During proliferative plant cell division, the new cell wall, called the cell plate, is first built in the middle of the cell and then expands outward to complete cytokinesis. This dynamic process requires coordinated movement and arrangement of the cytoskeleton and organelles.Here we use live-cell markers to track the dynamic reorganization of microtubules, nuclei, endoplasmic reticulum, and endomembrane compartments during division and the formation of the cell plate in maize leaf epidermal cells.Arabidopsis thaliana, it did not alter callose accumulation, or disrupt cell plate formation in maize. In contrast, CIPC treatment of maize epidermal cells occasionally produced irregular cell plates that split or fragmented, but did not otherwise disrupt the accumulation of cell-plate localized proteins.The microtubule plus-end localized protein END BINDING1 (EB1) highlighted increasing microtubule dynamicity during mitosis to support rapid changes in microtubule structures. The localization of the cell-plate specific syntaxin KNOLLE, several RAB-GTPases, as well as two plasma membrane localized proteins was assessed after treatment with the cytokinesis-specific callose-deposition inhibitor Endosidin7 (ES7) and the microtubule-disrupting herbicide chlorpropham (CIPC). While ES7 caused cell plate defects in Together, these markers provide a robust suite of tools to examine subcellular trafficking and organellar organization during mitosis and cell plate formation in maize. Division is an essential step in cell proliferation and contributes to plant development. The proper re-organization of the cytoskeleton, organelles, and endomembrane networks are essential for cell division. Before plant cells divide, chromosomes are duplicated while cells often reach a size threshold minimum . The nucArabidopsis thaliana, EB1 localizes to microtubule plus ends and accumulate in mitotic structures  . A subseructures .A. thaliana stomatal precursor cells depends on microtubules    , (Zhou e (MLKS2) . In maiz defects  due to d defects . A typicH2B:YFP) . In maizH2B:YFP) . The HISA. thaliana, RANGAP1 (AT3G63130) localizes to the division site throughout mitosis and cytokinesis . RANGAP increases RAN GTPase activity to generate the inactive GDP-bound form. In okinesis . RANGAP1okinesis . The GAPokinesis . In contetaphase .During metaphase, the dynamic movement of organelles and subcellular structures promotes their proper segregation into daughter cells. The spindle assembles after nuclear envelope breakdown in metaphase  and chroDuring cytokinesis, plasma membrane localized proteins are sometimes localized at the cell plate, potentially to aid partitioning into the plasma membrane after cytokinesis is completed. Two examples that localize to the cell plate are the related auxin efflux transporters PINFORMED1 (PIN1) and PIN2 in Arabidopsis. PIN1 localizes to the cell plate where it interacts with DYNAMIN RELATED PROTEIN1A (DRP1A) . SimilarOther proteins that localize to the cell plate are essential for cell plate formation. These include SNARE proteins that facilitate vesicle docking and fusion. Mutations in genes that disrupt cell plate formation lead to defects in cytokinesis that result in lethality or tiny club-shaped seedlings . KNOLLE RAB1A is most closely related to AtRABD2a  in brain, Rab) superfamily, play critical roles in vesicle transport and tethering throughout the endomembrane system and often localize to the forming cell plate . The RABAtRABD2a , which iAtRABD2a . The maill plate . Maize Rll plate , howeverguidance . In Arabthe cell . In maizthe cell .Here we characterize the localization of proteins and organelles during maize cell division using confocal microscopy and disruption of cell plate formation using two chemicals with distinct activities. Since these images were taken using confocal microscopy, we acknowledge that spatial resolution is limited. The well-described live cell microtubule marker lines (YFP-TUBULIN and CFP-TUBULIN) were imaged together with proteins that label microtubule plus ends, chromosomes, the ER, the plasma membrane, and the cell plate. We show that the ER aligns with mitotic structures. RAB-GTPases are required for vesicle-target docking, and the cytokinesis-specific syntaxin KNOLLE is required for vesicle fusion. Several RAB-GTPases and KNOLLE localize to the cell plate and motile particles. We incubated maize leaves with Endosidin 7 (ES7), a chemical that inhibits cytokinesis-specific callose deposition in Arabidopsis  and disr-2 s-1 from 1000Watt high pressure sodium bulbs (Gravita Pro Plus 1000W). Plants were grown for three to five weeks from seeds. Maize transgenic plants were identified by painting the leaf with 0.4% glufosinate  in 0.05% tween or by genotyping using specific primers, listed in Maize plants were grown in 2-quart pots in standard greenhouse conditions (temperature setpoint between 31 - 33 C) with supplementary lighting ~400 µE mwww.micromanager.org) built by Solamere Technology. Standard emission filters were used (Chroma Technology). For YFP–TUBULIN, PDI-YFP, YFP-KNOLLE, RANGAP-YFP, PIN1A-YFP, RAB1A-YFP, and RAB11D-YFP, a 514 nm laser with emission filter 540/30 was used. For CFP–TUBULIN, RAB1A-CFP, PIP2a-CFP, and aniline blue-stained samples, a 445 nm laser with emission filter 480/40 was used. For EB1-mCherry and GLOSSY8-RFP, a 561 nm laser with emission filter 620/60 was used. A 100X oil immersion lens (1.48 NA) was used with immersion oil  for leaf epidermal tissue and a 40X oil immersion lens (1.15 NA) was used with immersion oil  for root imaging.Microscopy was performed using an Eclipse TE inverted stand (Nikon) with a W1 spinning disk (Yokagawa), EM-CCD camera (Hamamatsu 9100c), standard solid-state lasers (Obis from 40 mW to 100 mW), and an ASI Piezo stage controlled with micromanager software (U test (GraphPad Prism) was used for statistical analysis for graphs in Time-lapse and other imaging experiments were performed using a Rose chamber or glass slides, vacuum grease, and coverslips with a temperature between 20 - 22°C . Three t2-y1)/(x2-x1)) before taking the absolute value of all the numbers. The values were then divided by the interval of the time lapse (in seconds) to account for the time elapsed between each frame. Afterwards, the speeds were averaged to generate an average speed of that particle. The particle speeds between non-telophase and telophase stages within the same marker and across markers were compared using a t-test with the Bonferroni correction.Time intervals of 1 to 4 seconds were used to capture YFP-KNOLLE, RAB11D-YFP, RAB1A-CFP, and RAB2A-YFP particle trafficking. Initial imaging intervals of 4 seconds were used and then changed to 1 and 2 second intervals for more efficient particle tracking. Time intervals: YFP-KNOLLE: 2 and 4 seconds, RAB11D-YFP: 2 and 3 seconds, RAB1A-CFP: 2 seconds, RAB2A-YFP: 1 and 2 seconds. 30-118 seconds timelapses were used for particle tracking. Particles were tracked with the FIJI plugin Mosaic with the following parameters: kernel radius = 2.0, cutoff radius = 0.2, percentile = 0.5, displacement = 10, link range = 2 and Brownian Motion . The parFluorescence intensity of YFP-KNOLLE, RAB11D-YFP, RAB2A-YFP and RAB1A-CFP was measured using a set ROI in Fiji based on the size of the smallest particle, a RAB2A particle. To avoid the photobleached parts of the time lapse or changes in the intensity, measurements were only taken from one frame per image. Five sets of data points were measured in different parts of the cell, including the background, cell plate, and particles found in dividing and non-dividing cells, respectively, and then averaged. The average of the cell plate or particle was subtracted from the average of the background, respectively. A log of the ratio of the cell plate and particle fluorescence intensities was generated to determine whether the cell plate or the particles had stronger fluorescence intensity.For maize pulse-treatment with CIPC and Endosidin 7 (ES7), matched samples were used in which one side of each leaf (from the midrib) was incubated with the treatment, while the other side was incubated with a corresponding amount of DMSO. For CIPC treatments, 4-week-old plants were used, and dissections were taken from symmetrically dividing leaf blades where the ligule was less than 2 mm in height. Leaf sections were then placed in a coverslip with either 20 µL of 0.02% DMSO or 2 µM CIPC for 2-4 hours at room temperature (~21 °C). For treatment with ES7, 1 mM ES7 was used, and matched negative control samples were incubated with DMSO (2%). Samples were then loaded into a Rose Chamber and the abaxial side was imaged.To measure the long-term effect of ES7 on maize roots, maize kernels were germinated for 5 days between two layers of germination paper soaked in 50 ml of 0.02% DMSO (negative control) or 10 µM ES7 in 6-quart bins (Sterilite). Three technical replicates were done, where one secondary root was chosen from 4 plants for each treatment. Secondary roots were imaged because primary roots were too thick to effectively image. Roots were stained for 5 minutes in propidium iodide  and loaded into a Rose chamber for imaging using a 40X objective (NA = 1.15).Arabidopsis thaliana ES7 treatments, wild-type (Col-0/Ws) seeds were sterilized with chlorine gas for 2 h at room temperature (∼21°C)  with CFP-TUBULIN grown on ¼ MS plates for 5 days with no treatment, then transferred into 2 ml of ¼ MS with either 50 µM ES7 or 0.1% DMSO treatment for 2 hours. After staining for 30 minutes in FM4-64 (2 µM), 3 replicates of 3-4 seedling roots were imaged. Three different cell plate morphologies were observed: normal, gap (gaps in the cell plate), and stub (when the cell plate is incomplete).For  (∼21°C) . ¼ stren (∼21°C) , 1% sucrTo assess the accumulation of callose in cell plates during pulse treatments, four day old Arabidopsis Col-0 seedlings were treated with 50 µM ES7  in 0.1% DMSO in ¼ Murashige and Skoog (MS) liquid medium, or 0.1% DMSO in ¼ MS liquid medium for two hours  and imagTo assess the accumulation of callose in maize, callose staining was performed as described . CIPC, EThe EB1-mCherry construct was assembled using triple template PCR  to generate the full genomic sequence  with the m-Cherry insert (in pDONR P3r-P4r) at the C terminal end, flanked by linker peptides to minimize folding interference. The PCR products were cloned using the Gateway system  into the donor vectors, pDONR P1-P4 and pDONR P3-P2.Agrobacterium tumefaciens strain EHA101 and transformed into maize A188/B73 hybrid embryo callus by the Iowa State Plant Transformation Facility. Transformed plants were crossed into the inbred line B73.YFP-KNOLLE was generated by a 939-bp genomic DNA fragment including the entire KNOLLE coding region and 5.7 kb of 5′ sequence amplified from B73 genomic DNA with primers KNOLLE-3GWp1 (Primer sequence = GGGGACAAGTTTGTACAAAAAAGCAGGCTCAGAGAGGAGGTGACCAAGC) and Knolle-3GWp4 (Primer sequence = GGGGACAACTTTGTATAGAAAAGTTGATCCAAATCTACAACCGGCAGG). A 305-bp fragment immediately 3′ of the KNOLLE coding region was amplified from B73 genomic DNA with KNOLLE-3GWp3 (Primer sequence = GGGGACAACTT GTATAATAAAGTTGATGAACGACCTCATGACCAAGTCCTTCATGAGC) and KNOLLE -3GWp2 (Primer sequence = GGGGACCACTTTGTACAAGAAAGCTGGGTATCCAGTGATCGGCACTATG). Citrine variant YFP was amplified as described previously . These tThe conserved microtubule plus end localized protein, END-BINDING1 , fused to a monomeric red fluorescent protein (EB1-mCherry), localized to all mitotic structures .). HISTONE1.1-YFP localized to the nucleus during interphase  and prophase (n = 24 cells), specifically labeling chromosomes  labeled the nuclear envelope and the cell periphery during most cell cycle stages in maize epidermal leaves . During Several proteins that label the endoplasmic reticulum (ER) accumulate near mitotic structures. To assess ER localization during mitosis, PROTEIN DISULFIDE-ISOMERASE 1 fused to YFP , an enzNext, we examined several proteins that accumulate in the cell plate, including the cell plate specific syntaxin KNOLLE fused to YFP  and RAB1A fused to CFP or YFP , localized as motile particles and in the cytoplasm in both interphase  and mitotic cells , and accumulated at the cell plate during telophase  and PLASMA MEMBRANE INTRINSIC PROTEIN 2-1 (PIP2A) are both plasma membrane localized proteins that are required for transport of molecules across the plasma membrane. In all cell cycle stages examined, PIN1-YFP  and PIP2A-CFP  localized to the plasma membrane. In addition, during telophase, both PIN1-YFP and PIP2A-CFP weakly accumulated in the cell plate as compared to parental cell wall sites. Accumulation at each cell was measured as a fluorescence intensity ratio of fluorescence at the cell plate: fluorescence at the plasma membrane. The ratio was 0.794 ± 0.634 for PIP2-CFP  and 0.461 ± 0.375 for PIN1-YFP . PIN1-YFP accumulated in the cell plate 100% in both early telophase (n = 7/7 cells) and 90% in late telophase . CIPC binds tubulin and generates fragmented phragmoplasts and fragmented or split cell plates in vivo . CIPC inin vivo . In CIPCin vivo Figure 6Since CIPC disrupted cell plate morphology but did not alter accumulation of cell-plate associated proteins, we treated maize epidermal cells with Endosidin 7 (ES7) a drug that inhibits callose deposition at the cell plate, affects KNOLLE localization at late cell plate stages in Arabidopsis and disrupts cytokinesis in algae . Maize eU test, p-value > 0.1). No cell wall stubs were seen in ES7 or DMSO treated maize roots stained with propidium iodide . Arabidopsis seedlings were stained with propidium iodide and cell wall stubs were frequently observed in roots of seedlings grown in 10 µM ES7 for 5 days  which encode proteins with 64% amino acid identity. Both maize EB1 homologs are most similar to AtEB1a and AtEB1b. Maize EB1(Zm00001eb068860) localization is more similar to AtEB1a and AtEB1b than AtEB1c, which localizes conspicuously in the nucleus in addition to the microtubule plus end , or vesicle populations. We used the well-characterized YFP-KNOLLE to assess cell plate formation and associated vesicle trafficking. In Arabidopsis, this mitotic syntaxin mediates homotypic cell fusion: knolle mutants have cytokinesis defects and an accumulation of unfused vesicles at the cell plate  is a chemical that inhibits cytokinetic specific callose deposition and cell plate maturation, often visualized as cell plate gaps. As a result, ES7 treatment disrupts the cell plate localization of KNOLLE and the RAB GTPase RABA2a during late cell stages of cell plate development . Of the okinesis . Althougokinesis , ES7 treokinesis , the tarAs demonstrated here, cell division in maize epidermal cells is a dynamic process that can be visualized at high spatial and temporal resolution. These markers highlight both conserved and potentially unique roles of proteins involved in cell division across the diversity of plants. Further experimental and functional studies using these markers will help clarify the role of these proteins and the spatial and temporal control of maize cell divisions.The original contributions presented in the study are included in the article/LA, MB, VH, MD, AC, AR, BV, SS, AU, SM, CR designed and performed experiments, prepared the figures, and wrote the draft. GD provided ES7 and edited the manuscript, RS took images for ES7 experiments in Arabidopsis. GS, BD, DBV, and AN contributed to interphase microtubule measurements. CR conceptualized and designed the research, provided supervision, acquired funding and wrote the final manuscript. HS, XZ, and AS designed and generated fluorescent protein constructs EB1-mCherry and YFP-KNOLLE. All authors contributed to the article and approved the submitted version.


Chlamydia trachomatis (CT) infection and vice versa. Coinfection of HPV and CT in women is a very common and usually asymptomatic finding that has been linked to increased risk of cervical cancer. It has been demonstrated that CT facilitates the entry of multiple high risk HPV genotypes, leading to damage of the mucosal barrier and interfering with immune responses and viral clearance, which ultimately favours viral persistence and malignant transformation. Although the facilitating effects elicited by CT infection on viral persistence have been reported, little is known about the consequences of HPV infection on CT development.Infection with Human Papillomavirus (HPV) is a recognized risk factor for Herein, we took advantage of a genetically modified human cervical cell line co-expressing HPV-16 major oncogenic proteins E6 and E7, as an experimental model allowing to investigate the possible effects that HPV infection would have on CT development.Our results show that CT infection of HPV-16 E6E7 expressing cells induced an upregulation of the expression of E6E7 oncoproteins and host cell inhibitory molecules PD-L1, HVEM and CD160. Additionally, smaller chlamydial inclusions and reduced infectious progeny generation was observed in E6E7 cells. Ultrastructural analysis showed that expression of E6 and E7 did not alter total bacterial counts within inclusions but resulted in increased numbers of reticulate bodies (RB) and decreased production of infectious elementary bodies (EB). Our results indicate that during CT and HPV coinfection, E6 and E7 oncoproteins impair RB to EB transition and infectious progeny generation. On the other hand, higher expression of immune inhibitory molecules and HPV-16 E6E7 are cooperatively enhanced in CT-infected cells, which would favour both oncogenesis and immunosuppression. Our findings pose important implications for clinical management of patients with HPV and CT coinfection, suggesting that screening for the mutual infection could represent an opportunity to intervene and prevent severe reproductive health outcomes, such as cervical cancer and infertility. Meta-anorldwide . In addiorldwide . This reorldwide . Some auorldwide . Moreoveorldwide .HPV and CT infections share similar risk factors such as young age and a high number of sexual partners as well as common clinical features such as their asymptomatic nature favouring transmission . In addiImmune evasion tactics utilized by CT include the entry into a persistent state in the presence of stressors, like exposure to cytokines or nutrient deprivation, which allows the bacterium to pause its developmental cycle and survive in an intracellular aberrant state for extended periods of time avoiding immune destruction and remaining able to resume normal propagation once the stressing condition is removed . In addiHPV can also evade the immune system by different strategies through the expression of HPV oncoproteins E6 and E7, which take advantage of their high binding affinity to cellular immune regulatory proteins, blocking immune-related gene expression and immune signalling pathways and creating an overall immunosuppressive environment . Indeed,Most individuals get infected with HPV during their lifetime and the immune system clears the virus within a couple of years post-infection . Howeverin-vivo occur after the integration of viral genome into host epithelial cells producing stable E6 and E7 expression  or the one containing the HPV-16 E6/E7 ORFs , kindly provided by Dr. Francisco Aguayo (University of Chile). Transfection assays were made using polyethyleneimine (PEI) as previously described  were continuously propagated for 2-3 weeks under selection and frozen stocks were prepared. G418 selection was removed for at least 3 passages before experiments (10-14 days). To verify stable transfection, PCR assays targeting the HPV-16 e6 and e7 genes were performed using primers specified in Cervical cancer-derived human epithelial cell lines C33-A , HeLa  and Ca Ski  were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% inactivated Foetal Bovine Serum (FBS) (Natocor), without antibiotics and incubated at 37°C in a 5% COescribed . Briefly2.2t (ΔCt) was used as quantitation method. GAPDH amplification was used for data normalization.Cells in basal, stimulated or CT-infected conditions were trypsinized and used to extract RNA at the indicated times. Cellular RNA was isolated using the extraction commercial kit “Gen Elute Mammalian Total RNA Miniprep Kit” (Sigma) according to the manufacturer’s instructions. RNA integrity was determined by direct visualization of samples electrophoresed in 1% agarose gel in TAE buffer . The RNA concentration and purity was determined using a Nanodrop 1000 spectrophotometer (Thermo Scientific). An aliquot of 2 μg RNA was treated with 1 μl of DNAse  for 15 min at RT and denatured at 65°C. cDNA was synthesized in a 10 μL reaction volume containing DNAse-treated RNA (2 μg), 1U RNAse inhibitor (Promega), 2 μg of random primers (Biodynamics), 2 mM of each dNTP (Promega) and 10U of M-MLV Reverse Transcriptase (Promega). The reaction mixture was incubated for 1 h at 42°C followed by incubation at 65°C for 10 min. The cDNA samples were subjected to PCR amplification with specific primers for HPV-16 E6, HPV-16 E7, TLR2, TLR3, TLR4, TLR5, TLR7, hBD1 and GAPDH Table 1.2.36 cells were stained for surface markers. Dead cells were excluded using Live-Dead fixable reagent (Invitrogen). The following antibodies from eBioscience  or Biolegend  conjugated with appropriate fluorochromes were used: anti PD-1, PD-L1, HVEM, CD160, CD39, CD73, BTLA, and HLA-I. To assess E6 or E7 protein expression, indirect intracytoplasmic stain was performed after surface labeling. For this purpose, cells were fixed with Cytofix (BD) according to manufacturer’s instructions and subsequently permeabilized using Perm Wash permeabilization buffer (BD). Anti-E7 or anti-E6 Mouse IgG monoclonal antibodies were used. These primary antibodies were gently provided by Dr. Prat Gay from the Leloir Institute, Buenos Aires, Argentina (® 594 (Abcam) was used as secondary antibody. Finally, cells were acquired in FACS-CANTO II or LSR Fortessa flow cytometers  and analysed using FlowJo™ v10.6.2 software. Proper compensation using Fluorescence Minus One (FMO) control was used.Single cell suspensions containing 1x10rgentina . Then, gFor cell cycle analysis, cells were collected and fixed with 70% ethanol overnight at 4°C, resuspended with 50 μg/ml RNase A plus 50 μg/ml propidium iodide, and then acquired by FACS-CANTO II. Cell cycle data analysis was done using FlowJo™ v10.6.2 software.2.42 humidified atmosphere at 37°C. Density gradient-purified EBs of CT serovar LGV-L2 strain 434/Bu ATCC VR-902B (L2 wt) or CT serovar E were obtained as previously described .For CT infection experiments, C33-Empty, C33E6E7 and Ca Ski cell monolayers were plated 24h before infection  in DMEM medium (Gibco) supplemented with 10% FBS (Natocor) without antibiotics, in a 5% COescribed . Infecti2.5To determine the generation of Inclusion Forming Units (IFUs), C33-Empty and C33E6E7 cells were seeded in 6-well plates and infected under the aforementioned conditions. At the time of infection, cell confluency was ∼80-90%. Forty hours later, cells were subjected to hypotonic lysis with water (800 μl/well) to release infectious particles and the appropriate volume of 5X sucrose–phosphate–glutamate (SPG) buffer  was added to obtain a final resuspension of the lysates in SPG 1X. Lysates were preserved at -80°C and later used to infect confluent monolayers of HeLa cells in order to assess the number of inclusion forming units (IFUs) per microliter as described . At 40 h2.6To determine CT inclusion size, C33-Empty and C33E6E7 cells were labelled with anti-CT043 antibody after 24 or 40 hpi as indicated above. The area of at least 100 inclusions per condition were measured and expressed as arbitrary units (a.u.) using the ImageJ software .2.7To evaluate the ultrastructural features of CT, monolayers of C33-Empty and C33E6E7 cells were obtained in 24-well plates and infected with CT (MOI:2). At 24 or 40 hpi monolayers were detached with a 0.5% Trypsin-EDTA solution for 30 sec. Subsequently, detached cells were centrifuged at 1500 rpm and the pellets obtained after centrifugation were fixed with Karnovsky fixative and stored at 4°C until processing. Samples were processed by the Electron Microscopy Center, School of Medical Sciences, National University of Cordoba. Post-fixation, the pellets were de-hydrated, embedded, cut, and examined as previously described  using Ze2.8Salmonella ser. Typhimurium (Sigma), at 100 μg/ml or 10 μg/ml, respectively, during 24h. After that, cells were trypsinized and RNA extracted to perform RT-PCR.C33-Empty and C33E6E7 cells were incubated with the TLR2 ligand Zymosan (Sigma), and the TLR4 ligand LPS from 2.9p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant, p > 0.5). Tests performed are detailed in the corresponding figure legends.All the experiments were done in triplicates and repeated at least three times. Statistical analysis was performed using the GraphPad Prism 9.1 software  or control (C33-Empty) epithelial cells were infected with CT LGV-L2 for 24 and 40 hours, as indicated. As shown in It is well known that CT has a biphasic developmental cycle involving non-replicative, infectious elementary bodies (EBs) and non-infectious replicative reticulate bodies (RBs) . InfectiTo evaluate the influence of E6 and E7 oncoproteins on different stages of CT development, we performed transmission electron microscopy of C33-Empty and C33E6E7 cells infected with CT LGV-L2. The ultrastructural analysis revealed that in both cell lines, inclusions contain a mixture of particles with typical morphological features of EBs, RBs and intermediate bodies (IBs), representing different CT developmental stages (3.4p=0.004). Although not significant, a similar trend was observed at 24 hpi (p= 0.08) (p=0.01), but not in C33-Empty cells. In addition, CT infection also induced increased E7 levels in Ca Ski cells, a cell line that has been reported to contain multiple copies per cell of integrated HPV-16 genomes (p=0.02) (p=0.06). However, in CT-infected condition, significantly higher frequencies of PD-1+ C33E6E7 cells were observed compared to C33-Empty (p<0.003). Regarding PD-L1 and CD160, a similar effect was observed with significant increments in PD-L1+ cells and CD160+ cells in C33-Empty and C33E6E7 after CT infection. On the other hand, CT infection did not induce significant HVEM changes in C33-Empty with respect to unstimulated C33-Empty cells. Nevertheless, when C33E6E7 cells were infected with CT, significant increments in HVEM+ cells were observed (p<0.0007) (p=0.03), while a high frequency of PD-L1+ cells was observed in both unstimulated and CT infected Ca Ski cells showing not significant differences between unstimulated and CT infected conditions (Human beta-defensin 1 (hBD1) is an antimicrobial peptide that is produced by several epithelial cells after stimulation with micro-organisms . We wondp= 0.08) , we then genomes . Finally4Coinfections are experiencing an increasing interest in clinical practice nowadays. Although they have been increasingly detected, their implication in disease development and their consequences remain to be unveiled. Certainly, HPV and CT coinfection has shown a considerable prevalence being each other mutual risk factors of infection and cervical pathology . Indeed,in-vitro model of coinfection, we demonstrate that during CT infection of C33E6E7 cervical epithelial cells and also Ca Ski cells, the expression of HPV-16 E6 and E7 oncoproteins increased even more suggesting that coinfection may have additive effects on cell cycle and cellular proliferation compared to each pathogen alone. In agreement, Challagunda et al. reported that CT infection of mouse epithelial cells expressing E6E7 increased E6E7 mRNA expression (in-vitro and in-vivo experimental models of CT infection and E6E7 overexpression, indicating that CT infection promotes cellular proliferation, survival and transformation (E6 and E7 oncoproteins from HR HPV act inhibiting tumour suppressors TP53, pRb and other molecular signals prolonging cell-cycle progression, delaying differentiation and inhibiting apoptosis in host cells . On the pression . Moreoveormation . Interesormation .in-vitro model of coinfection, we found that CT infection induces a higher expression of the regulatory molecules PD-L1, HVEM and CD160 in cells expressing HR HPV-16 E6E7 oncoproteins, further supporting the additive effects of these pathogens. Augmented expression of PD-1 is considered indicative of T cell exhaustion as evidenced in many types of chronic viral infections (On the other hand, it is well known that both HPV and CT manipulate and bias the immune system towards an immunosuppressive scenario . Using afections . PD-1 bifections . Liu et fections . It has fections . Herein,fections . Moreovefections . Indeed,fections . Thus, ifections . The molfections . Howeverin-vitro coinfection models, it was found that the interaction of an HSV glycoprotein was sufficient to trigger both suppressed production of infectious chlamydial progeny and stimulated formation of swollen, aberrantly shaped RBs (One of the most interesting results of the present work is the observation that expression of HR HPV-16 E6E7 oncoproteins in C33E6E7 cells altered CT development. Indeed, the presence of E6 and E7 proteins resulted in smaller CT inclusions and a significant reduction in infectious progeny generation. Additionally, ultrastructural analysis of CT-infected C33E6E7 cells revealed a decreased EB/RB ratio at the end of the CT developmental cycle, while the total number of bacterial particles was not significantly reduced compared to C33-Empty cells. These findings indicate that while HPV E6E7 expression does not seem to abolish CT replication, it clearly impairs RB to EB transition. All these observations are in agreement with a recent report by Koster et al., indicating that HPV-16 E6E7 expression interferes with the normal development of CT in a human-derived ectocervical organoid model of HPV-CT coinfection . In factaped RBs .A summary scheme illustrating a proposed model for HPV-16 E6 and E7-induced changes in CT developmental cycle and immune regulatory molecules is shown in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.VR and HS obtained funding, conceived and designed the experiments. CO substantially contributed to the conception and design of the work, acquired, analyzed, and interpreted the data. CO, JM, AA, GB, DP, FF and MM performed experiments. CO, VR, CC, JM, RM, HS drafted and critically revised the manuscript. All authors contributed to the article and approved the submitted version.


New disease targets and medicinal chemistry approaches are urgently needed to develop novel therapeutic strategies for treating pulmonary diseases. Emerging evidence suggests that reduced activity of protein phosphatase 2A (PP2A), a complex heterotrimeric enzyme that regulates dephosphorylation of serine and threonine residues from many proteins, is observed in multiple pulmonary diseases, including lung cancer, smoke-induced chronic obstructive pulmonary disease, alpha-1 antitrypsin deficiency, asthma, and idiopathic pulmonary fibrosis. Loss of PP2A responses is linked to many mechanisms associated with disease progressions, such as senescence, proliferation, inflammation, corticosteroid resistance, enhanced protease responses, and mRNA stability. Therefore, chemical restoration of PP2A may represent a novel treatment for these diseases. This review outlines the potential impact of reduced PP2A activity in pulmonary diseases, endogenous and exogenous inhibitors of PP2A, details the possible PP2A-dependent mechanisms observed in these conditions, and outlines potential therapeutic strategies for treatment. Substantial medicinal chemistry efforts are underway to develop therapeutics targeting PP2A activity. The development of specific activators of PP2A that selectively target PP2A holoenzymes could improve our understanding of the function of PP2A in pulmonary diseases. This may lead to the development of therapeutics for restoring normal PP2A responses within the lung. Chronic respiratory disorders affect the lungs, airways, and other structural components, leading to widespread illness and mortality. Lung cancer, asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) are some of these respiratory conditions, and are among the top causes of morbidity and mortality worldwide. From 1990 to 2017, there was an increase in the prevalence of chronic lung disease worldwide. In 2017, 544.9 million individuals worldwide were impacted by chronic respiratory diseases, which is a rise of 39.8% from the 1990 data [The most common lung cancer types, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), account for approximately 80–85% and 10–15% of all instances of lung cancer, respectively ,3. The 5One of the common molecular players observed to be altered in all of these pulmonary diseases is the serine/threonine (Ser/Thr) phosphatase, protein phosphatase (PP) 2A (PP2A). This review will outline the activation status of PP2A in pulmonary diseases and the subsequent impact on multiple pathways, discuss the potential means of targeting PP2A responses, and possible outcomes of targeting PP2A activity systemically.Reversible phosphorylation of signal mediators is a cornerstone of intracellular signaling and is primarily regulated by a delicate balance of intracellular and extracellular kinases and phosphatases . PhosphoPPP2R1A and PPP2R1B genes, respectively. This particular subunit serves as a backbone for holoenzyme recombination [The A subunit exists in two isoforms, PR65/Aα and PR65/Aβ, encoded by the bination . These ibination .PPP2CA and PPP2CB genes encode these isoforms, each forming a globular structure folding on itself in an α/β arrangement. PPP2CB has weaker promoter activity, resulting in its expression being ten-fold lower than PPP2CA [Similar to the A subunit, the C subunit has two isoforms that serve as PP2A holoenzyme’s catalytic domain. The n PPP2CA . It is tn PPP2CA ,30. The n PPP2CA . The catn PPP2CA .PPP2R2A, PPP2R2B, PPP2R2C, and PPP2R2D) [PPP2R5 (A-E) [PPP2R3A, PPP2R3B, and PPP2R3C) comprising of six isoforms [STRN, STRN3, and STRN4), each producing one isoform. The combination of all the different subunits, their subclasses, and isoforms leads to more than 80 different PP2A complexes, each with unique physiological functions. The B subunit attaches to repeats 1–10, while the C subunit occupies repeats 11–15 [B subunit isoforms bind in a mutually exclusive manner, providing subcellular localization, substrate specificity, and physiologic function of the final PP2A product ,34. As mPPP2R2D) ,37,38. TR5 (A-E) ,14,39,40isoforms . B‴  deficiency, lung cancer, asthma, and IPF ,49,50,51PP2A plays a major regulatory role in initiating and maintaining inflammatory disease seen in COPD . ModulatPtp1b deficient mice when exposed to cigarette smoke [Ptp1b deficient mice are more susceptible to COPD [Cigarette smoke enhances endogenous inhibitors to suppress PP2A and contributes to suppressing PP2A activators, such as protein tyrosine phosphatase 1B (PTP1B) ,52. PP2Ate smoke . Smoke i to COPD ,76. SmokA critical contributor to COPD disease pathogenesis is maladaptive airway remodeling. While the role of serine elastase and matrix metalloproteinases in the pathogenesis of COPD is rigorously described, there is emerging interest in the role of the cathepsin family of enzymes as a major initiator of pathologic airway remodeling . CathepsPP2A also plays a crucial role in mitigating and resolving established inflammatory responses via regulating the mRNA-destabilizing protein tristetraprolin (TTP). Enhancing TTP activity reduces the severity of cigarette smoke-induced experimental COPD . After sThe development of COPD in AAT deficiency individuals is the best described genetic associated link to COPD development. By examining PP2A activity levels in neutrophils from healthy MM and AAT deficiency ZZ individuals, our group observed diminished PP2A in the neutrophils from AAT deficiency subjects. Additionally, AAT supplementation in AAT deficiency individuals increases PP2A activity in neutrophils, monocytes, small airway epithelial cells, and A549 cells. Mechanistically, AAT increased PP2A activity in a PTP1B-dependent manner in vitro and in vivo. AAT stimulation leads to PP2A dephosphorylation and activation at Tyr307 in the catalytic C subunit, a specific target of PTP1B phosphatase . FurtherYoshiki and colleagues observed that severe asthmatic patients who are treatment resistant to conventional steroid therapy had increased phosphorylation of JNK-1 and glucocorticoid receptor (GR) Ser226 with concomitant decreases in PP2A activity . IFN-γ/LPatients with severe eosinophilic chronic rhinosinusitis characterized by local resistance to corticosteroids had decreased PP2A mRNA expression in the nasal epithelium, which was improved by PP2A plasmid transduction, omalizumab, and long-acting beta agonist ,68. NatiPpp2r1a (the gene responsible for subunit A) transgenic mouse models exposed to ambient particulate matter 2.5 (PM2.5) resulted in increased inflammation and pulmonary fibrosis in these animals [2.5 exposure can induce oxidative damage, fibrosis, and collagen deposition [2β1 integrin receptor concentrations lead to decreased PP2A levels, increased phosphorylated GSK-3β, and increased β-catenin and fibroblast proliferation.The deletion of PP2A is linked to the pathogenesis of IPF, as PP2A activation responses are blunted in fibroblasts isolated from IPF subjects . Myeloid animals . PM2.5 eposition . Similarposition . Direct position ,93,94,95position . In IPF,Tissue analysis of eight IPF patients’ lung biopsies revealed elevated MID1 and TRAIL expression and decreased PP2A activity . To furtLoss of PP2A subunits via gene mutations or its inhibition by various endogenous inhibitors is associated with the development of various cancers . ParticuMutations in genes encoding PP2A’s regulatory β-subunit are also implicated in lung adenocarcinoma cell proliferation. The PP2A-B56γ isoform is responsible for dephosphorylation and inactivation of ERK and thus aborting cancer cell growth and tissue invasion ,59. c-MYPP2A has multiple endogenous and exogenous inhibitors, but the major endogenous inhibitors are cancerous inhibitor of PP2A (CIP2A), inhibitor 2 of PP2A (I2PP2A/SET), endosulfine α (ENSA), ubiquitin E3 ligase midline 1 (MID1), and the protein phosphatase methylesterase 1 (PME-1) ,49.Our group has shown that cigarette smoke can enhance CIP2A expression resulting in PP2A inactivation . CIP2A h50 of 2nM in vitro [I2PP2A is a specific and potent inhibitor of PP2A displaying non-competitive kinetics with ICin vitro . I2PP2A in vitro ,111,112.in vitro . SET inhin vitro .MID1 interacts with the α4 regulatory subunit of PP2A and is required for its catalytic C subunit’s ubiquitin-specific modification and proteasome-mediated degradation . MID1 isPME-1 is upregulated in 3.1% (4/124) of lung cancer samples and is associated with PP2A demethylation at leucine 309 resulting in the inactivation of PP2A . ContrasENSA plays a significant role in regulating mitosis by inhibiting the activity of PP2A-B55 during the M phase. Ref.  ENSA intThe PP2A catalytic subunits are subject to ubiquitination and proteasomal degradation when not bound to an A or B subunit. α4 is a non-catalytically active protein that can protect PP2A activity via C subunit binding and protects the C subunits from ubiquitination. The overexpression of α4 leads to hasten resolution of stress associated with DNA damage and increased cell survival .Ceramide can activate PP2A in a stereospecific manner. In acute lung injury, TNFα induction leads to enhanced NFκB and MAPK responses, and IL8 mRNA production. TNFα activates sphingomyelinase, ultimately leading to ceramide production and accumulation. This accumulation results in PP2A activation leading to decreased inflammation . AdditioMost novel approaches aim to restore PP2A activity can be classified into direct strategies to activate the PP2A holoenzyme or its subunits and indirectly by counteracting the variety of endogenous negative regulators of PP2A see .®), an FDA-approved drug by Novartis, is a sphingosine analog with immunosuppressive actions used to treat multiple sclerosis. In vivo, FTY720 is phosphorylated to FTY720-phosphate, the active immunosuppressant compound, that later binds to the sphingosine phosphate receptor (S1P1), leading to internalization and destruction of this receptor [NDRG1 tumor suppressor gene, which drives epithelial to mesenchymal transition in cancerous A549/CDDP cells and impart chemoresistance [As already mentioned, one strategy to inhibit SET is using ceramide and its derivatives, endogenous bioactive sphingolipids that activate PP2A by disrupting SET/PP2A interaction ,128,129.receptor . FTY720 receptor . Inactivsistance . FTY720 sistance . Derivatsistance . WhetherIn addition to its immunosuppressive functions, FTY720 has an additional immunomodulatory role in modifying hypersensitivity response in reactive airway disease . In miceAnother possible option to inhibit SET involves apolipoprotein E (ApoE), a multifunctional protein that has a role in cholesterol transport and immunoregulation ,165,166.®) is an EGFR kinase inhibitor indicated for treating EGFR-mutant NSLC [Two FDA-approved drugs inhibit CIP2A: erlotinib and bortezomib. Erlotinib , a proteasome inhibitor first approved for treating multiple myeloma, has indirect PP2A-mediated activity potential [Bortezomib , a compound found in traditional Chinese medicine, and ethoxysanguinarine, a benzophenanthridine alkaloid extracted from stigated ,178,179.PME1 is a serine hydrolase that facilitates reversible demethylation and inactivation of PP2A at the leucine 309 residue . MethylaBachovin et al. identified several PME-1 inhibitors, including sulfonyl acrylonitrile base AMZ30 and aza-β-lactams-based ML174 . The mosTwo main classes of compounds target the scaffolding subunit A of PP2A and activate the holoenzyme: phenothiazines and small molecule activators of PP2A (SMAPs). Phenothiazines are FDA-approved medications traditionally used as potent dopamine receptor antagonists in treating various psychiatric disorders . For exaSMAPs DT-061, DT-382, DT-794, and DT-1154  outlined in Sangodkar et al. were tested in KRAS mutant lung cancer cells and demonstrated increased poly (ADP-ribose) polymerase cleavage, an indicator of DNA damage, and increased tumor cell death in a caspase-dependent manner . In vivoOur group has utilized DBK-1154 in a smoke-induced COPD mouse model and demonstrated reduced emphysematous airway remodeling compared to controls . In the Coleus forskohlii root that also activates PP2A and treatment with it results in the dephosphorylation of PP2A substrates [A select group of compounds are reported to activate PP2A, but without a suggested mechanism. Xylulose-5-phosphate (X5P), a nucleotide precursor in the pentose phosphate pathway, added to fractionated hepatic murine lysates, increased free phosphate resulting from X5P-induced PP2A activity . Carnosibstrates .There are several studies suggesting that inhibition of PP2A may be beneficial. For example, knockdown of PP2A’s catalytic c-subunit α (PP2Acα) can restore glycogen production in the liver, reduce serum glucose, and reduce hepatic fibrosis resulting from repeated hepatic injury ,195. LB-Recent studies demonstrated LB-100’s therapeutic potential in various disease contexts, including sensitization of malignant meningioma to radiation therapy and enhancing T cell-mediated immunity against glioblastoma ,201. In PP2A has different physiologic functions, including playing a role in the maturation of germ cells, maintaining homeostasis of various body organs, tumor suppression, and regulation of metabolic processes. Although this review primarily focuses on pulmonary functions of PP2A, we will briefly outline essential PP2A functions throughout the body as systemic treatment with PP2A activators could influence several critical responses within the body. A recent study suggests that antiphospholipid antibody recognition of β2 glycoprotein I promote thrombosis and the authors suggested that inhibition of PP2A could be a potential therapeutic mechanism for thrombosis .2+ channel, ryanodine receptor, phospholamban, troponin I, myosin light chain [2+ response pathway might be the common pathway involved in the pathogenesis of heart failure phenotypes [Dysfunction of PP2A signaling can lead to cardiac hypertrophy, increased levels of atrial natriuretic peptide and B-type natriuretic peptide, ventricular fibrosis, and impairment in the cardiac contractile function in mouse studies ,205,206.ht chain . Increasht chain . A studyht chain . Althougenotypes ,210. B56Recent work in patients with antiphospholipid syndrome (APS), an acquired autoimmune disease, suggest a possible association of PP2A in developing the prothrombotic state. APS pathogenesis involves an inciting factor triggering endothelial cell damage and apoptosis. This results in the formation of neoantigens formed from damaged cellular membranes bounded by serum proteins. These neoantigens sensitize an adaptive immune response by developing antiphospholipid antibodies (aPLs), mediating subsequent clinical manifestations of venous and arterial thromboembolism. Sacharidou et al.’s work on human aortic endothelial cells suggests that interaction between extracellular aPL and surface glycoprotein β2 glycoprotein I (B2GPI) culminate in downstream inhibition of eNOS, and subsequent production cessation of a key anticoagulant factor, nitric oxide, likely from PP2A mediated phosphatase activity resulting in eNOS S1177 dephosphorylation and subsequent de-activation. Of note, this investigation utilized aPL/B2GPI binding as a novel mode of PP2A activation. The group also observed that by knockdown of the PP2A-ɑ subunit (PR65ɑ), aPL suppression of eNOS was reversed. PP2A’s holoenzyme diverse heterogeneity stems from a large possible combination of variable subunits. A lack of data exists to inform whether differing modes of PP2A activation result in the common PP2A activation profile or distinct variability of PP2A activation dependent on the activation mechanism. Furthermore, whether PP2A activation contributes to systemic thrombophilia in individuals without serum antiphospholipid autoantibodies has yet to be determined .Insulin sensitivity is a hallmark of various metabolic diseases, including type 2 diabetes Mellitus. PP2A mRNA levels and activity levels were elevated in the liver and muscle tissue of the insulin-resistant Zucker Diabetic Fatty (ZDF) rat model . InsulinThere is mounting evidence that PP2A phosphatase responses are subdued in several pulmonary diseases which could plays an important role in altered inflammation signaling. The overlapping prevalence of some of these pulmonary diseases and the possible existence of common underlying molecular mechanisms, suggest that targeting similar pathways in pulmonary diseases may be a logical approach to future treatment and indicate that PP2A may be a key pathogenic link in the progression of several pulmonary diseases. Since PP2A is a key regulator of inflammation and proteolytic responses, reduced PP2A could alter the inflammation and extracellular matrix responses in the lungs triggering altered lung function, pathology, and promote tumor initiation, growth and possibly metastasis. Currently, there are several drugs that could be utilized to exert therapeutic effects on PP2A activation, directly or indirectly. However, further extensive research is required to determine the complex biology of PP2A and possibly improve the bioavailability of current PP2A-activating compounds. Equally, systemic effects of PP2A activation needs addressing in parallel with investigating the subsequent pulmonary outcomes. Clinical trials of these compounds are likely to take place first in cancer, where the balance of risk to benefit are more pronounced, though IPF which has a prognosis, after diagnosis, similar to lung cancer may be an exception to this. Determining the upstreaming regulators of PP2A and subsequent downstream effects of PP2A signaling at the initiation and through the progression of the disease needs to be thoroughly investigated.


Defensive responses to visually threatening stimuli represent an essential fear-related survival instinct, widely detected across species. The neural circuitry mediating visually triggered defensive responses has been delineated in the midbrain. However, the molecular mechanisms regulating the development and function of these circuits remain unresolved. Here, we show that midbrain-specific deletion of the transcription factor Brn3b causes a loss of neurons projecting to the lateral posterior nucleus of the thalamus. Brn3b deletion also down-regulates the expression of the neuropeptide tachykinin 2 (Tac2). Furthermore, Brn3b mutant mice display impaired defensive freezing responses to visual threat precipitated by social isolation. This behavioral phenotype could be ameliorated by overexpressing Tac2, suggesting that Tac2 acts downstream of Brn3b in regulating defensive responses to threat. Together, our experiments identify specific genetic components critical for the functional organization of midbrain fear-related visual circuits. Similar mechanisms may contribute to the development and function of additional long-range brain circuits underlying fear-associated behavior. Fear responses to threat represent an essential survival instinct. While previous work has focused primarily on the forebrain and limbic system, this study examines the midbrain visual circuitry, identifying specific molecular mechanisms underlying its organization and fear-related behavioral function. Behavioral responses to threat are crucial for both animal and human survival. In humans, the inaccurate interpretation of threat- and fear-related information can lead to devastating psychiatric conditions –4. ThreaBrn3b within the bottom layer of superficial SC. Neurons in this layer innervate the lateral posterior nucleus (LP) of the thalamus, and SC-LP connections apparently mediate innate fear responses to visual threat manifested by freezing behavior [Brn3b expression confined to the deep SC and PAG. The overall expression pattern of Brn3b led us to hypothesize that this transcription factor may play important roles in the development of subcortical circuits critical for fear-related defensive behaviors.To examine molecular mechanisms that control development of the circuits mediating visually triggered fear responses, we sought genes expressed by subsets of midbrain neurons. We focused on the superficial SC, which conveys visual information from the retina and visual cortex to other subcortical areas , and disbehavior ,10,14. WBrn3b in retina development [Brn3b functions in other parts of the nervous system. Here, we report multifaceted roles of Brn3b in the organization and function of visual threat-related circuits, involving both superficial SC and deep SC/PAG. Conditional deletion of Brn3b in the midbrain decreased axonal projections to the LP due to neuronal loss in the superficial SC and also decreased expression of the neuropeptide tachykinin 2 (Tac2) in the deep SC/PAG. Brn3b mutants exhibited diminished freezing responses to a threatening looming stimulus [Tac2 in the deep SC/PAG. These findings suggest that Tac2 acts downstream of Brn3b and that a change of Tac2 expression plays an important role in behavioral alterations in Brn3b mutants. Altogether, our findings define, for the first time, the molecular mechanism that regulates development and function of midbrain circuits conveying defensive responses to visual threat.Previous studies demonstrated a critical role for elopment ,17, but stimulus  followinOur initial screening for SC neuronal markers in the superficial SC showed that Brn3b is expressed at the bottom layer of the superficial SC . The orgBrn3b mouse carrying the Brn3b gene flanked by loxP sites and a human placental alkaline phosphatase (AP) coding region inserted immediately downstream of 3′ loxP , precommisural nucleus, hypothalamus, central lateral nucleus of the thalamus, adjacent parabigeminal nucleus, pons, and cuneiform nucleus . To rulere mouse . TogetheSlc17a6-Cre (VGluT2-ires-Cre) or Gad2-Cre to a Cre-dependent td-Tomato reporter line (Ai14). However, very dense immunolabeling of synaptic terminals and overly abundant filling of neuronal processes by td-Tomato signals precluded visualizing individual somas. Instead, we performed in situ hybridization using Slc17a6 and Gad1 probes. The brain tissue for the hybridization was obtained from a newly generated Brn3bGFP/+ mouse, in which an open reading frame of Brn3b was replaced with GFP by CRISPR-based genome editing  and confirmed Brn3b loss in midbrain by immunostaining  and mutants carrying conditional and null Brn3b alleles ; in both cases, mice carry 1 copy of En1-Cre and 1 copy of Brn3bflox, producing AP signals.To assess the role of staining . The con mutants ,26. TherBrn3b expression in both control and cKO mice. These signals disappeared in the superficial SC of the mutants, with no obvious changes in other layers. Thus, Brn3b deletion induces a selective loss of Brn3b+ neurons in the superficial SC  showed no changes in the survival of the superficial SC Brn3b+ neurons and projections to the LP, likely because Cre expression occurs too late during development in this Cre line , where LP-projecting neurons localize, which apparently explains the altered projections to LP. However, because some axonal projections to LP were still detectable in these cKO mice, we considered the possibility that Brn3b+ neurons in other layers of SC may also innervate LP. To test this idea, we first evaluated whether superficial SC Brn3b+ neurons project to LP using a second Cre line . We cros neurons ,28, but line see . TherefoBrn3bGFP/+ mouse to selectively label Brn3b+ neurons : GFP-dependent FLP recombinase (FLP-DOG) and FLP-dependent mCherry (fDIO-mCherry). The FLP-DOG (DOG: Dependent On GFP) is genetically engineered to be unstable and degrade, whereas binding to GFP prevents its degradation . Inside  neurons . Double  neurons . TogetheBrn3b deletion by En1-Cre completely eliminated innervation of the ventral LGN  and this labeling was mainly detected in superficial and intermediate SC, suggesting that Brn3b expression is necessary for neuronal survival in these layers.We next analyzed development of Brn3b+ neurons to define specific time points of neuronal loss and altered projections to LP. During development, Brn3b expression in the dorsal midbrain becomes detectable as early as E13 . Therefoelopment . To examaspase-3 . Indeed,Brn3b mutants indicates the death of Brn3b+ neurons. However, extensive AP signals in other layers made it difficult to distinguish somas from processes, preventing visualization of individual Brn3b+ cells. To assess possible changes of Brn3b+ neurons in other layers, we utilized the molecular markers ETV1 and Brn3a. ETV1 is expressed in Brn3b+ neurons occupying the intermediate SC layer  by immunostaining (Brn3bGFP/+ (control) and Brn3b-GFP cKO. Also, no changes in GFP+ signals were detected in deep SC/PAG at P1/P2. However, we found approximately 30% decrease of GFP+ cell number in superficial and intermediate SC, confirming that Brn3b deletion affects neuronal survival in these layers specifically. Collectively, these results suggest that the loss of Brn3b has no effect on neuronal survival in the layers of dorsal midbrain outside the superficial and intermediate SC, including the deep SC/PAG.Considering that ETV1 and Brn3a are expressed in only about half of Brn3b+ neurons in their respective areas of co-expression (intermediate SC and deep SC/PAG), we also examined survival of Brn3b+ neurons using the pression . We crosstaining . QuantifBrn3b mutants at E13, suggesting that Brn3b deletion had no effects on initial axonal growth . Altogether, our findings suggest that Brn3b deletion in the midbrain causes decreased projections to the LP due to the loss of superficial SC Brn3b+ neurons occurring around birth.We also examined axonal development during several embryonic and postnatal stages. We observed no obvious differences between control and l growth . Consistl growth , indicatBrn3b but did not show obvious morphological alterations in Brn3b mutants. Considering the diverse roles of Brn3b in retinal development [Brn3b loss could cause various transcriptional alterations, including dysregulation of the genes involved in visually triggered behavioral responses. To test this hypothesis, we conducted transcriptomic profiling of the dorsal midbrain and identified 1,992 genes that were significantly differentially expressed in Brn3b cKOs compared to controls  showed the highest level of down-regulation in the Brn3b mutants. Interestingly, Tac2 was previously implicated in fear learning [Tac2 expression was up-regulated by single housing-based social isolation, which caused enhancement of freezing responses to a visually threatening looming stimulus [Tac2 expression due to the loss of Brn3b could affect neural circuits mediating fear-related behavior.The deep SC/PAG highly expresses elopment ,30,31, wlearning ,33. Addistimulus . TherefoTac2 may act downstream of Brn3b, we performed reverse transcription-quantitative PCR (RT-qPCR) on independent biological samples and confirmed that Brn3b deletion indeed significantly reduces Tac2 expression  and corticotropin-releasing hormone (Crh), known to mediate effects of stress [Tac2, we performed in situ hybridization with Tac2 probes on brain tissues of the Brn3bGFP/+ mouse  and a main effect of housing condition  with no interaction . Specifically, the Brn3b cKO group froze less than the control group, and both groups froze longer following single housing. Follow-up post hoc analysis showed a significant difference between groups only in the single housing condition . All mouse groups behaved similarly in the light/dark exploration test (p = 0.323) and housing conditions . Overall, these results suggest that Brn3b cKO mice have overall normal vision; however, they exhibit impaired visually evoked freezing responses following single housing.Our looming stimulus assay evoked prolonged freezing responses . Indeed,ion test . A 2 × 2Brn3b deletion reduces Tac2 expression in the deep SC/PAG and that Brn3b mutants exhibit decreased freezing reactions to visual threat precipitated by single housing. Given that single housing was reported to enhance freezing behavior via Tac2 up-regulation [Tac2 expression in Brn3b mutants prevents visual fear augmentation induced by social isolation. However, due to a lack of direct evidence that increased expression of Tac2 in dorsal midbrain can affect visually triggered fear behavior, we first examined whether Tac2 up-regulation in the SC/PAG of WT mice is sufficient to enhance freezing responses to the looming stimulus  could have contributed to our macroscopic level measurement of LP area. This could explain the discrepancy that only approximately 23% of LP projecting cells were Brn3b+ but there was approximately 48% decrease of LP area covered by AP signals in Brn3b mutants. Sparser labeling of axons coupled with deletion of Brn3b at a single-cell level may provide additional insights into Brn3b function.ntiation ,17. Alsoesponses , althougesponses . MoreoveBrn3b mutants exhibited both reduced superficial SC-LP projections and down-regulated expression of Tac2 in the deep SC/PAG. What might be the relative contributions of these 2 phenotypes to the altered freezing responses of Brn3b mutants? Considering that Brn3b is expressed only in approximately 23% of Ntsr1+ neurons and that Tac2 overexpression in the deep SC/PAG of Brn3b mutants enhanced their freezing behavior following social isolation, Tac2 seems to play a fundamental role as a critical downstream effector of Brn3b in regulating defensive fear responses. Nevertheless, the contribution of the diminished superficial SC-LP projections to freezing behavior cannot be completely ruled out because of the apparent importance of SC-LP projections to freezing behavior [Brn3b mutants, we attempted to specifically delete Brn3b in the superficial SC using the Ntsr1-GN209-Cre line. However, this approach produced no obvious morphological changes. We speculate that Cre expression in the Ntsr1-GN209-Cre line may occur too late in development to cause significant anatomical alterations due to Brn3b loss. Further evaluation of the Brn3b-dependent phenotypes in the midbrain, including altered SC-LP projections and down-regulated Tac2 expression, will require additional genetic tools that enable more specific manipulations within individual midbrain layers. Additionally, it would be interesting to define specific molecular and cellular mechanisms linking Brn3b loss to Tac2 down-regulation.behavior ,10,14. TBrn3b mutants, the PBG innervation was intact. These findings suggest that Brn3b deletion is unlikely to have an obvious effect on escaping behavior. Indeed, an optogenetic activation of Tac2+ cells in the midbrain had no effect on the escaping responses [Drosophila gates visual aversion by increasing locomotion, similar to escaping [Defensive fear responses to visual threat include both freezing and escaping reactions . These 2esponses . Consideescaping .Additional projections from GABAergic neurons in the vLGN to SC are shown to regulate defensive fear responses to visual threat . Brn3b mBrn3b mutants in the light/dark exploration test  and were in compliance with federal guidelines  Committee).Both male and female animals were used in all studies. The age of the animals was specified in each experiment. In the experiments requiring timed pregnancy, the date of a copulation plug detection was considered to be E0.5.Brn3b knockout [Brn3b knockout [Brn3bGFP/+(this study); (4) engrailed1-Cre  global knockout ; (2) conknockout ; (3) Brnonfirmed ,23); (5) neurons ,28; (6) Brn3bGFP/+ mouse line was generated by the Yale Genome Editing Center using the CRISPR/Cas9 strategy. Briefly, 2 sgRNAs for Brn3b (for 5′ and 3′ ends) were designed using an online tool (http://crispor.tefor.net). A targeting vector, containing GFP with SV40 small t intron sequences flanked by homology arms to 162 bp upstream and to 100 bp downstream of Brn3b coding sequence, was synthesized by Genewiz. Two sgRNAs, the targeting vector and Cas9 were injected into fertilized zygotes that were then implanted in pseudo-pregnant females. PCR, followed by DNA sequencing analysis, identified 4 founders in which the GFP sequence replaced the open reading frame of Brn3b. All 4 founders were crossed to wild-type mice for 2 generations before conducting histological analysis. Double labeling showed approximately 95% overlap between GFP and Brn3b signals in the progeny of all 4 founders .The The sequences of the sgRNA were:for 5′ end, GCTCCGGCCGGGTACTTCTC; for 3′ end, GGAGAAGGGTCCCTAAATGC.The primer sequences for screening were:for 5′ end, forward-GGGGACTATAACTCCACCGC, reverse-TCGATGCCCTTCAGCTCGAT;for 3′ end, forward-AGGACGACGGCAACTACAGG, reverse-GCGAAACCGGTTCACAATCT.The sequences of homology arms and small t intron were:left arm: CGGAGCTAGCGGCCACACTGGGAGAACCGGGCCTGGAAGCAGTGGCGGCTGAGCACAACTTTGCAGTGTTGTTCCCTCTGCTGCTCCGGCCGGGTACTTCCTCAGAGGGTCGGGTAGCTGGGACCGGAGTGCGCCAGCGACGAGCGCGCCGCGCAAGGAAAG;small t intron: GTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTTTAG;right arm: GGACCCTTCTCCAGGGATGGCCCTTTCCCTTCGCCCTCTTTTTTTCTAACCCCCTTCTTGTCTCTTCTGCCTCTTTCCTTTCTACTTTGGCTATCAGAAA.Mice were anesthetized by intraperitoneal injection of 100 mg ketamine plus 10 mg xylazine/kg of bodyweight and perfused transcardially with 4% paraformaldehyde (PFA)/PBS. Following perfusion, the brains were dissected, postfixed overnight at 4°C, incubated successively with 15% sucrose/PBS and 30% sucrose/PBS overnight at 4°C, and sectioned with a cryostat (12 to 20 μm). For vibratome sectioning, the tissues were postfixed overnight at 4°C, washed with PBS, and sectioned (50 to 100 μm). For immunostaining, sections were washed twice with PBS, blocked with 3% donkey serum/0.1% Triton X-100/PBS for 30 min at room temperature, incubated with primary antibodies for 1 to 3 days at 4°C and then with secondary antibodies for 2 h at room temperature.Primary antibodies used were: rabbit anti-GFP , chicken anti-GFP , rabbit anti-cleaved caspase-3 , rabbit anti-calbindin D-28K , rabbit anti-Ki67 , rabbit anti-ETV1 , rabbit anti-DsRed , mouse anti-Brn3a , goat anti-Brn3a , mouse anti-calretinin , goat anti-Brn3b , goat anti-Brn3b . Secondary antibodies conjugated to Alexa Fluor-488, Cy3 or Alexa Fluor 647 were diluted at 1:500 .2/PBS twice, incubated for 2 h at 65°C to inactivate the endogenous AP activity and then washed 3 times with AP buffer ). AP staining was conducted in the AP buffer with 2 mM levamisole and NBT/BCIP  for 4 to 16 h. After staining was complete, the tissues were washed with PBS, postfixed with 4% PFA/PBS overnight and dehydrated through EtOH incubation (50%-75%-85%-95%-100%). Just before imaging, the tissues were cleared with benzyl benzoate and benzyl alcohol (2:1).For AP staining, brains were sectioned on a vibratome . The tissues were washed with 2 mM MgClTac2, a full-length Tac2 was amplified from P1 mouse brain cDNA by generating BamHI and EcoRI restriction sites at each end and subcloned into the AAV vector  [To generate an AAV vector carrying  #74291) . Generat #74291) . AAV-Cre #74291) . Detaile #74291) ,29.13 viral genome particles/ml in all preparations. Viral stocks were stored at −80°C.AAV production was based on a triple-transfection, helper-free method, and AAVs were purified as described . A plasmDorsal midbrains were dissected at P2-P4, snap-frozen on dry ice and stored at −80°C. Frozen samples were sent to the Yale Center for Genome Analysis for RNA extraction, library preparation, and RNA sequencing. Briefly, total RNA was prepared using the RNeasy PowerLyzer Tissue & Cell kit (Qiagen). RNA quality was assessed by estimating the A260/A280 and A260/A230 ratios by nanodrop and Agilent Tapestation 4200 RNA Screen Tape Assay. Samples with RIN values of 7 or greater were used for library prep.To generate cDNA libraries, 1 μg of total RNA per sample was enriched for polyA RNA and converted into libraries using the Kapa mRNA Hyper Prep Kit for Illumina Platforms (KAPA Biosystems). Briefly, mRNA was purified with oligo-dT beads and sheared by incubation at 94°C in the presence of Mg. Following first-strand synthesis with random primers, second strand synthesis and A-tailing were performed with dUTP for generating strand-specific sequencing libraries. Adapter with 3′ dTMP overhangs were ligated to library insert fragments. Library amplification amplified fragments carrying the appropriate adapter sequences at both ends. Strands marked with dUTP were not amplified. Indexed libraries that meet appropriate cut-offs for both were quantified by qRT-PCR using a commercially available kit and insert size distribution was determined by the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/μl were used for sequencing. The samples were sequenced on an Illumina NovaSeq S2. Briefly, the sample concentrations were normalized to 1.2 nM and loaded onto an Illumina NovaSeq flow cell at a concentration that yields 25 million passing filter clusters per sample. Samples were sequenced using 100 bp paired-end sequencing on an Illumina NovaSeq according to Illumina protocols.Brn3b mutants were used for further analysis. Differential expression analysis was performed using DESeq2, which produced fold changes and statistical significance of changes between samples [Low-quality base calls, sequences with low-complexity tails and adaptor sequences were removed using fqTrim2.py . Trimmed paired-end (PE) reads were aligned to the mouse genome (GRCm38.p5) using Tophat2/Bowtie 2 . Raw rea samples .GAPDH and expression level of each gene relative to that of controls was calculated by ΔΔCt method. Primers used for qPCR were as follows: GAPDH, 5′-GTGGAGTCATACTGGAACATGTAG-3′ and 5′-AATGGTGAA GGTCGGTGTG-3′; Tac1, 5′-TTTCGTAGTTCTGCATCGCG-3′ and 5′-TGGCCAGATCTCTCAC AAAA-3′; Tac2, 5′- GATGTCTCCTTTGGTCCCAC-3′ and 5′-AGGGAGGGAGGCTCAGTAAG-3′; Crh, 5′-ATCTCTCTGGATCTCACCTTCC-3′ and 5′-CCCGATAATCTCCATCAGTTTCC-3′; Brn3b, 5′-ATTGAAGAGCTCCGGCTTAG-3′ and 5′-CTGTCACACAACAACATGATCG-3′.Midbrains including SC and PAG  were isolated. Total RNA was prepared using the RNeasy Mini Kit (Qiagen) and cDNA was synthesized using the Superscript III First-Stand Synthesis SuperMix . RT-qPCR was performed in duplicates using iQ SYBR Green Supermix (Bio-Rad) on the CFX96 real-time system (Bio-Rad). The Ct values of the samples were normalized to that of Tac2, Slc17a6, and Gad1 expression in brain tissues was detected by in situ hybridization following the method previously described [Tac2  and Slc17a6 (NM_080853) or the partial coding sequence of Gad1 (NM_008077) from the brain cDNA and subcloned into pCR8/GW/TOPO TA vector (Invitrogen) or pGEM Teasy (Promega). Antisense riboprobes were synthesized using digoxigenin-labeled UTP. Signals were detected by peroxidase-conjugated anti-digoxigenin antibodies and by the tyramide signal amplification system . To examine overlap between Tac2+ and Brn3b+ cells in Brn3bGFP/+ mouse brain, the signals were first amplified by anti-digoxigenin antibody to Tac2 and then by antibodies to GFP .escribed . BrieflyAnimals received analgesic buprenorphine (0.05 to 0.1 mg/kg of body weight) before surgery and were then anesthetized with a mixture of 100 mg ketamine plus 10 mg xylazine/kg of bodyweight. All drugs were administrated intraperitoneally. To visualize axonal distribution of RGCs in the SC, a small hole was made in an eye with an insect pin (size 00) to release intraocular pressure. Cholera toxin B subunit conjugated to Alexa Fluor-555  was injected through the same hole using a Hamilton syringe. Intraperitoneal injections of buprenorphine (0.05 to 0.1 mg/kg of body weight) were given for 48 h after the surgery.For enucleation, the mice were treated with buprenorphine and meloxicam and anesthetized with a mixture of ketamine and xylazine described above. Before removal of eyes, lidocaine (<7 mg/kg of body weight) was administrated around the surgical area by subcutaneous injection. The eyes were removed using sterile surgical scissors. Eyelids were sutured to prevent unwanted infection using surgical specialties. Animals then received intraperitoneal injection of buprenorphine (0.05 to 0.1 mg/kg of body weight) and meloxicam (1 to 5 mg/kg of body weight) for 48 h after surgery.Brn3bflox/+ mice. The mice received intraperitoneal injection of buprenorphine (0.05 to 0.1 mg/kg of body weight) before surgery and were then anesthetized by intraperitoneal injection of a mixture of 100 mg ketamine plus 10 mg xylazine/kg of bodyweight before being mounted on a stereotaxic apparatus. Lidocaine (<7 mg/kg of body weight) was administrated around the surgical area by subcutaneous injection before incision. A small craniotomy was made over the SC using a dental drill. Coordinates used for injection were bregma −4.5 mm, lateral ± 0.6 mm, dura −1.2 mm , and −2.4 mm (for the deep SC/PAG). AAVs (approximately 50 nl) were unilaterally injected with a glass pipette at a rate of approximately 10 nl/min, and the pipette was left in place for 5 min after injection. Enucleation was conducted after the brain injection on the same day when AAVs were delivered into the intermediate SC. Intraperitoneal administration of buprenorphine (0.05 to 0.1 mg/kg of body weight) and meloxicam (1 to 5 mg/kg of body weight) was given for 48 h after the surgery. Three to 4 weeks later, the brain tissues were dissected and processed to visualize axonal projections.To trace axonal projections from distinct layers of the midbrain, we injected AAV-mCherry (or AAV-YFP) into wild-type mice or AAV-Cre into To overexpress Tac2, we injected a mixture of AAV-Tac2 and AAV-mCherry, or AAV-mCherry alone for control into the deep SC and PAG. General surgical procedures were performed as described above. Coordinates used for injection were bregma −4.5 mm, lateral ± 0.6 mm, dura −2.4 mm. Four to 6 weeks later, the animals were single-housed for 2 weeks before conducting behavioral tests. After completion of the tests, the brain tissues from all tested animals were dissected, sectioned, and imaged to confirm mCherry expression in the deep SC and PAG.Brn3bGFP/+ mouse at P1. Mice received oral injection of meloxicam (0.3 mg/kg of bodyweight) before surgery and were anesthetized by chilling on ice. Pups had no direct contact with ice, as they were placed on a barrier covering the crushed ice. A small incision (maximum length 3 mm) was made on the skin over the SC. The skull and brain tissue were penetrated at the same time by disposable glass pipettes (<0.1 mm outer diameter) containing AAVs. A pressure injector was used to deliver approximately 50 nl of AAVs unilaterally. The pipette was gently withdrawn after injection and the skin was sealed using cyanoacrylate glue. Oral injection of meloxicam was given for 48 h after surgery. Three to 4 weeks later, the brain tissues were dissected and further processed for histological analysis.To label superficial Brn3b+ neurons by FLP-DOG strategy, we injected AAV-FLP-DOG and AAV-fDIO-mCherry into the SC of Both male and female mice (16 to 20 weeks old) were utilized in the behavioral experiments; the subject numbers were sex-adjusted. Different behavioral assays were conducted 3 to 4 days apart for group-housed mice and 5 to 7 days apart for single-housed mice to allow for additional post-experimental recovery of the animals that experienced social isolation. All behavioral tests were performed and analyzed in a double-blind fashion.-1). The stimulus was generated in MATLAB. Mouse freezing responses were tested in the absence of a shelter and analyzed using Boris 7.13, an open-source event tagging software [Visually triggered defensive behavior was tested using a looming stimulus assay as described in Yilmaz and Meister. Briefly, the behavioral apparatus consisted of an open field-type arena with a computer monitor mounted on the top of the plexiglass chamber. All sidewalls of the chamber (40 × 40 × 30 cm) were coated with a matte finish (Krylon) to create a unidirectional visual perspective. The mouse was initially habituated to the test room for approximately 45 min and then placed into the arena to acclimate for approximately 10 min. During this time, the computer monitor illuminated the arena with a gray screen (mean luminance). Mouse behavior in the arena was recorded by a camera mounted on the chamber and the video was stored on a computer using video capture software. Following acclimation, the computer screen, regulating illumination of the test area, began presenting an expanding dark spot—a negatively contrasted circle expanding from 2 to 20 degrees of visual angle over 250 ms and then remaining at its largest size for an additional 250 ms. After a 500-ms interstimulus interval, during which the gray screen was presented, the looming stimulus repeated for a total of 10 times  or 4 μm steps using a 10× objective (NA 0.45). AP images were acquired using a Zeiss Axioskop equipped with SPOT software . Data analysis was conducted by ImageJ software .Fluorescent images were collected using a Zeiss Imager M2 fluorescence microscope and a Zeiss LSM 800 confocal microscope. The To indicate anatomical location of brain sections/images used for analysis, the Bregma levels were indicated on the figures depicting sections obtained from approximately 24-day-old mice. For younger animals when the brain is still developing and/or a clear Bregma is lacking, the following localization and visualization approaches were employed. For coronal images used for analysis, diagrams of the coronal sections showed their specific brain positions and the corresponding sagittal images depicted the levels where each section was obtained. Anatomical structures of the coronal sections were defined using information from the Atlas of the Developing Mouse Brain . For sagTo quantify axonal projections to the LP, AP images were taken using 50-μm thick coronal sections, approximately 300 μm apart, extending from the suprachiasmatic nucleus to the rostral cerebellum. Sections containing the LP adjacent to the dorsal lateral geniculate nucleus (dLGN) were chosen for further analysis to ensure the correct LP location. The poor quality of antibodies to AP made it difficult to conduct double immunostaining with an antibody to calretinin, which demarcate LP . TherefoTo assess and compare the total number and distribution of cells stained with specific antibodies, sagittal sections anatomically matched in controls and mutants, were used unless noted otherwise. Images were taken from the sections located at approximately 100 μm from the medial edge and continued laterally, approximately 50 μm apart by E16, and approximately 75 μm apart by P1/P2. For cell counting, the images were cropped to 350× up to 450 μm at E15; to 350× up to 500 μm at E16; to 350× up to 800 μm at E18; and to 350 × 1,000 μm at P1/P2. Such cropping covers an area from the pia to the dorsal PAG at each developmental stage. For ETV1+ neurons, the entire image was used for the analysis. To assess the distribution of Brn3a+ neurons and GFP+ neurons, the counted cells were grouped within 50 μm intervals for E15/E16 analysis and within 100 μm intervals for P1 analysis. To analyze the number of Tac2+ neurons, the images were taken from coronal sections located just rostrally to the inferior colliculus and continued rostrally approximately 240 μm apart at P13-P18; the images were cropped to 700 × 700 μm for counting.Analysis of axonal projections was conducted in 2 to 3 different sections per animal and averaged for 4 to 5 animals per condition. The number and distribution of cells were analyzed using 2 to 3 different sections per animal and averaged for 3 to 5 animals per condition. Animals of either sex were utilized for the analysis.t test when comparing means between 2 groups or two-way ANOVA followed by Tukey post hoc test when comparing means between multiple groups. The number of animals used per condition, the mean values ± SEM for each data set, and statistical significance were provided in each figure/figure legend. Statistical significance was determined when p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).All data were reported as mean ± SEM and analyzed using GraphPad Prism software . Statistical analyses were conducted using an unpaired two-tailed Student’s S1 FigA–C) Schematic diagrams of coronal images showing the brain area (boxed) used for analysis (left) and sagittal images depicting the level (dashed line) where the coronal section was obtained (right) at P1 (A) and P9-P12 (B and C). (D–G) Brn3b expression in Brn3bflox/+:: En1-Cre mouse, visualized by immunostaining at P1 (D) and P10 (F) and by AP labeling at P1 (E) and P12 (G). A magnified view of the boxed areas (i–iv) showed that Brn3b+ neurons are located close to the pia at P1 and become confined to the SO layer at P10/P12 . Due to the poor quality of AP antibody staining and the required tissue clearing for AP staining, immunostaining and AP labeling were conducted using different sections/animals.  Brn3b immunostaining in WT mouse at P9. Brn3b expression is missing in the upper layer of superficial SC (SGS: H), consistent with observation in the Brn3bflox/+:: En1-Cre mice. No Brn3b signals were detected in LP (I). Immunostaining with anti-Brn3b was conducted at P9 because of very weak signals produced by Brn3b antibody beyond P12 (n = 3 mice). To demarcate dorsal lateral geniculate nucleus (dLGN) and LP in the thalamus, calbindin antibody (green) was used (Grubb and colleagues [J) No AP signals were detected in the retina of Brn3bflox/+:: En1-Cre mouse . Dashed lines indicate either the pia surface or boundary of dLGN and LP (D–I). Blue (DAPI). Scale bar: 200 μm.(lleagues ). (J) No(TIF)Click here for additional data file.S2 FigA) CRISPR/Cas9-mediated genome-editing strategy to generate Brn3bGFP/+ (Brn3b-GFP) knock-in mouse line. Two sgRNAs (for 5′ and 3′ ends) were utilized. The targeting vector contains the GFP with a small t intron flanked by homology arms (indicated by gray boxes) to 162 bp upstream and 100 bp downstream of Brn3b open reading frame. This strategy produces a mouse in which the GFP sequence replaces an open reading frame of Brn3b. (B) Schematic diagrams of a coronal image showing the brain area (boxed) used for analysis (top) and a sagittal image depicting the level (dashed line) where the coronal section was obtained (bottom). (C) (Left) A section of Brn3bGFP/+ mouse brain labeled with antibodies to GFP (green) and Brn3b (red) at P10. (Right) Magnified view of the boxed areas . Quantification revealed that approximately 95% of Brn3b+ cells express GFP  and approximately 93% of GFP+ cells express Brn3b , confirming that GFP+ cells faithfully represent Brn3b+ neurons. Orange arrows indicate examples of overlapping signals. DAPI (blue). (D) Schematic diagram showing the brain areas of the Brn3bGFP/+ mouse analyzed using double labeling by in situ hybridization with probes to Slc17a6 and Gad1 and immunostaining with anti-GFP antibody. (E–L) Brn3b+ neurons  express Slc17a6  but not Gad1 . Quantification of overlap between Slc17a6 and GFP  or between Gad1 and GFP . Orange arrows indicate examples of overlapping signals; white arrows indicate examples of non-overlapping signals. Scale bars: 250 μm (C), 50 μm (G–L). The data underlying this figure can be found in ((TIF)Click here for additional data file.S3 FigA) Schematic diagrams showing the brain area (boxed) of a coronal section used for analysis (top) and a sagittal diagram depicting the level (dashed line) where such coronal section was obtained (bottom) at P1 (i) and at P10-P13 (ii). Brn3b expression (red) in control (B and D) and cKO (C and E) brains, visualized by immunostaining (n = 3 mice/group/development stage). DAPI (blue). Scale bars: 200 μm.((TIF)Click here for additional data file.S4 FigA–D) Ntsr1-GN209-Cre line expresses Cre in the superficial SC neurons projecting to LP  The retina of Ntsr1-GN209-Cre:: Brn3bflox/+ mouse showing AP signals. (B) Brn3b+ neurons in superficial SC  and projections to LP (right), visualized by AP signals in control brain (Ntsr1-GN209-Cre:: Brn3bflox/+). (C) No structural changes were detected in the mutants (Ntsr1-GN209-Cre:: Brn3bflox/-). To ensure that the labeled axons in the LP originated from the SC, and not the retina, both eyes were enucleated. Additional AP covered areas were detected in the midbrain, suggesting that Cre expression pattern in the Ntsr1-GN209-Cre line differs from the pattern in En1-Cre (n = 3 mice). (D) (Left) Schematic diagram showing the brain area analyzed to examine Brn3b expression in Ntsr1-GN209-Cre:: Ai14 mice expressing Cre-dependent td-Tomato. (Right) Overlap between Brn3b (green) and td-Tomato (red). Quantification revealed that approximately 23% of td-Tomato+ cells were Brn3b+ . Arrows indicate examples of overlapping signals. (E) Schematic diagram depicting the FLP-DOG/ fDIO-mCherry strategy. FLP-DOG is unstable and degrades in the absence of GFP. Binding of GFP stabilizes FLP-DOG, which coverts fDIO-mCherry to an active form resulting in mCherry expression . (F) Schematic diagram of AAV-FLP-DOG and AAV-fDIO-mCherry delivery into the SC of Brn3bGFP/+ mouse. (G) Representative image of mCherry expression in the SC. Magnified view of the boxed areas showing overlap between mCherry+ and GFP+  in superficial SC (i) but no mCherry signals in deep SC/PAG (ii). Arrows indicate examples of overlapping signals. (H) Quantification revealed that approximately 98% of mCherry+ cells express GFP . (I) Projections of mCherry+ axons to LP. Dense GFP labeling in dLGN and vLGN indicates RGC axonal innervation. Scale bars: 500 μm  and 50 μm (D). The data underlying this figure can be found in (d Murphy ). (A) Th(TIF)Click here for additional data file.S5 FigA) Schematic diagram of AAV-mCherry injection into deep SC/PAG. (B–D) Representative images showing mCherry expression in deep SC/PAG (B) and mCherry+ neuronal projections to LP (arrow in C). Magnified view of the boxed area (D). LP was identified by immunostaining with calretinin (CR) antibody ). (E) Schematic diagram of AAV-Cre injection into intermediate SC of Brn3bflox/+ mouse.  Representative images of AP signals in intermediate SC (E) and projections to ventral LGN (arrow in G), indicating that Brn3b+ neurons in the intermediate SC project to ventral LGN (n = 2 mice). Very faint signals in LP (G) might originate from neurons in other layers of the dorsal midbrain. However, if other layers beyond the targeted areas were obviously labeled by the AAV, such animals were excluded from analysis (A–G). Scale bars: 500 μm.(lleagues ) (n = 3 (TIF)Click here for additional data file.S6 FigA, B) Schematic diagram illustrating the level of the sagittal sections as a lateral distance from the midsagittal line at E15, E16, P1, and P2 (A) and the layer distribution of Brn3b+ versus ETV1+ neurons and Brn3b+ versus Brn3a+ neurons in the dorsal midbrain (B). (C–F) Sections stained for Brn3b (red) and ETV1 (green) at E16  and P2 . Neither Brn3b expression nor ETV1+/ Brn3b+ neurons in superficial SC were detected at E16 in control (C). Segregation of Brn3b+ neurons into superficial SC and intermediate SC was detectable at P2 in control (E). Some Brn3b+ neurons in the intermediate SC but not in superficial SC express ETV1 (arrows). (G) Quantification of ETV1+ neurons shows no clear difference between control and cKO . Unpaired two-tailed Student’s t test . (H–K) Sections stained for Brn3b (red) and Brn3a (green) at E15  and P1 . No double Brn3b/Brn3a staining was conducted at E15 due to incompatibility of Brn3b and Brn3a antibodies (both raised in goat). Mouse antibody to Brn3a, used at P1, did not produce any signals at E15, likely due to a low level of Brn3a at this stage.  Quantification of Brn3a+ neuronal number and position at E15 and P1 showed no obvious difference between control and cKO . Unpaired two-tailed Student’s t test . The dashed lines delineate the pial surface (C–F and H–K). DAPI (blue). Scale bars: 50 μm. The data underlying this figure can be found in ((TIF)Click here for additional data file.S7 FigA) Schematic diagram of a coronal image showing the brain areas (boxed) used for analysis (left) and a sagittal image depicting the level (dashed line) where a coronal section was obtained (right) at P9/P10. (B) Schematic diagram illustrating the level of the sagittal sections as lateral distances from the midsagittal line at E16 and P1/2.  Double immunostaining with anti-Brn3b (red) and anti-GFP (green) shows no Brn3b expression in the Brn3bGFP/flox:: En1-Cre at P9/P10. Magnified view of the boxed areas showing the loss of GFP+  neurons in the superficial SC , indicated by arrows . DAPI (blue).  Representative images of sections stained for Brn3b (red) and GFP (green) at E16. DAPI (blue). (G) Quantification of GFP+ neurons (iii) and their distribution (iv) showed no obvious differences at E16 . Unpaired two-tailed Student’s t test .  Representative images of sections stained for Brn3b (red) and GFP (green) at P1/2. DAPI (blue). (J) Quantification of GFP+ neurons (v) and their distribution (vi) revealed that Brn3b loss decreases the number of neurons in the superficial and intermediate layers . Superficial and intermediate layers were defined as located within 350 μm from the pia. Unpaired two-tailed Student’s t test . Magnified view of the boxed area (vii). No clear difference was detected in the deep SC/PAG . Unpaired two-tailed Student’s t test . The dashed lines delineate the pial surface (C–F and H–I). DAPI (blue). Scale bars: 200 μm (C–D) and 50 μm (E–F and H–I). The data underlying this figure can be found in ((TIF)Click here for additional data file.S8 FigA, B), E16 , and P1/P2 . Projections to the ventral midbrain, including tegmentum, pons, and medulla, were detected during embryonic development and disappeared after birth. Overall, no differences were found between control and cKO . The images were taken using consecutive sections covering the entire superior colliculus, 200 μm apart at E13, 200 μm apart at E16, and 600 μm apart at P1/P2. Scale bars: 500 μm.Neuronal projections were visualized by AP signals at E13 ((TIF)Click here for additional data file.S9 FigA) Schematic diagram of a coronal image showing the brain area (boxed) used for analysis (top) and sagittal images depicting the level (dashed line) where each coronal section was obtained (bottom) at P1-P5 and P8-P13. (B) (Left) Axonal branches in LP were barely detectable at P1/P2 in control and cKO mice (n = 4 mice/group). (Right) Magnified view of the boxed areas. (C) (Left) Axonal branches in LP were clearly noticeable at P4/P5 in control but barely visible in cKO mice (n = 4 mice/group). (Right) Magnified view of the boxed areas showing the LP regions used for analysis. (D) Quantification was presented as a relative difference between the AP-covered LP area in controls and mutants . (E) (Left) Axonal branches in LP became abundant at P8/P9 in control and cKO mice, but the LP area covered by AP signals was smaller in cKO (n = 4 mice/group). (Right) Magnified view of the boxed areas. (F) Quantification . (G) (Left) Axonal innervation in the LP is complete at P12/P13 in control mice, however, clearly decreased in cKO animals (n = 4 mice/group). (Right) Magnified view of the boxed areas. (H) Quantification ; unpaired two-tailed Student’s t test . Scale bars: 250 μm. The data underlying this figure can be found in Axonal projections were visualized by AP signals. ((TIF)Click here for additional data file.S10 FigA) Schematic diagrams of a coronal image showing the brain areas (boxed) used for analysis (left) and a sagittal image depicting the level (dashed line) where each coronal section was obtained (right).  Brain sections of Brn3bGFP/+ mouse labeled with anti-GFP  and in situ probe to Tac2 (red) at P18. (B) (Left) No Tac2+ signals were detected in the superficial and intermediate SC layers. (Right) Magnified view of the boxed areas . (C) (Left) Tac2+ cells are detectable in deep SC/PAG. (Right) Magnified view of the boxed areas . Quantification revealed that approximately 57% of Tac2+ cells are Brn3b+ . Arrows indicate examples of overlapping signals. DAPI (blue). Scale bars: 250 μm.((TIF)Click here for additional data file.S11 FigA) Schematic diagram of AAV-mCherry and AAV-Tac2 + AAV-mCherry delivery into the deep SC/PAG of WT mice. (B) Increased level of Tac2 mRNA after overexpression . (C) Schematic diagrams of the behavioral tests.  Representative images of mCherry expression  and Tac2 + mCherry co-expression . The sections were collected following the behavioral analysis. DAPI (blue).  Quantification of freezing responses  and of the time spent in the brightly lit area . Tac2 overexpression increased the total freezing time. No statistical difference was found during the light/dark exploration test. Unpaired two-tailed Student’s t test . Scale bars: 250 μm. The data underlying this figure can be found in ((TIF)Click here for additional data file.S1 TableBrn3b mutants. Table displays total reads, the percentage of unique reads, the percentage of unmapped reads, and the percentage of coding UTR.RNA seq was conducted with 3 controls and 4 (XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.