terrarium provided lizards with a thermal gradient (23 to 35°C) 6h a day (09:00 to 12:00 and 14:00 to 17:00) with a light bulb (25W) at one end of the terrarium. Animals were fed with house crickets three times per week  and provided with water ad libitum. We therefore assumed that they were fully hydrated at the start of experimentation and physiologically dependent on water supplementation. Animals were maintained for a minimum of two weeks in captivity before the start of experiments.Experiments were performed under permit 17/DDPP/SPAE/57 to CEREEP-Ecotron and permit 77–01 to J.-F. Le Galliard. We sampled yearling common lizards  were simulated in two 13 m3 climatic chambers during daytime. Air humidity  was adjusted to a constant level that was monitored continuously with a calibrated capacitive sensor . Inside each chamber, we installed 12 thermal gradients  warmed at one end with a heat bulb (25 W) located 15 cm above each thermal gradient. Thermal gradients were provided with dry and sterilized peat soil as a substrate, a Petri dish placed at the center of the box to manipulate water availability, and a wood plate on the warm side of the box to enhance basking efficiency. We recorded the range of substrate temperatures from the cold to the warm range of the box before and at the end of each daily trial. Extreme temperatures ranged from 22–25°C on the cold side to 45–50°C on the warm side, thus allowing behavioral selection of optimal body temperatures for common lizards [between 25–40°C during field activity, Measurements of thermal preferences (see 2.3) were conducted in a controlled environment facility allowing precise regulation of air temperature, air humidity, lighting and gas concentrations in the atmosphere . Constanad libitum access to water between each trial. Fasting ensured that most animals were post-absorptive during the tests [Animals were first tested for their thermoregulation behavior inside the climate chamber in a pilot study (see below), which further allowed them to familiarize with the set up. Then, we designed three independent experiments performed with the same sample of lizards. In order to evaluate the hypothesis that lizards exposed to acute restrictions on water availability are expected to reduce body temperature, we manipulated air relative humidity and free water availability in the thermal gradient (Experiment 1). During this first experiment, performed between June 9 and 16, we contrasted a “dry treatment”:(20% RH) with a “wet treatment” (80% RH) and further manipulated at the same time free water availability in the thermal gradient (Petri dish filled with water or empty during the day). Environmental conditions were thus equivalent to a water vapor pressure deficit  of 1.87 kPa and 0.46 kPa at 20°C in the dry and wet treatments, respectively. All animals were tested four times sequentially with just one daily trial per treatment condition, the sequence of four treatment conditions being pseudo-randomly determined to avoid confounding treatment effects with trial number. Thus, lizards were observed for 1 full day in each treatment condition. Lizards were fed right after each trial and maintained in the laboratory during a minimum rest period of 2 days without food but with he tests , but we ad libitum access to water between each trial.In order to evaluate the hypothesis that lizards exposed to a milder change in air humidity also reduce their body temperature, we manipulated air relative humidity in a moister range of atmospheric conditions (Experiment 2). During this second experiment, performed between June 28 and July 1st, we compared dry and wet conditions ranging from 70% RH (“wet treatment”) to 95% RH . This range of moisture is probably more relevant to the natural variability seen across micro-habitats exploited by this species inside natural populations . LizardsIn order to evaluate the hypothesis that micro-habitat selection plays an important role in the thermoregulatory behavior of active lizards, we repeated the second experiment with a set-up where lizards could use a cold, moist shelter in the thermal gradient (Experiment 3). During this third experiment, performed between July 5 and July 9, we enriched the thermal gradient with a shelter  located above the substrate on the cold side of the thermal gradient. Soil below the shelter was made fully wet at the start of each trial to create a cold and wet microhabitat during the day . When lizards were inside the shelter, we did not disturb them and did not record their body temperature. We manipulated air relative humidity in the same way than in the second experiment  and did not provide free-standing water during experimentation. All animals were tested two times sequentially with one daily trial per treatment condition, the sequence of two treatment conditions being pseudo-randomly determined to avoid confounding treatment effects with trial number.Post-absorptive lizards kept without food for 2 days in the laboratory prior to each trial were removed from their home cage, measured for body mass at 8:00 am and then placed alone in each gradient at least 1:30 hours prior to the start of observations. This procedure was chosen to eliminate effects of digestion on body temperatures and to decrease the effects of handling stress . We acknb of lizards ranged from 25 to 35°C during the day and matched qualitatively the values recorded in earlier studies  using laboratory thermal gradients or performed in outdoor conditions [b tended to decrease during the day and lizards were predominantly observed basking (53% of records) and in the warm side of the box (41% of records), especially at the beginning of the day. This parallels qualitatively the thermoregulatory behavior seen in a previous laboratory study [Surface body temperature was measured with a calibrated infrared thermometer  at ca. 20–30 cm from the target. Cloacal body temperatures measured with a K-type thermocouple are highly correlated with these surface skin temperatures . Similarnditions ,50,51. Iry study , which inlme package for mixed model fitting [All statistical analyses were done using the software R version 3.4.3 and the  fitting . We fittb of each daily trial and its SD was calculated from the 12 data points of each daily trial to provide summary statistics for the thermal preferences and the precision of thermoregulation. We then analyzed the variation of each of these three variables with a linear mixed model accounting for repeated measurements on the same lizards over consecutive trials. In the full linear mixed models, we included the main fixed effects of sex, treatment groups and their interactions to take into account the factorial ANOVA design. We further included individual identity as a random intercept factor. These analyses were based on 192 records in Experiment 1 and 96 records records in Experiments 2 and 3.We did these analyses because this is the classical approach to quantify thermal preferences statistics in lizards.First, we calculated for each individual and each daily trial the PBT, the SD of PBT  and the body mass change . The PBT was calculated as the mean Tb from each individual and daily trial to characterize better intra-individual variation in body temperature during the daytime. We analyzed Tb with a linear mixed model including fixed effects of sex, treatment groups and time of the day and their interactions to take into account the factorial ANCOVA design. The covariate time of the day was calculated as the number of minutes since the start of the experiment at 10:00 am and was centered prior to analysis to ease interpretation of parameter estimates. Time of the day might represent a dehydration and/or starvation effect or some intrinsic diurnal variation in thermal preferences of lizards. We used a linear rather than a curved relationship with time of the day based on graphical explorations of the data. We further included individual identity as a random intercept factor. These analyses were based on 2320 records of body temperature in Experiment 1, 1138 records in Experiment 2 and 776 records in Experiment 3.We did these analyses because this allowed us to test the hypothesis that treatment influenced daily changes of body temperature instead of mean body temperature.Second, we independently used repeated measurements of Tb were fitted using the lme function. The Tb data were transformed with a Box-Cox power transformation to ensure the normality and homogeneous variance of the residuals of the model [All linear mixed models for PBT, SD of PBT, body mass change and The model . Model she model . This pr1,46 = 6.74, P = 0.01) and when free water was available than in water-restricted conditions  and a two-way interaction between water availability and air humidity . The SD of PBT was higher when air humidity was high and when free water was not provided  and when air humidity was wet . In males, body temperature was also higher on average when free water was available  and it decreased with time of the day  independently from free water availability and air humidity. Animals lost on average 0.16 g (± 0.11 SD) during the experiment. Body mass loss was significantly influenced by a two-way interaction between air humidity and water availability  after controlling for differences between sexes. Availability of free water canceled out body mass loss as well as the negative effect of air dryness on body mass change  against high (RH = 80%) humidity conditions in the presence or in the absence of free water. A complex three-way interaction model between sex, air humidity treatment and water availability best explained daily variation in Tb . In femab  and it dange see .b included a two-way interaction between air humidity treatment and time of the day and a two-way interaction between sex and time of the day  during the experiment, and body mass loss was higher in dry than in wet conditions  after correcting for effects of sex  and initial body mass  and very high (RH = 95%) humidity conditions without access to free-standing water. The best model for intra-individual variation in T the day . On aver the day . The decnditions . Howeverb included a two-way interaction between air humidity treatment and sex and a two-way interaction between sex and time of the day  while females had higher Tb  than in dry conditions. In addition, a decrease of Tb during the day was seen in males  but not in females . Animals lost on average 0.05 g (± 0.07 SD) during the experiment, but body mass loss was not influenced by air humidity  nor by sex  and very high (RH = 95%) humidity conditions without free-standing water but in the presence of a wet and cold shelter. The best model for intra-individual variation in T the day . In wet Taking into account the water balance and its interaction with thermoregulation is crucial because the hydration status of an animal is expected to modify its thermoregulatory behavior, even in dry-skinned ectothermic species more resistant to water loss ,54. We pSceloporus undulatus [Podarcis [Anniella pulchra [The mean daily thermal preference of common lizards changed significantly in the absence of free-standing water in the thermal gradient but was not influenced by atmospheric humidity even when we compared an extremely dry and wet air. In the absence of drinking water, the lizards lost significantly more body mass during the day, in the order of 7–10% of their initial body mass depending on the ambient humidity. The preferred body temperature, calculated as the daily average, decreased significantly by about 0.5 to 1°C and the statistical dispersion of body temperatures tended to narrow around this lower average in the absence of drinking water. These effects of free water availability on thermal preference are weak but qualitatively similar to those observed in the lizard ndulatus  and in fPodarcis  as well  pulchra . These d pulchra ,56,58.Podarcis lizards by Sannolo and Carretero [a priori because the common lizard is a species less resistant to water loss than Podarcis lizards [Podarcis lizards and this foraging mode should be sensitive to water restriction, with lizard moving less and saving more water by reduced movement effort in more desiccating environments [Podarcis are species with a more sit-and-wait foraging mode, they might have less opportunities to adjust their behavioral activity and their “only” behavioral solution is therefore to lower importantly their body temperature in the absence of water. Unfortunately, we did not record movement activity in this study but previous investigations with lizards have shown that dehydration can reduce locomotor activity [Quantitatively, the decrease in body temperature observed in this study remains relatively small compared to that observed (2–4°C) in four species of arretero . This re lizards ,38,59. Ironments ,61. Sincactivity ,43. ThesThe analysis of daily thermoregulation patterns reveals subtler effects of water conditions. In general, lizards bask at higher temperatures at the beginning than at the end of the day, as highlighted in our previous work using the same protocol with a thermal gradient ,46 or wiPodarcis lizards might be sensitive to the perception of dehydration risk. Indeed, in these species, adults exposed to water restriction adopt more cautious thermoregulatory behaviors from the beginning of the day, at a time when they are very unlikely to be physiologically dehydrated. We suggest that more studies should be performed to understand the behavioral rules and threshold functions dictating the behavioral conflicts between water balance and body temperature regulation in lizards [e.g., The daily patterns of thermoregulation are broadly consistent with the hypothesis that thermoregulatory behavior adjusts to individual variation in hydration status rather than to environmental stimuli concerning the presence of water  or air humidity [via hygrosensation, ds e.g., .The thermoregulatory behaviors are modified by the presence of a cold, damp shelter that limits water losses in drying conditions and allows lizards to reduce their thermoregulation behavioral activity. Lizards used the wet refuge ca. 30% of the time during the day. However, the observed changes in thermoregulatory behaviors are not consistent with our hypothesis that the presence of a cold and wet shelter should amplify the trade-off between hydroregulation and thermoregulation. First, we found that more desiccating conditions were not associated with higher lizard mass losses when we provided lizards with a shelter contrary to the two other experiments. This suggests that the use of shelter allows lizards to buffer effects of air humidity on the water balance. Second, we found a lower body temperature of females under the most dehydrating conditions, which was of the same order of magnitude as the response to complete water deprivation in Experiment 1. Surprisingly, however, the opposite result is observed in males. One possibility for interpreting these results could be sex differences in the prioritization of shelter use versus active thermoregulation, perhaps also in response to the repeated stress caused by the presence of an experimenter to measure body temperatures. For example, males might prioritize strategies to reduce water loss by using more shelters in drier conditions and this could allow them to capitalize on activity phases to increase their body temperature. These explanations remain very speculative insofar as the frequency of shelter use does not seem to be related to the treatment conditions in this experiment (data not presented), but they confirm earlier observations of sex-specific responses to water restriction in this species .Natural populations of ectotherms are exposed to joint changes in water and thermal conditions, the consequences of which can only be predicted with studies that integrate the interactions between these two factors ,36. In r