Hoxc8 gene is expressed by pSNs and motor neurons (MNs) targeting distal forelimb muscles, and sensory-specific depletion of Hoxc8 in mice disrupts sensory-motor synaptic matching, without affecting pSN survival or muscle targeting. These results indicate that the diversity and central specificity of pSNs and MNs are regulated by a common set of determinants, thus linking early rostrocaudal patterning to the assembly of limb control circuits.Relay of muscle-derived sensory information to the CNS is essential for the execution of motor behavior, but how proprioceptive sensory neurons (pSNs) establish functionally appropriate connections is poorly understood. A prevailing model of sensory-motor circuit assembly is that peripheral, target-derived, cues instruct pSN identities and patterns of intraspinal connectivity. To date no known intrinsic determinants of muscle-specific pSN fates have been described in vertebrates. We show that expression of Hox transcription factors defines pSN subtypes, and these profiles are established independently of limb muscle. The  Sensory-motor circuits within the spinal cord are essential for the coordinated control of limb muscle. In mammals, proprioceptive sensory neurons (pSN) process a continuous stream of data from approximately 50 forelimb or hindlimb muscles, and relay this information to the appropriate circuits tasked to orchestrate motor behaviors . The ordThe simplest input pathway to MNs is the monosynaptic reflex arc, composed of a limb muscle innervated by a pool of alpha MNs and type Ia pSNs with stretch-sensing mechanoreceptor endings embedded within muscle spindles. Type Ia pSNs are uniquely poised to provide direct and immediate muscle status information through the monosynaptic connections they establish with MNs. During development, pSN axons navigate through the spinal cord, preferentially contacting MN pools innervating the same peripheral target, while avoiding MNs of functionally antagonist muscles . These cNrtk3, and the calcium binding protein Parvalbumin (PV). Genetic studies in mice indicate that Runx3 and Etv1 are essential for establishing and maintaining core features of pSN identity, including their survival and ability to extend central axons into the ventral spinal cord  neurons dedicated to limb control . Mutatio and MNs . In additing MNs . By contnal cord . HoweverNrtk3 receptor renders pSNs sensitive to peripheral neurotrophin-3 (Ntf3) signaling, and both Nrtk3 and Ntf3 are essential for the differentiation and survival of pSNs  segments  while Hoxc8 expression was restricted to caudal cervical and rostral thoracic DRG (C6 to T2)  . Thus, HHox genes is initiated by secreted morphogens acting on progenitors along the rostrocaudal axis   . As in mn mouse) . Co-stain mouse) . The redHox gene expression have been established, but prior to the appearance of postmitotic pSNs and MNs (~E8.5 in mouse) (+ SNs expressing Pea3 was reduced to 5.5 ± 0.1% (mean ± SEM), compared to 31.8 ± 3.6% in controls   16–18, a phase when the initial rostrocaudal profiles of n mouse) . After ln mouse) . After fn mouse) . The fra t-test) . In addi t-test) . The dected limb .+ SNs expressing Hoxa5 , Hoxc6 , and Hoxc8  was not significantly changed  of Hoxc8 pattern . At thespulation . Between neurons . We foune Hoxc8- .+ LMC neurons typically innervate more anterior/proximal forelimb muscles, while caudal cervical Hoxc8+ MNs project to distally and/or posteriorly located forelimb muscles  and examined Hoxc8/βGal protein expression in retrogradely labeled neurons. We performed retrograde tracing assays on nine specific forelimb muscles, ranging from proximal to distal positions, and varying in size, but sharing a common role in controlling arm, wrist, or digit movement . Spinal cords with attached DRG were then isolated at P7 to assess representative populations of sensory neurons innervating the injected muscle. Injections were performed no earlier than P4 due to the thin size of the distal forelimb muscles as well as the inefficiency of neonatal CTB labeling, a probable outcome of lower expression levels of the CTB receptor in neonates , flexor carpi ulnaris (FCU), palmaris longus (PL), and flexor digitorum profundus (FDP). For the PM, FCU, PL, and FDP,~70% or more of the total CTB labeled pSNs were Hoxc8+ . In SN∆Hoxc8 animals, expression of Hoxc8 protein is selectively removed from SNs but maintained by neurons within the spinal cord  and postnatally (P4-P7). To clearly visualize Hoxc8+ populations at postnatal stages, we used the inserted LacZ reporter which expresses βGal in lieu of Hoxc8, enabling us to track the fate of pSNs lacking Hoxc8. We compared the number of βGal+ cells between LacZ/+Hoxc8 and SN∆Hoxc8 animals in DRG C8, where a subset of Hoxc8+ pSNs reside. At P7, the percentage of PV+ pSNs that expressed βGal was similar between control and SN∆Hoxc8 animals    . We also t test) . Moreove animals . All βGa neurons . These oHoxc8 is necessary for the ability of cervical pSNs to innervate their normal forelimb muscle targets, we examined the formation of muscle spindles in the palmaris longus (PL) and flexor carpi ulnaris (FCU), two distal forelimb flexor muscles that normally receive input from Hoxc8+ pSNs. We found no discernible difference in the pattern of PV or vGlut1, which accumulate on the peripheral terminals of pSNs, indicating that PL and FCU muscle connectivity is unaltered in the absence of Hoxc8   . Loss ofRunx3 or Etv1, leads to marked reduction in the extension of pSN central afferents into the ventral spinal cord   . CollectHoxc8 disrupts the pattern of central connectivity between muscle-specific pSNs and MNs. To examine pSN synaptic specificity, we employed a modified rabies labeling strategy which directs monosynaptically-restricted anterograde transfer of virus from pSNs to neurons within the spinal cord (SN∆Hoxc8 mice with a Cre-dependent line (CAG-loxp-STOP-loxp-rabies-G-IRES-TVAGt(ROSA)26Sor mice, henceforth referred to as RGT) expressing two rabies helper proteins: TVA, an avian-specific receptor protein, which permits infection to rabies virus pseudotyped with EnvA, and a rabies glycoprotein, which allows transsynaptic transfer of the virus, both produced in sensory neurons following recombination using the PLAT::Cre line  or the palmaris longus (PL) muscles with RabV, and mapped the location of transynaptically-labeled MNs, marked by Choline Acetyltransferase (ChAT). We then generated scatter plot and contour maps of the distribution of labeled RabV+ ChAT+ neurons . In control mice, injections into the FCU or PL labeled discrete clusters of RabV+/ChAT+ neurons located in a dorsal region of the caudal LMC   . This re: N = 4) .SN∆RGT Hoxc8 mice we observed ectopic connections originating from distal flexor pSNs onto distal extensor MNs  , compared to 0 ± 0% in control animals  , compared to that of 0.8 ± 0.8% in control animals   . We quan t-test) . Similar t-test) . The per+ sensory boutons at the soma of synaptically-coupled MNs. Thus, after muscle injection we can compare pSN CTB labeling of vGlut1 synapses on Rh-Dex labeled MNs. We injected the FCU with CTB and distal extensor muscles with Rh-Dex in RGT control and SN∆RGT Hoxc8 mice. Similar to the results of the rabies tracing assay, we observed the presence of ectopic synapses from CTB/vGlut1 labeled distal flexor pSNs onto distal extensor Rh-Dex+ MNs . After intramuscular injection, Rh-Dex is taken up by MNs, as well as pSN afferents, but is not transported transganglionically, thus restricting central tracing to MN soma. CTB, however, transfers into the central sensory axon, and accumulates in vGluT1Dex+ MNs . TogetheHox genes are critical in generating appropriate patterns of central connections between pSNs and MNs. We suggest that the coordinate activity of Hox genes in multiple neuronal classes plays a key role in establishing synaptic specificity within developing limb control circuits.Animals rely on internal neural representations of muscle position and activity in order to execute coordinated motor behavior . In vertHox genes have neuronal class-specific functions associated with the development of limb sensory-motor circuits. Our findings reveal that a subset of Hox genes are selectively expressed by pSNs generated at specific segmental levels, and these patterns parallel the rostrocaudal profiles of Hox genes in the CNS. We found that the Hoxc8 gene is preferentially expressed by pSNs targeting distal forelimb flexor muscles, important for wrist and digit movement, reflecting the Hoxc8 expression domain in MNs. While our studies focused on a single Hox gene, it is likely that other forelimb-specific pSN subtypes can be similarly delineated by specific combinations of Hox4-Hox8 genes.Hox transcription factors are well known intrinsic determinants of patterning and cellular identities along the rostrocaudal axis of metazoans . Our resHoxa and Hoxc gene clusters, while Hoxb genes appear to be expressed by broader populations of sensory neurons, most of which are likely cutaneous. These patterns are reminiscent of the differential expression of Hox genes within the spinal cord, where Hoxb genes are typically expressed in dorsal populations, containing cutaneous sensory relay interneurons, while Hoxa and Hoxc genes are expressed by motor-related interneurons and MNs  as well as homozygous mutant mice (SN∆Hoxc8). LacZ-flox/+Hoxc8 and Cre negative Hoxc8 flox mice were used as controls. No gross phenotypic differences between LacZ-flox/+, Hoxc8 floxHoxc8 and wild-type mice were observed. Breeding combinations of homozygous RGT mice were mated with homozygous Hoxc8 flox mice to obtain Hoxc8 flox-RGT mice. SN∆Hoxc8 and LacZ-flox/+Hoxc8 were then bred with Hoxc8 flox-RGT to generate mice heterozygous for both Hoxc8 flox and SN∆-RGT flox/+RGT, Hoxc8 as well as LacZ-flox/+-RGT flox/+. Hoxc8 floxHoxc8 mice were crossed with Olig2::Cre mice to generate MN-specific mutants (MNΔHoxc8 mice). Animal work was approved by the Institutional Animal Care and Use Committee of the NYU School of Medicine in accordance with NIH guidelines.LAT::Cre , Hoxc8 fxc8 flox , Olig2::ig2::Cre , and RGTCre (RGT  mouse lihttp://www.med.nyu.edu/dasenlab/).Mouse embryos were harvested between E11.5 to E18, fixed in 4% paraformaldehyde (PFA) for 1.5–2 hr, cryoprotected in 30% sucrose/PBS overnight and cryosectioned at 10 um or 16 um thickness. Postnatal day P4-P10 mice were perfused with PBS and 4% PFA and post-fixed for 2 hr at 4C prior to cryoprotection and cryosectioning. Primary antibodies against Hox proteins, Foxp1, Isl1/2, Meis2 and Pea3 have been previously described . AdditioUnilateral limb ablations were performed between stages 16–18  and embrMice were sacrificed at P12 by transcardial perfusion and whole muscle dissections were performed with the animal preparation submerged in ice cold 1X PBS solution. Following removal, each muscle was pinned down in a sylgard plate and immersed in 4%PFA for 2 hr at 4C followed by cryoprotection in 30% sucrose solution overnight. Muscles were embedded in mounting media and cryosectioned at 16 um thickness.8 IU/mL.Local stocks of virus were used to amplify, purify, and concentrate rabies virus (RV∆G-mCherry-EnvA) according to established protocols . RV∆G-mc1% CTB (Sigma-Aldrich) solutions were injected with a glass capillary into a forelimb muscle of anesthetized mice at P4-P5 and examined after 3 days. Pups were perfused with PBS and 4% PFA. Spinal cords with attached DRG were isolated and post-fixed for 2–3 hr at 4C. Tissue was cryosectioned at 16 um following cryoprotection.For anterograde labeling of sensory and motor neurons,~0.8 uL of RV∆G-mCherry-EnvA virus was injected with a glass capillary into either the FCU or PL of anesthetized mice at P5-P6 which were then perfused with PBS and 4% PFA 5 days later. Spinal cords were isolated, processed, and cryosectioned at 16 um or 30 um. Muscle injection specificity was verified post-mortem by the exclusive presence of fluorescent labeling in muscle of interest.To anterogradely label sensory neurons and monosynaptically connected motor neurons, RV∆G-mCherry-EnvA virus was injected into either the FCU or PL of mice at ~P5, as described above, for analysis of ectopically labeled motor neuron soma. Concurrently,~10–50 nl of 1% CTB solution was injected into the ECR and ED muscles. Animals were perfused with PBS and 4% PFA 5 days later. Spinal cords were isolated, processed, and cryosectioned at 16 um.To anterogradely label sensory neuron synapses onto motor neurons,~30–50 nl of 1% CTB solution was injected into either the FCU or PL muscle of ~P5 mice, as described above. Concurrently, TMR-Dextran (Rh-Dex) was injected into the ECR and ED muscles to retrogradely label motor neurons. Animals were perfused with PBS and 4% PFA ~3 days later. Spinal cords were isolated, processed, and cryosectioned at 30 um.+ CTB-labeled sensory neurons. Tissue sections of 16 um thick muscle tissue were imaged to analyze muscle spindle projections. Sensory endings within muscle spindles were identified based on the presence of vGluT1+ terminals with characteristic annulospiral morphology. While each unique spindle of an entire muscle was not counted, a series of sections of the whole muscle was profiled to obtain a representative sample of muscle spindles for each muscle. For both cell counts and spindle analysis, serial sections throughout the entire tissue sample were collected into 8 and 5 parallel series of sections respectively and at least one full series of sections was compared between controls and mutants or limb ablated chick embryos. Analyses were performed on N ≥ 3 mice/genotype or per muscle type and N = 3 limb ablated chick embryos.Neuronal cell counts were performed on 10 or 16 um cryosections obtained from caudal cervical DRG. Images were acquired using an LSM 700 Zeiss confocal microscope and cell counts were calculated using the Fiji/ImageJ cell counter feature. For chick limb ablation assays, neuronal cell counts were compared between DRG of the ablated and non-ablated sides of an individual chick embryo. Neuronal cell counts in which forelimb muscles were injected with 1% CTB were performed on 16 um cryosections from caudal cervical DRG and processed/analyzed as described above. Quantifications were done based on comparable labeling efficiency between all injected animals for each muscle type and was required to have a minimum of 10 PVQuantitative analysis of pSN fiber density in the ventrolateral region of the spinal cord was performed on collapsed confocal Z-stacks using Fiji/ImageJ analysis software. The total PV+ collateral area  was measured within a confined lateral region of the ventral spinal cord at the segmental level of DRG C8 set by the borders of the midline and the ventral and lateral gray matter and white matter boundaries. An ROI was set to cover the ventrolateral region to be quantified and measured 45,832 pixels. The threshold was designated based on PV labeling only in the ventrolateral area and the same threshold value was used across all animals. For each genotype, N = 3 animals were analyzed.Plotting of labeled motor populations was performed on 30 um cryosections. Tiled images were acquired with a Zeiss confocal microscope (LSM 700) at 10X. X-Y coordinates for motor neuron soma measured in um units were determined with respect to the central canal using IMARIS software. Contour plots were generated from the X-Y scatter plots and six isolines were automatically assigned in Matlab.+ MNs and 10 RabV/ChAT+ MNs. Serial sections throughout the entire tissue sample were collected into 10 parallel series of sections and three full series of sections were compared for each animal.RV∆G-mCherry-EnvA/CTB/ChAT labeled motor neurons were analyzed from 16 um serial cryosections of the cervical spinal cord. Coincident labeling of soma was quantified using the cell counter feature in ImageJ. For each genotype/forelimb flexor muscle type, at least N = 3 animals were analyzed. Only animals with comparably efficient labeling were used for analysis. Efficient labeling was designated as a minimum of 20 CTB/ChAT+ sensory bouton contacts with P7–P9 motor neuron soma and ∼100 μm proximal dendritic arbor was performed using 0.4 μm confocal z stacks of 30 μm thick sections using a 63X oil objective lens. Gamma-motor neurons were excluded from analysis. Distance of boutons on dendritic arbor from soma was assessed using the scale bar set by Zen software. Images were analyzed using Fiji/ImageJ.Analysis of vGluT1Samples sizes were determined based on previous experience and the number of animals and definitions of N are indicated in the main text and figure legends. In figures where a single representative image is shown, results are representative of at least two independent experiments, unless otherwise noted. No power analysis was employed, but sample sizes are comparable to those typically used in the field. Data collection and analysis were not blind. Graphs of quantitative data are plotted as means with standard error of mean (SEM) as error bars, using Prism 8 (Graphpad) software. Significance was determined using unpaired (Student’s) t-test and was calculated using Prism eight software. Exact p values are indicated in the main text and figure legends. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:hox genes regulate synaptic specificity in sensory-motor circuits. We think that eLife readers will be interested in the findings, and we thank the authors for their detailed attention to the reviewer criticisms, most importantly the addition of quantification of the results for the benefit of our readers.The paper presents a new model of extrinsic and intrinsic control of sensory-motor circuit development, and how Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Intrinsic control of neuronal diversity and synaptic specificity in a proprioceptive circuit" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Summary:Hox genes in sensory-motor circuit assembly is largely unknown. The role for pSN Hox codes in synaptic specificity has been poorly explored because 1) Hox deletion needs to be conditional to prevent MN defects or lethality, and 2) pSNs innervating different muscles are intermingled inside a dorsal root ganglion (DRG), making it difficult to individually study synaptic targeting of each pSN subtype. This is a careful study addressing intrinsic mechanisms of neuronal specificity and the role of Hox transcription factors circuit generation-in this case matching proprioceptive sensory neurons to their targets both in muscle and spinal cord neurons. The authors have overcome these challenges by utilizing a Cre line specific to spinal sensory neurons, and also by introducing a new anterograde labeling technique. This virus-mediated labeling approach is particularly remarkable because this enables selective visualization of entire MNs that a given sensory subtype synapses on, despite their sparse distribution inside a DRG. With this strategy, the authors successfully demonstrated that Hoxc8 is required for caudal pSNs to form specific monosynaptic connections with their appropriate MN targets. This study demonstrates that Hoxc8 in subsets of dorsal root ganglia neurons is not required for survival of the neurons, matching to muscles, or extending to the appropriate region within the spinal cord. But Hoxc8 is required in the sensory neurons for precisely targeting the motor neurons innervating limb flexor muscles, and in its absence, the sensory neuron forms inappropriate synapses on motor neurons for limb extensor muscles. These findings are convincingly shown and carefully reported. Although this is shown for one Hox factor, this may have broader impact as a mechanism for sensory-motor matching. Results from this study suggest that the coordinated regulation of the same Hox gene in pSN and MNs is essential for the specificity of connections in proprioceptive circuits. This paper suggests a novel molecular code that defines muscle- specific proprioceptive sensory neurons subtypes, shows that proprioceptive sensory neurons subtype identities are intrinsically programmed in early development, and that Hox genes are required for proprioceptive sensory neurons central afferents to target the appropriate postsynaptic targets within the spinal cord. In terms of significance the work is appropriate for publication in ELife.The role of Both reviewers had misgivings about the work. For example, in its current form, reviewers point out many places where there is insufficient data to support the conclusions that are being made. Also, where the data is sufficient to make a conclusion, other interpretations that could be equally valid and are not ruled out.Essential revisions:Hoxc8 in sensory neurons doesn't alter pSN identity .1) While the most challenging experiments in the paper  are quantified, many of the foundational experiments in the earlier figures are represented by a single image, with no n's and consequently no statistical analysis. In some cases, these are key negative findings, such as in the limb bud extirpation experiment in Figure 2 and Figure 2—figure supplement 1 (see below) or the very important finding that conditional knockout of Hoxc8 in SNs or MNs in Figure 7—figure supplement 2 is presented anecdotally. In some cases where quantitative data has been obtained, such as in Figure 6, it is not determined whether differences are statistically significant. Although the intrinsic Hox code model for sensorimotor circuit formation is an appealing one and the experiments are perfectly designed to test it, the lack of quantitation of much of the data presented in the paper make it difficult to assess whether the model is supported.2) The important positive result of the altered localization of pSN synapses on motor neurons after conditional KO of Hox family factors in the dorsal root ganglia. This is important for setting up ideas on how they make function. However, as presented, it is not clear which Hox factors were actually assessed  and what the characteristics are for each? The Materials and methods section doesn't help either because it just states the antibodies for the Hox factors have been described. This information needs to be more precise. We suggest a table summarizing all the Hox factors tested and a description of the characteristics would be valuable (supplement is fine). These should include the points made in the first few figures like is it expressed in DRG and does it overlap with markers of proprioceptive identity, what is the rostral caudal restriction if any, which Hox factors are co-expressed.3) The first few figures describe the expression of the Hox program in pSNs, this paper contradicts previous reports showing that muscle-derived cues specify pSN identity and connectivity with MNs. The key finding in the current paper supporting an intrinsic mechanism is the chick limb bud extirpation experiments in Figure 2 and Figure 2—figure supplement 1. However, the conclusions need to be better supported. While Figure 2I gives the sense that Hoxc8 expression is unaffected after limb bud removal, Figure 2—figure supplement 1C reveals that only a small fraction of Hoxc8-expressing neurons is in fact pSNs (this is different from mouse). If most of the Hoxc8-expressing sensory neurons are cutaneous, it is expected that they would be unaffected by removal of limb bud muscles. Figure 2—figure supplement 1C is thus the more important finding if it shows that the number of Hoxc8-expressing pSNs is unaffected after limb bud ablation. However, the number of +Runx3+Hoxc8 neurons is small (2-3) in both images. More n's and quantification of +Runx3+Hox neurons are needed to support the conclusion that pSN Hox expression is unaffected by removal of the limb bud.4) Limb bud removal experiments: In arguing for an autonomous Hox expression in DRGs along the rostro-caudal axis but the data only shows where the various Hox genes are expressed, not where they are not expressed, so we have to trust that the schematic accurately represents data that is not shown. All the rostro-caudal levels that are included in the schematic should be shown for all four Hox genes, as these authors did very beautifully in Catela et al., 2016 Figure 1.5) Figure 1—figure supplement 1A: schematizes Hoxc8. Rather than being presented as a single figure with three or four co-expressing neurons, this experiment should be done in Hoxc8 SN∆ mutants as quantitatively as the wildtype data presented in Figure 4.6) Figure 5E is an important figure showing that pSN identity is unaffected by conditional KO of Hoxc8 mutant pSNs inappropriately target extensor-innervating motor neurons, the authors conclude that a Hox matching mechanism required for synapse formation between pSNs and their appropriate MN targets has been disrupted. While this is an appealing possibility, other interpretations are equally possible. For instance, Hoxc8 may be important for the segregation of pSN axon subtypes in the sensory nerve, or for their axon guidance within the spinal cord. A prediction of the Hox matching mechanism is that flexor-innervating pSNs will fail to synapse on flexor-innervating MNs in Hoxc8 MN∆ animals and may inappropriately innervate extensor-innervating MNs. A single example of the latter result is shown  – more examples of this outcome would strengthen the Hox matching model.7) Based on the finding in Figure 7 showing that  Essential revisions:1) While the most challenging experiments in the paper  are quantified, many of the foundational experiments in the earlier figures are represented by a single image, with no n's and consequently no statistical analysis. In some cases, these are key negative findings, such as in the limb bud extirpation experiment in Figure 2 and Figure 2—figure supplement 1 (see below) or the very important finding that conditional knockout of Hoxc8 in sensory neurons doesn't alter pSN identity .We have addressed each of these issues with additional quantification, further analyses, and provision of specific information on: 1) the number of animals analyzed for each experiment, including those in which a single representative image is shown, 2) clarifying the specific statistical methods used in the paper, and 3) further quantifying the results presented in Figures 2, Figure 2—figure supplement 1, and Figure 5. New quantifications are now plotted in Figures 2F, J, K, L, Figure 5F and Figure 2—figure supplement 1D. See details below.2) The important positive result of the altered localization of pSN synapses on motor neurons after conditional KO of Hoxc8 in SNs or MNs in Figure 7—figure supplement 2 is presented anecdotally. In some cases where quantitative data has been obtained, such as in Figure 6, it is not determined whether differences are statistically significant. Although the intrinsic Hox code model for sensorimotor circuit formation is an appealing one and the experiments are perfectly designed to test it, the lack of quantitation of much of the data presented in the paper make it difficult to assess whether the model is supported.Hoxc8 SN mutant. The corresponding quantitative results, which required retrograde labeling of extensor MNs is shown in Figure 7C, E. We have clarified this in the revision. We have also revised our presentation of the data shown in Figure 7—figure supplement 2, and have limited our claims in the paper with respect to a pSN-MN “molecular matching” model (see point 7 below).In the revision we now provide a more thorough quantification of key experimental data, added additional statistical analyses, and provided specific information on the statistical methods used. We have quantified the results shown in Figure 2 (see point 4 below) and Figure 5F (point 6 below). The data shown in Figure 6 is intended to be qualitative, in showing mislocalization of rabies-labeled neurons in the 3) The first few figures describe the expression of the Hox family factors in the dorsal root ganglia. This is important for setting up ideas on how they make function. However, as presented, it is not clear which Hox factors were actually assessed  and what the characteristics are for each? The Materials and methods section doesn't help either because it just states the antibodies for the Hox factors have been described. This information needs to be more precise. We suggest a table summarizing all the Hox factors tested and a description of the characteristics would be valuable (supplement is fine). These should include the points made in the first few figures like is it expressed in DRG and does it overlap with markers of proprioceptive identity, what is the rostral caudal restriction if any, which Hox factors are co-expressed.Hox genes were tested as well as those that were untested. We have included in this table: 1) whether the Hox protein is expressed in SNs, 2) its rostrocaudal domain of expression, and where possible, 3) whether any other Hox protein is coexpressed with the Hox protein under consideration (this is constrained by the host species of certain Hox antibodies).We thank the reviewers for this excellent suggestion and apologize for neglecting to include this important information. In the revision we have added a table indicating specifically which Hox4-Hox8 paralog genes were analyzed. We have added precise information on the Hox antibody reagents used in the Key Resources Table.We have also clarified in the main text that not all + Runx3+ neurons is small (2-3) in both images. More n's and quantification of Hox+Runx3+ neurons are needed to support the conclusion that pSN Hox expression is unaffected by removal of the limb bud.4) Limb bud removal experiments: In arguing for an autonomous Hox program in pSNs, this paper contradicts previous reports showing that muscle-derived cues specify pSN identity and connectivity with MNs. The key finding in the current paper supporting an intrinsic mechanism is the chick limb bud extirpation experiments in Figure 2 and Figure 2—figure supplement 1. However, the conclusions need to be better supported. While Figure 2I gives the sense that Hoxc8 expression is unaffected after limb bud removal, Figure 2—figure supplement 1C reveals that only a small fraction of Hoxc8-expressing neurons is in fact pSNs (this is different from mouse). If most of the Hoxc8-expressing sensory neurons are cutaneous, it is expected that they would be unaffected by removal of limb bud muscles. Figure 2—figure supplement 1C is thus the more important finding if it shows that the number of Hoxc8-expressing pSNs is unaffected after limb bud ablation. However, the number of Hoxc8+ sensory neurons that express each of the three Hox proteins we analyzed in these experiments, both in controls and after forelimb ablation. We find that the fraction of SNs expressing Hox proteins is unchanged after limb bud ablation. However, consistent with recent studies , the number of SNs expressing the proprioceptive marker Runx3 is reduced by ~50% - making it difficult to assess the fraction of pSNs that maintain Hox expression. It has also been previously demonstrated that the pSN-class specific marker Er81 is markedly reduced after limb bud ablation . Nevertheless, while the fraction of SNs that express Hoxc8 is relatively small in chick relative to mouse, we find that this percentage is unchanged after limb ablation . We added these results to main text as follows:In the revision we have quantified the fraction of all Isl1+ SNs expressing Hoxa5 , Hoxc6 , and Hoxc8  was not significantly changed .”“The fraction of Isl1Hoxc8 is expressed by both cutaneous and proprioceptive SNs in chick, this pattern also appears to be target-independent.We would also like to note that these ablation experiments not only remove limb muscle, but all limb-derived tissues (e.g. limb mesenchyme) that might provide instructive signals to both muscle and cutaneous SN subtypes. As the reviewers are likely aware, recently studies from the Ginty lab  provide evidence that target-derived cues play important roles in specifying the transcriptional identity of both cutaneous and proprioceptive SNs. Thus, while Hox-dependent program that operates in conjunction with limb-dependent signaling programs. We have modified our Discussion to highlight this:We would also like to clarify that our study in no way contradicts the important role that target-derived cues play in pSN differentiation and connectivity. Our results provide evidence for a crucial limb-independent and undoubtedly required to establish the full molecular profile of pSNs and their functional characteristics.”“While these results indicate a limb-independent mechanism of early neuronal differentiation, target-derived cues are 5) Figure 1—figure supplement 1A: schematizes Hox expression in DRGs along the rostro-caudal axis but the data only shows where the various Hox genes are expressed, not where they are not expressed, so we have to trust that the schematic accurately represents data that is not shown. All the rostro-caudal levels that are included in the schematic should be shown for all four Hox genes, as these authors did very beautifully in Catela et al., 2016 Figure 1.Hoxa5 and Hoxc8 expression in DRG and spinal cord, which includes DRG that lack expression of these proteins. In the revision we have added top-down expression analyses for Hoxc4, Hoxc6, and Hoxa7 in Figure 1—figure supplement 1A that similarly show the DRG lacking these proteins.The results in Figure 1I show a top-down view of Hox expression data for the serial sections shown in Figure 1—figure supplement 1A. Nevertheless, the data shown in Figure 1—figure supplement 1A were taken from a single embryo in which the relative rostrocaudal position for each section was recorded for each Hox antibody stain. Thus, the relative positioning of the rostrocaudal domains for Hoxc4 and Hoxc6 are accurate with respect to Hoxa5 and Hoxc8 in these panels, but only approximate relative to segmental level (i.e. C1-T1). We have clarified this in the figure legend, and indicate that similar results were obtained from a total of N=3 embryos analyzed at this stage.Unfortunately, we did not collect the negative 6) Figure 5E is an important figure showing that pSN identity is unaffected by conditional KO of Hoxc8. Rather than being presented as a single figure with three or four co-expressing neurons, this experiment should be done in Hoxc8 SN∆ mutants as quantitatively as the wildtype data presented in Figure 4.We thank the reviewer for this suggestion. In the revision we have quantified the results from the retrograde tracing experiments and now present them in Figure 5F and the main text, as follows:SN∆Hoxc8 mice at P4 and collected spinal cords with attached DRG at P7. We found that the fraction of pSNs that were βGal+ CTB+ was similar between controls and SN∆Hoxc8mice  .”“We injected CTB into the FCU of 7) Based on the finding in Figure 7 showing that Hoxc8 mutant pSNs inappropriately target extensor-innervating motor neurons, the authors conclude that a Hox matching mechanism required for synapse formation between pSNs and their appropriate MN targets has been disrupted. While this is an appealing possibility, other interpretations are equally possible. For instance, Hoxc8 may be important for the segregation of pSN axon subtypes in the sensory nerve, or for their axon guidance within the spinal cord. A prediction of the Hox matching mechanism is that flexor-innervating pSNs will fail to synapse on flexor-innervating MNs in Hoxc8 MN∆ animals and may inappropriately innervate extensor-innervating MNs. A single example of the latter result is shown  – more examples of this outcome would strengthen the Hox matching model.Hoxc8 MN mutant, which would enable a more rigorous quantitative and definitive assessment of synaptic specificity deficits, as we were able to do with the SN-restricted knockout. We are also currently unable to repeat the synaptic tracing assay, due to the effects of the Covid19 shutdown on our mouse colony. We have therefore moved the Hoxc8 MN mutant analysis from the main results to the Discussion, and explicitly state that analysis of Hoxc8 MN mutants are preliminary and require additional supporting data. This section now reads as follows:In the revision, we have reworded our interpretation of these results, and only suggest a “matching model” as one possibility in the Discussion. We have also included other interpretations of the results as the reviewers have thoughtfully suggested. Unfortunately, due to technical constraints, we cannot perform the rabies-mediated transynaptic assays on the Hox gene contributes to sensory-motor specificity. […] Although a Hox-specific molecular matching model for pSN-to-MN connectivity is provocative, Hoxc8 could be required in pSNs, independent of Hoxc8 in MN, for the segregation of axonal subtypes within the sensory nerve, or for their axon guidance within the spinal cord.”“Further studies will be necessary to definitively assess whether the coordinate regulation of MN and pSN connectivity by the same Hox genes are needed in MNs during sensory-motor connectivity is additionally supported by the analyses of the Hox accessory factor Foxp1  and MN-Hoxc9 mutants . We agree it remains to be determined whether there is an exact matching between muscle-specific pSNs and MN pools subtypes, but feel it is not an overstatement to say Hox genes are needed in both neuronal classes during sensory-motor circuit development.We believe the idea that


The order Odonata (dragonflies and damselflies) is among the most ancestral groups of winged insects with drastic morphological changes upon metamorphosis, and thus important for understanding evo-devo aspects of insects. However, basic developmental descriptions of Odonata have been scarce. In an attempt to establish the foundation of developmental and experimental biology of Odonata, we present an unprecedentedly comprehensive survey of dragonflies and damselflies, in total 158 larvae representing 49 species and 14 families, wherein morphological changes of all the final and/or penultimate instar larvae were photographed and monitored everyday. Although their morphology and development were diverse, we consistently identified two visually recognizable morphogenetic events in the final larval instar, namely start of wing expansion and onset of melanization on the wing sheaths, thereby categorizing the final instar into three stages. While the duration of the first stage ranged 4–66 days across diverse Odonata species, the second or third stages exhibited relatively small variation ranging 3–22 days or 1–8 days, respectively, probably reflecting the steady and irreversible metamorphosis process after stage 2. We also described other characteristic morphological changes during the larval development, although they were observed only in some Odonata species and lineages. In Odonata, while adults show an impressive diversity in their body colors and patterns, larvae exhibit a remarkable diversity in their body shape, which reflect their habitats and ecological niches8. Because mayflies (order Ephemeroptera), which also represent the most ancestral winged insects with Odonata, undergo unique subimago stage between the final instar larva and the adult, elucidation of the developmental progress of the last three stages of Odonata  is important for understanding the evolution of insect metamorphosis. Despite the potential biological importance, the basic information on morphological changes of Odonata during larval-adult metamorphosis has been quite fragmentary, mainly because aquatic and carnivorous Odonata larvae are difficult to maintain in the laboratory. The majority of previous studies focused on the morphological changes during the final larval instar of a single species as follows: Coenagrionidae , Aeshnidae , Gomphidae (Asiagomphus pryeri17) and Libellulidae . In these studies, the final larval instar was categorized into 3 to 8 developmental stages defined by morphological traits. However, since different characteristics were used as indicators of morphological changes among different species and most papers did not describe the precise timing for each stage, it is very difficult to comparatively analyze these morphological and developmental data from different Odonata species in a common framework.Dragonflies and damselflies (order Odonata) are among the most ancestral winged insectsHence, in an attempt to establish the foundation of developmental and experimental biology of Odonata, we performed an unprecedentedly comprehensive morphological survey of dragonflies and damselflies. We everyday recorded the morphological changes during final and/or penultimate larval instar using 158 individuals of 49 Japanese Odonata species representing 14 families. From the enormous amount of morphological data based on approximately 5000 photos, the following two morphological changes, namely “the start of wing expansion” and “the onset of melanization on the wing sheaths”, emerged as easily and commonly recognizable developmental indices across the diverse Odonata species. Based on these indicators, we propose that the final larval instar of Odonata could be generally classified into three developmental stages. Other morphological changes in the compound eyes, heads, larval labia, and wing sheaths were also described in detail, although they were observed in more specific Odonata lineages and species.12. We started daily photographing of each larva on the day of ecdysis unless otherwise mentioned  and the penultimate larval instar as F−1 (= F minus 1), because the number of larval instars is not fixed in many Odonata speciesed Table , where tArtemia brine shrimps, whereas dragonfly larvae (suborders Anisozygoptera and Anisoptera) were fed with Chironomus midge larvae. Soon after feeding cessation of the F−0 instar larvae prior to the melanization on the wing sheaths, we transferred them individually to a plastic cage for adult emergence as described previously12.All the 158 larvae of 49 Odonata species representing 14 families Fig. . The lar9, while some species were identified by their nuclear DNA sequences as described below. We extracted DNA from a caudal gill of Zygopteran larvae or a pair of forelegs of newly-emerged Anisopteran adults by using QIAamp DNA Mini Kit (Qiagen). Nuclear ITS1 region was PCR-amplified from the DNA samples using the primers ITS-F0 (5′-GGA AAG ATG GCC AAA CTT GA-3′) and 5.8S-AS1 (5′-GCC GGC CCT CAG CCA G-3′)21. PCR-amplified products were subjected to DNA sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3130xl Genetic Analyzer (Applied Biosystems). Nuclear ITS sequences of all the Japanese Odonata species have been previously deposited in the public database22.Most Japanese Odonata species were identified by their morphological charactersF−0 and/or F−1 instar larvae were photographed everyday using a stereoscopic microscope S8APO (Leica Microsystems) with a digital high definition microscope camera MC120HD (Leica), a stereoscopic microscope S9D (Leica Microsystems) with a digital high definition microscope camera MC190HD (Leica), or a stereoscopic microscope SZ-6850T (Relyon) with a digital high definition microscope camera TrueChrome Metrics (Relyon). Most larvae were photographed from the day of ecdysis, whereas some larvae were photographed from the day of collection, when the insects were already F−0 instar (See Table Epiophlebia superstes and Sinogomphus flavolimbatus), because their entire body is very dark in color (Tables Lestes (Lestidae) did not exhibit black dots on the wings but could be classified as stage 3 by melanization at the base of the wing sheaths  of 49 species everyday Fig. , and allr Tables ; Fig. S117. In these species, notably, while the total durations of F−0 larval instar were generally and conspicuously long . Recently, in I. senegalensis (Coenagrionidae), we categorized F−0 larval instar into five developmental stages that entail the following two-step expansions of the compound eyes: the dorsal expansion  species, the expansion of the compound eyes was observed less obviously, which was partly due to the melanization on their body surface and thus not suitable as an indicator for developmental staging  , Aeshnidae larvae exhibited the relatively complicated expansion patterns in their compound eyes as previously describedine Fig. A–D. Duriine Fig. Y. This aa) Figs. ; Table 1Rhyothemis fuliginosa), and thus cannot be regarded as common developmental indicators 19. On the day before adult emergence, the color of the compound eyes resembled that of adult  as an indicator of metamorphosis in Odonata18. In general, the wrinkles appeared a few days after the onset of stage 2  during F−0 instar has not been described in detail26. Considering that precise larval staging is critical for effective gene knockdown26, this study will facilitate the identification of appropriate developmental stages for RNAi, thereby facilitating the molecular genetic studies on various topics in Odonata.Recently, we established a gene knockdown method by electroporation-mediated RNAi in OdonataSupplementary Legends.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Table S1.


To evaluate effects of age and simulated and real cataractous changes on color vision as measured by the high-definition cone contrast test (CCT).Twenty-four healthy volunteers from two cohort studies performed CCT using best-corrected visual acuity, filters, mydriasis, and pinhole correction. Retrospective cross-sectional study of patients seen in eye clinics evaluated the relationship between age and color vision, and age and lens status in 355 eyes. Last, 25 subjects underwent CCT before and after cataract surgery.CCT scores were most reliable in the nonmydriatic condition without pinhole correction. Progressively dense brown filters produced small decreases in S-cone sensitivity. Linear regression analysis of phakic subjects showed a decline for all cone classes with age. Rate of decline was greater for S-cones  than M-cones  and L-cones . CCT scores increased for S-cones but reduced for L- and M-cones in pseudophakic subjects compared with phakic patients. CCT scores after cataract surgery increased for S-cones, M-cones, and L-cones by 33.0 , 24.9 , and 22.0 , respectively.CCT assessment allows for clinically practical quantitation of color and contrast vision improvement after cataract surgery and aging patients who note poor vision despite good visual acuity.CCT testing, which quantifies hereditary and acquired color deficiency, can also quantify the degree of cataract severity and, combined with other parameters, can provide more precise guidance for cataract extraction to optimize patient care. The anomaloscope, considered the quantitative gold standard” for assessing color vision deficiency (CVD), is a matching test that quantifies abnormalities along the deutan and protan (red–green) axis.The Farnsworth–Munsell 100-Hue arrangement test is more discriminative than pseudoisochromatic plates. It requires subjects to arrange 85 different colored caps in four different groups by order of their hue.Computerized programs, like the custom system used by Fristrom and Lundh,,The more recent introduction of the cone contrast threshold (CCT) test answered the challenges posed by traditional color vision tests by allowing for faster and easier testing while producing accurate quantitation and characterization of CVD.CCT testing differentiates itself from prior testing modalities because it rapidly yields continuous quantitative data for all three cone classes. Recent advances in precise computer-based testing and system affordability have yielded logistically practical and readily available clinical visual function test devices to quantify and report quantitative changes in color and contrast vision. This work presents findings from a systematic investigation of media opacity on color vision using a conventional and clinically practical CCT system, ColorDx CCT HD . The CCT HD was a collaborative development under the Cooperative Research and Development Agreement with the US Air Force, School of Aerospace Medicine, Operational Based Vision Assessment Team at Wright Patterson Air Force Base, to provide increased reliability and resolution of color vision testing with an expanded low-contrast range not available with the original RCCT. Precision pilot assessment was the original focus of CCT HD. However, efficient and highly granular assessment of acquired deficiencies, even in normal color vision ranges, may have useful clinical implications for functional vision testing in disease and drug or substance toxicity conditions.This study sought to quantify the influence of age and cataractous media opacities on color and contrast vision. Color vision changes were first quantified in healthy subjects with simulated media opacities and in normal healthy patients from all age groups. A cohort of healthy patients across a wide age range was studied to evaluate color vision changes with age. Finally, color vision changes associated with cataract surgery were quantified before and after surgery.This study received Institutional Review Board approval from the University of California, Irvine, and was conducted in accordance with the Declaration of Helsinki. Studies performed were compliant with the Health Insurance Portability and Accountability Act of 1996 and all participants enrolled in the perioperative CCT testing provided informed written consent. Three separate analyses were performed to evaluate the effects of cataracts and age on color vision.CCT testing conditions were optimized to determine if mydriasis affected CCT results, and if pinhole correction could be used to account for mydriasis or presbyopia during CCT testing. NDs were evaluated to confirm reduction in CCT scores with increased filter density. Thirty-six phakic eyes of 18 subjects  between the ages of 23 and 38 years (25.3 ± 3.3 years) were recruited for this study. Testing was performed in triplicate under the following conditions: best-corrected visual acuity (no filter), distance best-corrected visual acuity with mydriasis (dilated), ND 0.3 (2× decrease in luminance), ND 0.9 (8× decrease), ND 1.5 (32× decrease) (Bernell Corp), and pinholes. ND 0.3, ND 0.9, and ND 1.5 have optical densities of 03, 09, and 15, respectively. Testing using ND was performed in order of decreasing luminance. One eye was temporarily patched while the fellow eye underwent testing. The inclusion criteria were (1) age 18 years or older, (2) no history of ocular disease or procedures, and (3) best-corrected visual acuity of 20/20 or better in the study eye.Cataract-like media opacities were simulated using brown filter lenses with increasing optical density. The inclusion criteria were the same as described for CCT testing optimization. Six phakic eyes of six additional healthy subjects  between the ages of 23 and 29 years without any ocular history were recruited for this study. Right eyes were tested under the following conditions: no filter, filter 1, filter 2, filter 3, filter 4 (a combination of filter 2 and filter 3 in series), and “blue blocker” filter . The left eye remained patched throughout testing.The spectral transmission for the different ND and brown filters was obtained using a compact spectrometer . Indirect sunlight from clear skies was used to create a baseline spectrum without photobleaching the sensor. The spectra from each filter were collected while maintaining the same position and angle of the sensor. Relative transmission of each filter was calculated. The spectral emission of the CCT monitor for red, green, and blue screens were also measured.n = 256) eyes were identified from the electronic medical record. Inclusion criteria for phakic subjects included: (1) age between 6 and 90 years, (2) no history of cataract extraction or surgery in study eye, and (3) no history of ocular disease in study eye other than age-related cataract formation. A second analysis evaluated color vision in older patients. Forty-two phakic and 57 pseudophakic eyes were identified and evaluated with CCT. Inclusion criteria for the second older age analysis included: (1) age 50 years or older, (2) history of age-related cataract surgery in the study eye, and (3) no history of ocular disease aside from cataract in study eye. Testing in pseudophakic subjects was performed well after (>3 months) the perioperative period from cataract surgery.The relationship between age and color vision in phakic and pseudophakic subjects was retrospectively reviewed in healthy patients attending optometry and medical clinics. Healthy phakic  from 25 patients  between the ages of 51 and 84 years were recruited for this prospective study. The inclusion criteria were (1) age older than 50 years, (2) no history of retinal or macular pathology affecting central vision, and (3) completion of a postoperative visit. Subjects were excluded if their visual acuity decreased by more than two lines on Snellen at their postoperative month 1 visit. Visual acuity and color vision were assessed before surgery and 1 to 2 months after cataract extraction with intraocular lens (IOL) insertion to minimize the occurrence or retinal or corneal edema that may have developed in the proximal perioperative period. At their postoperative visit, pseudophakic subjects with a monofocal IOL targeted for distance vision performed CCT with near add correction, whereas patients with IOL selection for near vision, multifocal lens or extended depth of field lenses wore no reading add. Information regarding IOL type and color was obtained for 30 eyes and is listed in 2). Subjects indicated the perceived direction of the gap by pressing a keypad with arrows, with gated visibility of 5 seconds per stimulus as a forced choice to the subject, whether discernable or not. The optotype contrast, as LogCS, for each stimulus was recalculated based on the subject's prior answers using a Bayesian thresholding method, the Psi-Marginal Adaptive Technique accounting for nuisance parameters like guessing and finger entry errors.The ColorDx CCT HD  is an adaptive visual function testing device that selectively stimulates retinal L-cones, M-cones, and S-cones. A white Dell desktop all-in-one computer and a 21.5” LG LED backlit IPS, 60 Hz color-calibrated anti-glare monitor with a maximum resolution of 1920 × 1080 pixels were used. The size of each Landolt C projected onto the center of the screen has a Snellen equivalent of 20/317, with a viewing angle of 1.43°. A series of tumbling Landolt-C optotypes were presented, with the gap in the “C” facing one of four randomized directions  against a neutral (grey) background  without any ocular history performed CCT using brown filters simulating cataractous media opacity. n = 256) from subjects between the ages of 6 and 90 years were included in this retrospective study based on having completed CCT performance during their clinical care. Phakic eyes (Thirty eyes from 25 subjects between the ages of 51 and 84 years (68.9 ± 6.9 years) were recruited to prospectively study changes in color vision with cataract surgery. Changes in CCT scores before and after cataract extraction are presented in CCT is a modern quantitative color vision and contrast assay that takes approximately 7 minutes for two monocular assessments and is practical for use in clinical evaluation. It has the capability of detecting early stages of systemic, neurologic, and ocular diseases,CCT scores significantly changed depending on testing conditions. Decreases in cone isolation under mydriasis produced the greatest variability in CCT results, whereas decreases in retinal illuminance using pinhole optics may explain the decreased CCT values. Both mydriasis and pinhole optics increase optical aberration which may necessitate CCT testing without mydriasis and without pinhole correction b. CCT sc,,,CCT scores for all cone classes decreased with advancing age in phakic subjects, as previously found by Fujikawa et al.,Prior studies comparing color sensitivity between pseudophakic and phakic eyes found either diminished cone contrast sensitivity for the S-cones,,In patients who underwent cataract surgery, cataracts were noted to decrease color sensitivity for all three cone classes to values below normal, with the greatest decrease seen for S-cone class. After cataract surgery, color vision increased to normal levels with the blue cone showing the greatest improvement. This finding is similar to previous studies using quantitatively discrete and continuous assays.,,A limitation in our study is small sample size for studying progressively dense brown filter lenses. However, test subjects were healthy and reliable test takers. Another limitation is that specific time periods between filter assays were not explicitly controlled. Subjects were tested on one filter, and the subsequent filter was used 30 seconds after normal binocular vision in photopic conditions. This factor may have a small effect by allowing gradual dark adaptation as filters became progressively darker. However, subjects were in photopic conditions during the test and between tests with the total testing time being well below 30 minutes, so dark adaptation would likely be negligible. Additionally, testing with the ND and brown filters could have been initiated using the lowest luminance (darkest filter) first, allowing for a specified adaptation time to adapt to this luminance before testing. This strategy would minimize learning effects by proceeding from worst case to best case scenario. Because light adaptation is rapid, no adaptation would be required when switching to a lighter filter. Another limitation is that the cohort of cataract surgery patients did not exclude early disease. A history of glaucoma,–In conclusion, lenticular senescence and cataract formation diminish color sensitivity for all three cone classes, with the greatest decrease for S-wavelength sensitive cones. Cataract surgery can recover a significant proportion of color and contrast vision. CCT can be used to quantify the effect of cataracts and age impose on vision beyond black and white visual acuity testing. Future investigation may reveal the ways CCT testing might be used to guide recommendations for surgery or optimizing lighting conditions during activities of daily life and mitigate fall risks in the elderly.


Standardized uptake values (SUV-mean) of the region of interest and energy expenditure (EE) were measured. Plasma free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) were measured before and 2 hours after each intervention. Subjects were divided into groups based on the presence (n = 12) or absence (n = 7) of BAT after cold exposure. 12 of 19 subjects were classified as BAT-positive. Subjects with BAT had higher baseline FT3 concentration, baseline FT3/FT4 ratio compared with subjects without BAT. Controlling for body fat percentage, FT3 concentration at baseline was associated with EE change from baseline after cold exposure (P = 0.037) and capsinoids (P = 0.047). Plasma FT4 level significantly increased associated with reciprocal decline in TSH after acute cold exposure and capsinoids independently of subject and treatment status. Circulating FT3 was higher in BAT-positive subjects and was a stronger predictor of EE changes after cold exposure and capsinoids in healthy humans. BAT activation elevates plasma FT4 acutely and may contribute towards augmentation of thermogenesis via a positive feedback response.Thyroid hormones (TH) control brown adipose tissue (BAT) activation and differentiation, but their subsequent homeostatic response following BAT activation remains obscure. This study aimed to investigate the relationship between cold- and capsinoids-induced BAT activation and TH changes between baseline and 2 hours post-intervention. Nineteen healthy subjects underwent  Regulating thermogenesis is one of the major tasks of TH in adult humans2. TH are important for heat generation during shivering and non-shivering cold adaptation. Furthermore, oxygen consumption and heat production at rest and during activities are sensitive to small alterations of TH3. The mechanisms for thyroid thermogenesis are complex4 and not fully elucidated, but one of its key thermogenic effectors operate through brown adipose tissue (BAT)5. With respect to thermoregulatory control, BAT is a critical organ that evolved primarily in mammals to defend against hypothermia through non-shivering thermogenesis (NST). The physiological link between BAT and TH is regulated by the sympathetic nervous system (SNS), while the molecular basis linking TH and SNS to BAT is now understood to operate via the 5’-flanking region of the uncoupling protein 1 (UCP1) gene under the control of both thyroid hormone response element (TRE) and cAMP response element (CRE)7. Effective adaptive thermogenesis in BAT requires TH8, adrenergic stimulation, and uncoupling protein-1 (UCP-1) expression9. TH mediation of adaptive thermogenesis involves both thyroid hormone receptor-α (TRα) and –β (TRβ). While the sympathetic response of BAT depends on TRα, Ribeiro et al.9 reported that TRβ is required for UCP-1 dependent adaptive thermogenesis in BAT of mice that underscored the importance of TH for BAT function. Lopez et al.10 reported that TH are able to up-regulate BAT via the brain as central administration of triiodothyronine (T3) resulted in BAT activation in rats. Adequate circulating concentrations of unbound, free T3 (FT3) derived from intrathyroidal and peripheral deiodination of T4 are necessary for recruitment of brown adipocytes and up-regulation of UCP-1. More recently, we have demonstrated that T3 also contributes to BAT activation via mitochondrial biogenesis and MTOR-mediated mitophagy11. T3 is thus one of the key essential hormones in the process of BAT activation and browning14.Thyroid hormones (TH) including triiodothyronine (T3) and thyroxine (T4) have long been well established to play an important role in energy expenditure (EE) and basal metabolic rate15. TH has been extensively shown to play an important role in cold adaptation in rodents and humans16. As TH can influence BAT mass and activity with resulting augmentation of white adipose tissue (WAT) fat catabolism18, then TH probably exert a physiological role in mitigating against obesity and metabolic syndrome. Conversely, while NST to preserve core temperature is a primary function of BAT activation, it remains unknown if active BAT in turn affects thyroidal TH secretion through some form of homeostatic feedback. In our previous report, we found that EE was significantly increased after cold exposure and capsinoid ingestion in BAT-positive subjects assessed through 18F-FDG PET19. Although this can be attributed purely to BAT activation, might the thyroid also contributed to the persistence and maintenance of EE elevation via TH? To gain further insights regarding BAT activation effects on TH and vice versa in adult humans, this study evaluated TH changes after cold and capsinoids exposure in BAT-positive and BAT-negative healthy participants. The participants’ BAT status was classified by BAT activity via 18F-FDG PET/MR scan. The relationship between BAT and TH before and after cold and capsinoids exposure were evaluated.Cold stress increases the expression and activity of tissue cAMP-dependent type 2 iodothyronine deiodinase (DIO2), a selenoenzyme which stimulates the conversion of T4 to active T32) completed all study procedures  session and were thus excluded; 19 of them . The participants were asked to refrain from caffeine, alcohol intake and any vigorous physical activity on the day before the study. The study protocol and flow chart were described previously19.The subjects attended two separate visits for PET/MR and two separate visits for whole body calorimetry /infrared thermal imaging (WBC/IRT), to assess the effects of cold exposure and capsinoids ingestion. Visits were separated by a minimum of 48 hours washout period in order to minimize carryover effects. Capsinoids capsules  were provided by Ajinomoto Inc. . The composition details were described previously18F-FDG (3 mCi), 20 min before the start of the scans. The scans were performed on a hybrid PET-MR system  for 80 minutes. One experienced researcher blinded to the allocated intervention assessed FDG uptake on both sides of neck and supraclavicular regions excluding bone, muscle and blood vessels. BAT activity was quantified by calculating SUV mean, SUV max and BAT volume. The cut-off value of SUV mean for dividing subjects into BAT-positive and BAT-negative groups was 2.0. Detectable BAT volume was calculated as the sum of pixels meeting this classification criteria multiplied by the pixel volume. The imaging analysis was described as previously21.After fasting for 10–12 hours, the participants arrived at the Clinical Imaging Research Centre. An indwelling cannula was inserted into a forearm vein by a registered nurse after a 10-min rest, and fasting blood glucose was measured. At the first visit, subjects were instructed to ingest capsinoids 30 minutes before the PET/MR scan. During the second visit, subjects donned on the cold vest upon arrival at the research centre. For both visits, subjects were administered an intravenous injection of 22. During each test session, after 45 minutes of resting metabolic rate (RMR) measurement, the subjects were asked to wear the cooling vest or ingest 12 mg capsinoids inside the WBC room for up to 2 hours.After an overnight fast of 10–12 hours, EE was assessed through gaseous exchanges using a dual-chamber whole body calorimeter (WBC) facility located at the Clinical Nutrition Research Centre. The WBC chambers are open-circuit air-tight indirect calorimeters and provide EE, fat oxidation and respiratory quotient (RQ) measurement based on oxygen consumption and carbon dioxide production. The WBC has been described in detail in our previous published paper-assay coefficient of variation (intra-assay CV) of the Cobas immunoassays for the analytes was less than 3%.Screening blood samples were sent to the National University Hospital referral laboratory for biochemical assessment of renal, liver and thyroid function. Fasting and 2 hours post-intervention blood samples during the WBC session was collected from venous samples to measure TH. Plasma concentration of free TH  and TSH were measured using immunoassay analyser . The intrawww.Clinical Trials.gov identifier NCT 02964442). Details on power calculations were shown in our previous publication23. EE was calculated based on volume of O2 consumption (VO2) and CO2 production (VCO2) using the Weir equation24. RMR was defined as the average 45 minutes EE during the 45 min measurement period. The change in EE and fat oxidation (FOX) in 2 hours was calculated as average 2 hours EE and FOX minus RMR. Paired-t tests were used to compare the differences between baseline and 2 hours post-intervention in the concentrations of free, unbound circulating TH. The data were checked for normality using Shapiro-Wilk test and visually using histogram and Q-Q-plot. The data analyzed by parametric statistics were normally distributed. Pearson correlations were performed to identify relations between variables. Data were presented as means ± SEM, unless otherwise stated. A P-value ≤ 0.05 was considered statistically significant. Statistical analysis was performed by using SPSS software version 23 (IBM SPSS Inc.).This was a secondary analysis of data acquired from participants in a protocol that investigated BAT activation and energy expenditure by cold and capsinoids stimulation  and those with detectable 18F-FDG uptake with mean SUV ≥ 2 . No significant differences in anthropometric measurements were found between the two groups , REE  and triglyceride level  but not with other baseline measurements Table . FT4 conIn all of the participants, both cold exposure and capsinoids ingestion significantly increased FT4 concentrations coupled with decreased TSH concentrations compared with baseline Table . There w25. PET SUV was positively associated with EE change from baseline after cold exposure . There was a trend towards a positive association between FT3 concentration at baseline and PET SUV . Also, FT3 at baseline is correlated to EE change post-cold and post-capsinoids  BAT status, (2) modality of BAT stimulation (ie. cold versus capsinoids), (3) pre-stimulated versus post-stimulated time points, and (4) FT3 versus FT4.26. As well, the TH elevations observed were consistently associated with significant reciprocal TSH reductions, which implies that the primary ‘driver’ of the TH secretion resides within the thyroid gland itself rather than being mediated at the central, hypothalamus-pituitary level.Firstly, two potential physiological processes may account for the observation of baseline TH levels  being significantly higher among BAT-positive compared to BAT-negative subjects. One hypothesis is that the presence of BAT within an individual accentuates TH secretion by the thyroid gland via some yet undefined positive feedback interactions and mechanisms. The other possibility is that a higher TH output by the thyroid results in greater brown adipocyte differentiation and BAT activation, which in turn increases the probability of higher TH being found among those who are BAT-positive. Both processes are not mutually exclusive and may co-exist in any given individual to produce this finding. It would require dynamic experiments using appropriately labelled tracers to decipher the kinetics involved and which mechanism predominates. Secondly, while both cold and capsinoids stimulation evoke changes in circulating TH levels regardless of BAT status, the magnitude of increase in TH was greater in FT4 compared to FT3. Because T4 is specifically produced by the thyroid whereas T3 can be derived from both the thyroid and peripheral tissues possessing 5’-deiodinase activity, it is likely that cold and capsinoids directly act on the SNS which then triggers the secretion of thyroidal secretion of TH, with T4 predominating over T3 as per established thyroid physiologyGiven that TSH is an exquisitely sensitive biomarker and regulator of ambient TH levels, the fact that TSH declined inversely to the TH escalations was itself remarkable as it suggested that these “quantitatively miniscule” changes in TH are physiologically significant. This crucial result implied, as a necessary corollary, that TSH should not have altered to any detectable extent had the magnitude of those TH changes been clinically irrelevant. Notably, the pattern of TSH change was similar in both cold and capsinoids stimulation of BAT, and TSH varied inversely with all these TH elevations.27. Apart from differences in thyroidal biosynthetic rate of T4 and T3, it is also known that the thyroid gland harbours a reservoir pool of preformed TH consisting of far more T4 and T328. Hence, it is conceivable that the FT4 level would escalate far more than FT3 in response to any stimulus that increases net thyroid glandular secretory output of TH. Importantly, we observe that the increase in FT3 in the circulation evoked by cold and capsinoids regardless of BAT status is miniscule compared to FT4 despite the documented 10- to 50-fold upregulation of type 2 iodothyronine deiodinase (D2) selenoenzyme during cold stress in BAT15. This is probably because the accentuation of D2 activity led to locally generated T3 that is mainly intracellular within BAT adipocytes with much lesser T3 being secreted into the blood. On the other hand, as the activity of T3 is 3–8-folds higher than T429, this could explain the strong correlation of FT3 changes to EE whereas FT4 changes showed no obvious correlation to EE.Apparently, FT4 rose much more so than FT3 as a result of cold stimulation, associated with a decreased TSH. This is consistent with the fact that at any time, the secretory rate of T4 by the thyroid is nearly 20-fold higher than that of T330. Nevertheless, the finding that the increase in FT4 is 2.3-folds greater in BAT-positive relative to BAT-negative subjects seemingly support the presence of a tentative undefined feedforward factor emerging from activated BAT which has a capacity for stimulating T4 secretion. Finally, as the changes in TH and TSH were all significantly correlated to both PET-SUV and EE, our discovery lends credence to the hypothesis that the positive feedback loop bridging between the thyroid gland and BAT is not trivial but reveals an intriguing aspect of homeostatic regulation that probably plays a definite role in normal thermoregulation biology and in the maintenance of metabolic health. This shows that both BAT and the thyroid, being organs hardwired for thermogenesis, can be simultaneously activated independently by appropriate external stimuli with ensuing TH/TSH changes that reflected the existence of an intrinsically positive homeostatic feedback loop between the two organs  is a novel adipokine secreted by both classical brown and beige adipocytes37. Plasma NRG4 levels have also been shown to be inversely related to the risk of obesity, metabolic syndrome and type 2 diabetes40. Interestingly, serum NRG4 has been demonstrated recently to be elevated in hyperthyroidism, a clinical state of heightened fat catabolism41. Hence, BAT exosomal NRG4 may potentially be one of several critical links between BAT and the thyroid gland42. Our own research also revealed the presence of selenoprotein P within cold-stimulated BAT exosomes (unpublished data). This finding adds further evidence that suggests BAT may regulate thyroid hormone synthesis because selenoprotein biosynthesis defects were shown to cause abnormal thyroid hormone production45. Such exosomal batokines may augment TH output via the observed short (acute) BAT-thyroid feedback loop mediated by pre-existing brown fat, and possibly a long (chronic) positive feedback loop mediated by beige fat derived from browning of WAT  delivered to the thyroid packaged in exosomes. The recent literature has also shown that BAT exosomes contain non-coding RNA such as microRNAs and long non-coding RNAs which can exert gene regulation effects on distant targets, including the thyroid47 that a substantial number of people living within the tropics/equatorial belt with ambient temperatures up to 35 degrees Celsius can still be demonstrated to possess much activatable BAT by cold or capsinoids. Hence, we surmise that this BAT-thyroid axis is physiologically critical in maintaining normal metabolism in addition to defending against hypothermia. While speculative at this point, it is possible that a defect in this BAT-thyroid axis could be medically impactful and predispose the affected individuals to metabolic disorders, such as obesity and type 2 diabetes mellitus. No additional PET-MR scan at ambient temperature was performed on any subjects as a control condition due to ethical constraints and the need to abide by the ALARA principle of keeping ionization radiation exposure to the minimum while fulfilling any clinical/research objective for which we hereby acknowledge as a limitation of our study.It may be argued that this novel axis may seem functionally negligible in humans living in persistently warm climates. However, we have elegantly shown hereIn summary, we postulate that part of the purpose for activated BAT triggering an increase in TH output by the thyroid gland is an evolutionary adaptation as such a biological response should translate, in accordance to fundamental control systems engineering principles, to a far more efficient and optimized thermogenic system if the resulting higher TH output in turn augments BAT activity and development (classic brown fat and beige fat via browning of WAT). We therefore predict that a similar ‘reflex’ feedback loop ought to occur in other mammalian vertebrates of comparable biology, a hypothesis awaiting further proof by future research. This positive feedback cycle within a BAT-thyroid axis favours the successful sustenance of thermogenesis and metabolic homeostasis more robustly than either organ operating independently without any linkage. Therefore, such a BAT-thyroid interaction conceivably exists to confer a biological survival advantage.The trial was registered at clinicaltrials.gov as NCT02964442.


HEPH) show increased hypothalamic and pituitary gene expression related to hypothalamo-pituitary-gonadal (HPG) axis stimulation as well as increased in vitro responsiveness to gonadotropin releasing hormone (GnRH) stimulation in the pituitary when compared to low egg producing hens (LEPH). Transcriptome analysis was performed on hypothalamus and pituitary samples from LEPH and HEPH to identify novel regulators of HPG axis function.High egg producing hens (DEGs) were identified between LEPH and HEPH, with 2021 genes up-regulated in LEPH and 2623 genes up-regulated in HEPH. In LEPH, up-regulated genes showed enrichment of the hypothalamo-pituitary-thyroid (HPT) axis. Beta-estradiol was identified as an upstream regulator regardless of tissue. When LEPH and HEPH samples were compared, beta-estradiol was activated in HEPH in 3 of the 4 comparisons, which correlated to the number of beta-estradiol target genes up-regulated in HEPH. In in vitro pituitary cell cultures from LEPH and HEPH, thyroid hormone pretreatment negatively impacted gonadotropin subunit mRNA levels in cells from both LEPH and HEPH, with the effect being more prominent in HEPH cells. Additionally, the effect of estradiol pretreatment on gonadotropin subunit mRNA levels in HEPH cells was negative, whereas estradiol pretreatment increased gonadotropin subunit mRNA levels in LEPH cells.In the hypothalamus and pituitary, 4644 differentially expressed genes (Up-regulation of the HPT axis in LEPH and upstream beta-estradiol activation in HEPH may play a role in regulating HPG axis function, and ultimately ovulation rates. Thyroid hormone and estradiol pretreatment impacted gonadotropin mRNA levels following GnRH stimulation, with the inhibitory effects of thyroid hormone more detrimental in HEPH and estradiol stimulatory effects more prominent in LEPH. Responsiveness to thyroid hormone and estradiol may be due to desensitization to thyroid hormone and estradiol in LEPH and HEPH, respectively, due to up-regulation of the HPT axis in LEPH and of the HPG axis in HEPH. Further studies will be necessary to identify possible target gene desensitization mechanisms and elicit the regulatory role of the HPT axis and beta-estradiol on ovulation rates in turkey hens. LEPH) being more expensive per egg produced than high egg producing hens (HEPH). At the neuroendocrine level, egg production is regulated by the hypothalamo-pituitary-gonadal (HPG) axis. Proper function of the HPG axis involves feedback on the hypothalamus and pituitary by gonadal steroid hormones and can be impacted by inputs from other neuroendocrine axes, such as the hypothalamo-pituitary-thyroid (HPT) axis.Egg production within the turkey industry is necessary for hatching poults for meat production and has been negatively impacted by intensive selection for carcass traits. Egg production varies within commercial flocks, with low egg producing hens (PS) of luteinizing hormone (LH) and progesterone, resulting in follicle ovulation, but the role of estradiol feedback during the PS is not well characterized in the turkey hen. In the chicken, estradiol reduces gonadotropin inhibitory hormone (GnIH) production and exerts positive and negative feedback on gonadotropin releasing hormone (GnRH) production in the hypothalamus, indicating that estradiol feedback may play a role in ovulation timing [Within the HPG axis, feedback mechanisms of progesterone and estradiol are instrumental for follicle ovulation to occur. Progesterone feedback on the hypothalamus and pituitary triggers a preovulatory surge . Roughly one-fourth of the common DEGs in the hypothalamus and pituitary were unannotated as well. Of the DEGs common to both groups of hens during the ovulatory cycle, a majority showed similar expression patterns in LEPH and HEPH   (RPKM> 0.2) , TSHB, transthyretin (TTR), prolactin (PRL), hydroxysteroid 17 beta dehydrogenase 2 (HSD17B2), and aromatase (CYP19A1), while differentially expressed target genes of beta-estradiol in the pituitary outside of the PS included the androgen receptor (AR), glycoprotein hormones alpha subunit (CGA), steroidogenic acute regulatory protein (STAR), and solute carrier family 7 member 5 (SLC7A5) , prolactin receptor (PRLR), STAR, and TTR, whereas differentially expressed target genes of beta-estradiol in the pituitary of HEPH included CGA and TSHB  or estradiol pretreatment (E2) combined with GnRH treatment. Pituitary cells from LEPH and HEPH responded differently to each pretreatment in terms of gonadotropin subunit mRNA levels, indicating functional differences in the response of the HPG axis to thyroid hormone and estradiol that could be related to differences seen in egg production levels between the two groups of hens , and CGA mRNA levels in cells from LEPH and HEPH, however the effect was more prominent in HEPH cells. T3 decreased LHB, FSHB, and CGA mRNA levels compared to no pretreatment in HEPH pituitary cells, regardless of GnRH treatment concentration. T3 also decreased LHB, FSHB, and CGA mRNA levels in LEPH pituitary cells, but only at 10− 9 M GnRH for LHB, 0 M and 10− 8 M GnRH for FSHB, and 0 M and 10− 9 M GnRH for CGA. Generally, T3 negatively regulated gonadotropin production, independent of GnRH treatment concentration, with a higher negative response from HEPH. These findings suggest that HEPH are more sensitive to the effect of T3 on gonadotropin production, whereas LEPH are more resistant to the effects of T3.T2 decreased LHB mRNA levels in HEPH pituitary cells compared to no pretreatment at 10− 8 M GnRH. E2 also decreased FSHB mRNA levels in HEPH pituitary cells relative to no pretreatment at 0 M GnRH and increased FSHB mRNA levels in LEPH pituitary cells at 10− 9 M GnRH. E2 in HEPH pituitary cells decreased FSHB mRNA levels at lower GnRH treatment concentrations but decreased LHB mRNA levels at higher GnRH treatment concentrations. In contrast, E2 upregulated FSHB in pituitary cells from LEPH at 10− 9 M GnRH. Overall, E2 had varied impacts on gonadotropin production, depending on the rate of egg production of the hens.EExpression patterns of 8 genes per tissue were confirmed through RT-qPCR. Confirmation genes were equally distributed to have one of four expression profiles: genes showing up-regulation in HEPH compared to LEPH (both outside and during the PS), genes showing up-regulation in LEPH compared to HEPH (both outside and during the PS), genes showing up-regulation in one hen group outside of the PS and up-regulation in the other hen group during the PS, and genes showing no changes in expression between hen groups (both outside and during the PS). Each of the confirmation genes examined in the hypothalamus Fig. a and pitFA2H) and somatostatin (SST). FA2H, which was up-regulated in both groups of hens during the PS, is involved in myelin production, which is essential for proper nerve conduction [SST, which was also up-regulated in both groups of hens during the PS, is the main inhibitory hormone of the somatotropic axis but has been shown to inhibit GnRH neuron activity in mice [PRPF19). PRPF19, which was down-regulated in both groups of hens during the PS, has been shown in mouse models to impact the splicing of gonadotropin subunits [Though 4419 DEGs were identified between LEPH and HEPH collectively in the hypothalamus and pituitary outside and during the PS, analysis of the gene expression changes during the PS in each egg production group provided 242 common genes showing similar expression patterns in both egg production groups. Among the genes in the hypothalamus showing similar expression patterns during the PS in both groups of hens was fatty acid 2-hydroxylase  displayed up-regulation in HEPH and down-regulation in LEPH during the PS. In mice, mutations in PSMD2 have been associated with decreased thyroid hormone production [ND4) and cyclooxygenase-2 (COX2) have been previously associated with reproductive functions [ND4 and COX2 showed up-regulation in HEPH and down-regulation in LEPH during the PS. Swine selected for high ovulation rates displayed higher pituitary ND4 gene expression when compared to control lines [COX2 encodes the rate limiting enzyme in prostaglandin production, and deletion of COX2 in mice results in decreased ovulation [On the other hand, analysis of the gene expression changes during the PS in each egg production group revealed 32 common DEGs displaying inverse expression patterns in LEPH and HEPH. Of the hypothalamic common DEGs showing inverse expression patterns between LEPH and HEPH, proteasome 26S subunit, non-ATPase 2 , which encodes avian gonadotropin inhibitory hormone (GNIH) and of gonadotropin releasing hormone 1 (GNRH1)  Fig. a. GnIH n to HEPH . In the  of NPVF . Up-reguFSH) and LH during the PS . Decreased LH during the PS is consistent with decreased mRNA levels for the beta-subunit of LH (LHB) seen in average egg producing hens during the PS [CSNK2A2) is down-regulated in the pituitary of HEPH during the PS. CSNK2A2 encodes an uncharacterized protein in avian species but this protein was shown to be decreased in laying geese pituitaries when compared to non-laying geese, indicating a possible role in egg production or ovulation [GNRHR) during the PS, which was also seen in average egg producing turkey hens during the PS [Endocrine functions of the pituitary, in terms of reproductive hormone production and the regulatory feedback loops involved, showed differential expression between LEPH and HEPH. When comparing HEPH outside and during the PS, HEPH showed down-regulation of follicle stimulating hormone  during the PS compared to levels seen outside of the PS  and solute carrier organic anion transporter family member 1C1 (SLCO1C1) relative to HEPH  in contrast to HEPH  and integrin (encoded by ITGAV and ITGB3) relative to HEPH  relative to LEPH  acts on the thyroid gland to promote the synthesis of thyroid hormones [TSHB during the PS in HEPH could indicate lower circulating levels of TSH, ultimately impacting circulating thyroid hormones. Furthermore, under in vitro culture conditions, thyroid hormone treatment impacted gonadotropin subunit mRNA levels in the pituitary  and at the University of Maryland, College Park . A total of 400 turkey hens over two equal sized flocks from the same commercial line were provided by a poultry primary breeding company  and housed at BARC individually in wire cages during two separate time periods 6 months apart (200 hens per flock). Hens were managed with artificial lighting (14 L:10D) and were provided feed and water ad libitum to NRC standards . These samples were also used to confirm gene expression results obtained from RNA sequencing through RT-qPCR (n = 3 per group). For this experiment, the hypothalamus and pituitary were isolated from each hen, snap frozen as whole tissues in liquid nitrogen, and stored at − 80 °C prior to assessment through RNA sequencing and confirmation of RNA sequencing results as described below. The second flock  was used to perform follow up in vitro pituitary cultures, with three LEPH and three HEPH sampled exclusively outside of the PS (n = 3 per group). For the follow-up experiment, the pituitary was isolated from each hen and placed in ice cold Dulbecco’s Modified Eagle Medium (DMEM) until dispersion, cell culture, and RT-qPCR as described below. For both experiments, blood samples were taken from the wing vein immediately before sampling to measure plasma progesterone levels to confirm correct sampling outside or during the PS. Blood samples were collected and fractionated as previously described [All hens were euthanized by cervical dislocation prior to tissue isolation. Additionally, all hens were sampled on the second day of the hen’s sequence. From the LEPH and HEPH groups, a total of 12 hens were sampled from the first flock and 6 hens were sampled from the second flock, based on the progression of the hen’s sequence and the timing of the preovulatory surge. Experimental replicate numbers per group were determined through a power analysis , with a recommended sample size of three replicates per group. The first flock  was used to perform transcriptome analysis of the hypothalamus and pituitary, with six LEPH and six HEPH, half sampled outside of the PS and half sampled during the PS (escribed . Plasma RIA) kit  to confirm that sampling occurred at the correct time during the ovulatory cycle, based on experimental group assigned. All protocols were performed as directed by the supplier and samples were assayed in duplicate in a single RIA. Ether extraction of plasma samples prior to progesterone assessment and standard curve assessment were performed as previously described [Plasma progesterone levels were measured using a coated tube radioimmunoassay , including on-column deoxyribonuclease digestion. Quantification of RNA was performed as previously described . AmplifiSequencing libraries were generated using a Nextera XT DNA Library Prep kit  with input of 150 pg of amplified cDNA per library, following the manufacturer’s procedure. For each sample, two libraries were produced (from the same amplified cDNA), with a unique index pairing for library. Libraries were purified using Agencourt AMPure XP kit  and were quantified using an Agilent 2100 Bioanalyzer and High Sensitivity DNA Kit. For sequencing 24 libraries (2 tissues) were pooled (10 nM). Libraries were pooled so that set 1 for each tissue was sequenced in a different pool than set 2. Pools were submitted to NC State’s GSL facility for paired-end sequencing (75 bp reads) on an Illumina NextSeq 500.https://usegalaxy.org/) suite. Adapter sequences and low-quality sequences (Phred < 20) were removed from FASTQ files using the TrimGalore tool. Trimmed reads were mapped to the Meleagris gallopavo reference genome . TopHat was used to analyze mRNA libraries. DEGs were determined using the Cuffdiff tool. Pairwise comparisons were made between LEPH and HEPH for each timepoint in the ovulatory cycle as well as between timepoints in the ovulatory for each egg production group. Due to poor annotation of the turkey genome, the protein sequences for DEGs that were unannotated in the turkey were subjected to orthologous comparisons in human, mouse, and chicken protein sequences using Ensembl Biomart (https://useast.ensembl.org/info/data/biomart/index.html). Unannotated DEGs were assumed orthologous if greater than 50% identity to the human, mouse, and chicken was seen at the protein level.All FASTQ sequencing files have been deposited to the NIH Short Read Archive (accession numbers SAMN11624488-SAMN11624511). Processing and analysis of sequencing data was performed using the Galaxy (IPA)  was performed on the differential expression data. IPA was used to construct gene networks as well as to predict upstream biological regulators for each pairwise comparison [2 transformed RPKM values across all of the comparisons examined. The threshold of DEGs was set at P < 0.05 and absolute fold change ≥1.5. Pathways and predicted upstream regulators with P-value < 0.05 (Fischer’s exact test) were considered to be statistically significant. For upstream regulators, published findings in the Ingenuity knowledge database were used to calculate the activation z-score to infer activation or inhibition of transcriptional regulators. Upstream regulators with a z-score greater than 2 or less than − 2 and P < 0.05 were considered to be significantly activated or inhibited. Networks and legends derived from Ingenuity Pathway Analysis are subject to copyright owned by Qiagen Inc.Ingenuity pathway analysis (mparison . Only DESMEM) or DMEM as noted below. Media supplementation, dispersion of pituitary cells, and plating of pituitary cells were performed as previously described [NPT) (10 μl DMEM/F12 added), T3 (1.5 ng/mL of thyroid hormone), or E2 (1.5 ng/mL of estradiol) for 12 h, followed by treatment with chicken GnRH-I  at 0, 10− 9, or 10− 8 M for 6 h . Incubation settings, cell retrypsinization, and cell storage were performed as previously described [All cell isolation procedures were performed using Minimum Essential Medium, Spinner modification  was used to conduct a three-way ANOVA and a two-way ANOVA, respectively. The test of least significant difference (PDIFF statement) was used to compare the least squares means for each group when an overall significance level of P < 0.05 was indicated.SAS software  was used to analyzed all logAdditional file 1: Figure S1. Transcriptome alignment and mapping. (A) The number of reads obtained for each sample. The portion of mapped reads for each sample is represented in blue, whereas the portion of unmapped reads for each sample is represented in orange (mapped to Turkey 2.01 using ENSEMBL annotation release 98). (B) The number of aligned pairs obtained for each sample. The portion of aligned pairs with proper alignment is represented in blue, with discordant alignment in orange, and with multiple alignments in gray (mapped to Turkey 2.01 using ENSEMBL annotation release 98). Figure S2. Hypothalamic network analysis. Ingenuity® Pathway Analysis  was used to generate networks to biologically interpret the expression data. Copyright permission has been obtained from QIAGEN for use of the images presented. (A) Hypothalamic network comparing low egg producing hen (LEPH) and high egg producing hens (HEPH) gene expression during the preovulatory surge (PS) . Green represents genes up-regulated in LEPH, whereas red represents genes up-regulated in HEPH. (B) Hypothalamic network comparing LEPH and HEPH gene expression during the PS . Green represents genes up-regulated in LEPH, whereas red represents genes up-regulated in HEPH. Figure S3. Pituitary network analysis. Ingenuity® Pathway Analysis  was used to generate networks to biologically interpret the expression data. Copyright permission has been obtained from QIAGEN for use of the images presented. (A) Pituitary network comparing low egg producing hen (LEPH) and high egg producing hens (HEPH) gene expression during the preovulatory surge (PS) . Green represents genes up-regulated in LEPH, whereas red represents genes up-regulated in HEPH. (B) Pituitary network comparing HEPH gene expression outside and during the PS . Green represents genes up-regulated outside of the PS, whereas red represents genes up-regulated during the PS.Additional file 2:. Supplement tablesAdditional file 3:. Supplemental file 1Additional file 4:. Supplemental file 2Additional file 5:. Supplemental file 3Additional file 6:. Supplemental file 4