Sporisorium reilianum f. sp. zeae. This alternative respiratory component has been previously identified in other eukaryotes and is essential for alternative respiration as a response to environmental and chemical stressors, as well as for developmental transitionaoxs during the life cycle of an organism. A growth inhibition assay, using specific mitochondrial inhibitors, functionally confirmed the presence of an antimycin-resistant/salicylhydroxamic acid (SHAM)-sensitive alternative oxidase in the respirasome of S. reilianum. Gene disruption experiments revealed that this enzyme is involved in the pathogenic stage of the fungus, with its absence effectively reducing overall disease incidence in infected maize plants. Furthermore, gene expression analysis revealed that alternative oxidase plays a prominent role in the teliospore developmental stage, in agreement with favoring alternative respiration during quiescent stages of an organism’s life cycle.The mitochondrial electron transport chain consists of the classical protein complexes (I–IV) that facilitate the flow of electrons and coupled oxidative phosphorylation to produce metabolic energy. The canonical route of electron transport may diverge by the presence of alternative components to the electron transport chain. The following study comprises the bioinformatic identification and functional characterization of a putative alternative oxidase in the smut fungus  Sporisorium reilianum is a phytopathogenic basidiomycete with two formaespeciales, able to infect maize  and sorghum . Much like its better known relative, Ustilago maydis, SRZ displays a dimorphic lifestyle, existing as yeast-like haploid sporidia that reproduce asexually by budding [U. maydis, however, SRZ causes systemic infection on the plant host, emerging as teliospore sori on both male (tassel) and female (ear) inflorescences and causes additional developmental abnormalities, such as phyllody and irregular branching architecture [ budding . Sexual  budding . Unlike itecture ,4.1 or complex III and cytochrome c oxidase or complex IV) that can function as proton pumps (with the exception of complex II) to help maintain the electrochemical gradient that drives ATP synthesis via oxidative phosphorylation. Pumping of protons is facilitated by the fixed movement of electrons through the protein complexes, from NADH as the initial donor to molecular oxygen as the final acceptor.The bioenergetics and intermediary metabolism of fungal systems are poorly understood, despite the relevance of mitochondria and the associated electron transport chain (ETC) as the main source of metabolic currency. Like its mammalian counterpart, the fungal “powerhouse” is comprised of the four classical protein complexes  and are able to use NADH and succinate as substrates for uncoupled respiration, with significantly reduced ATP production [Alternative respiratory pathways recently received special attention, in which additional components diverge electron movement from the classical cytochrome c pathway. For instance, plants were known to deviate from the norm in response to specific metabolic needs, as is the case of the peanut plant, oduction . Anotheroduction . InsteadTrypanosoma brucei  [Cryptosporidium parvum (CpAox), the causative agent of cryptosporidiosis, that lacks membrane-bound organelles and is solely dependent on Aox-mediated respiration limited to mitochondrion-like compartments (mitosomes) [The alternative respiratory component first identified in the peanut plant is a quinol oxidase, also known as alternative oxidase (Aox), which functions like complex IV and catalyzes the reduction of oxygen into water but does not function as a proton pump . In thisse, TAO) , in whicse, TAO) ,18. Aox tosomes) ,20.Botrytis cinerea [Magnaporthe grisea [Mycosphaerella graminicola [Aspergillus species [Blastocladiella emersonii, in which respiratory capacity is higher in highly motile zoospores, compared to vegetative cells that favor Aox-mediated respiration due to their lower metabolic demands [In the case of fungi, Aox has been identified in most annotated genomes, including a variety of agriculturally relevant phytopathogens like  cinerea , Magnapoe grisea , Mycosphminicola  and seve species ,25,26, m demands .U. maydis [U. maydis in the way it infects maize, with symptoms appearing during the late stages of maturity of the plant. This peculiarity makes SRZ an excellent model to determine if Aox is involved in the long-term survival of the fungus in the plant tissue. Additionally, expression analysis of distinct developmental stages of the fungus indicated increased aox transcript levels in teliospores, corresponding with the use of a less energy-providing respiratory pathway during quiescent stages of the life cycle. The aim of this study was the identification and functional characterization of a putative Aox in SRZ, based on previous studies involving the related species . maydis ,29,30, wEscherichia coli DH5α strain  was grown in Lysogeny Broth (LB) on a rotary shaker at 200 rpm or on solid LB agar at 37 °C. Plasmid selection was achieved by supplementing the LB medium with ampicillin at a final concentration of 200 µg/mL or gentamicin at a final concentration of 10 µg/mL.The SRZ strains used and generated in this study are listed in U. maydis Aox  was used in a BLASTp search against the genome of SRZ2 (GCA_000230245.1). The resulting amino acid sequence was then used for Multiple Sequence Comparison by Log-Expectation (MUSCLE) in SnapGene 5.3.2 . For comparison, the amino acid sequences of Aox in other species were included: Sporisorium scitamineum Aox (CDU22616.1), Sporisorium graminicola Aox (XP_029737873.1), Ustilago bromivora Aox (SAM82122.1), U. maydis Aox1 (XP_011389130.1) and Pseudozyma hubeiensis Aox (XP_012186999.1).The amino acid sequence of the U. maydis [E. coli. DNA isolation from SRZ and transformation protocols were carried out as previously described for . maydis . Primers. maydis  with com. maydis . Primer aox deletion construct, the primer pair oHM39/40 was used to generate a single fragment containing both an ampicillin resistance cassette and an origin of replication element using pUC19  DNA as template. Subsequently, the primer pairs oHM35/36 and oHM37/38 were used to generate upstream and downstream flanking regions, respectively, to aox using SRZ2 genomic DNA as template. The primer pair oHM33/34 was used to amplify the hygromycin resistance cassette from pUMA1507 [E. coli.For the development of the aox deletion construct (4582 bp), made up of the flanking upstream region, followed by the hygromycin resistance cassette and the flanking downstream region. The amplified construct was used in the transformation of different strains of SRZ facilitated by homologous recombination. Preliminary verification of deletion mutants was achieved by polymerase chain reaction (PCR) using the primer pair oHM33/68, which resulted in the synthesis of a 4582 bp fragment in successful aox deletion mutants. Wildtype (WT) strains were simultaneously used as controls, in which production of a 4005 bp fragment corresponded to the intact aox locus.The final plasmid was then isolated and linearized with EcoRV . Subsequently, the primer pair oHM36/38 was used to amplify the aox complementation construct, the primer pair oHM40/146 was used to generate a single fragment containing both the ampicillin resistance cassette and origin of replication element using pUC19  DNA as template. The primer pairs oHM147/148 and oHM39/155 were used to generate upstream and downstream flanking regions, respectively, to aox using SRZ2 genomic DNA as template . The primer pair oHM149/156 was used to amplify the carboxin resistance cassette fragment from pMF4-1c [E. coli. For the development of the  pMF4-1c  DNA. Theaox complementation construct (5670 bp), made up of the flanking upstream region containing aox in its entirety, followed by the carboxin resistance cassette and the downstream flanking region. The amplified construct was used in the transformation of aox deletion mutants facilitated by homologous recombination. Preliminary verification of successful transformants was achieved by PCR using the primer pair oHM146/156, which resulted in the production of a fragment 4400 bp in size (WT strains were used as negative controls).The final plasmid was then isolated and linearized with EcoRV . Subsequently, the primer pair oHM30/147 was used to amplify the aox, using SRZ2 genomic DNA as template . The primer pair oHM102/103 was used to generate a fragment containing eGFP from pMF4-1c [E. coli. For the investigation of mitochondrial localization of Aox, a fusion construct with a fluorescent protein was designed. The primer pair oHM39/40 was used to generate a single fragment containing both the ampicillin resistance cassette and origin of replication element using pUC19 DNA as template. The primer pairs oHM70/104 and oHM37/38 were used to generate upstream and downstream flanking regions, respectively, to  pMF4-1c  DNA. The pMF4-1c  DNA. Theaox-eGFP fusion construct (6835 bp), made up of the flanking upstream region containg the aox sequence without the STOP codon, followed by eGFP fragment, the hygromycin cassette and the flanking downstream region. The amplified construct was used in the transformation of different strains of SRZ facilitated by homologous recombination. Preliminary verification of successful transformants was achieved by PCR using the primer pair oHM34/102, which resulted in the production of a 3101 bp fragment in succesful transformants . Subsequently, the primer pair oHM38/70 was used to amplify the PCR was carried out in a T100 Thermal Cycler  with Ex Taq Hot Start DNA Polymerase , PrimeSTAR Max DNA Polymerase  or DreamTaq Hot Start PCR Master Mix . PCR cycling conditions for Hot Start DNA polymerases involved an initial denaturation step at 94 °C for 4 min, followed by 34 cycles of a three-step process  and a final extension step at 72 °C for 10 min. PCR cycling conditions for PrimeSTAR Max DNA Polymerase involved an initial denaturation step at 98 °C for 2 min, followed by 34 cycles of a three-step process  and a final extension at 72 °C for 5 min. In preparation for Gibson Assembly, all PCR products were generated using PrimeSTAR Max DNA Polymerase. Gibson Assembly was carried out using the Gibson Assembly Master Mix kit  and according to the manufacturer’s instructions.5 cells/mL. Control treatments included PD broth supplemented with the respective respiratory inhibitor solvent . Cells were incubated overnight on a rotary shaker at 200 rpm at 28 °C and subsequently sub-cultured onto PD agar for determination of total viable colonies (TVC) per mL of medium. One-way ANOVA followed by Tukey’s Multiple Comparison test was performed in GraphPad Prism 9.2.0  to determine statistical significance, with p < 0.05 = significant and p > 0.05 = not significant.Haploid SRZ strains were grown overnight in 4 mL of PD broth on a rotary shaker at 200 rpm at 28 °C. Cells were harvested by centrifugation at 14,000 rpm, quantified using a hemocytometer and adjusted with fresh PD broth, supplemented with antimycin A  and/or sailcylhydroxamic acid , to 10600 of 0.5–0.8 was reached. Cells were harvested by centrifugation at 3500 rpm and resuspended in ddH2O to a final OD600 of 2. The desired strain combinations were then mixed in a 1:1 ratio and used to inoculate 7-day old Tom Thumb maize  seedlings. Disease evaluation was performed 7–8 weeks post infection (wpi), in which plant height was recorded and symptoms were scored for the calculation of disease incidence indexes and according to preestablished criteria [p < 0.05 = significant and p > 0.05 = not significantStrains were grown in 50 mL of PD broth on a rotary shaker at 28 °C until an ODcriteria . One-wayTwo-step reverse transcription quantitative PCR (RT-qPCR) was used for the verification of gene deletions and differential gene expression analysis. Nucleic acids were isolated from SRZ haploid sporidia, diploid teliospores and mated cells by homogenization in a frozen mortar and pestle and treatment with TRIzol reagent . Following cell disruption, RNA was isolated using the Direct-zol RNA Miniprep kit . Subsequent cDNA synthesis was carried out using Super Script IV  and followed the manufacturer’s instructions. qPCR was carried out in a StepOne Real-Time PCR System  using EvaGreen dye  following the manufacturer’s instructions. Cycling conditions involved an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. gapdh, sr109402) was used as endogenous control for the calculation of relative expression levels using the 2−ΔΔCt (Livak) method [p < 0.05 = significant and p > 0.05 = not significant.Melting curve analysis was performed at the end of each cycle to ensure specificity of the reaction. Gene-specific primers were designed using the open access Primer3 software  and are ) method . Ct valu600 of 1. Ten-fold dilutions of each culture were made and 500 µL were placed in the wells of 35-mm CELLview culture dishes . eGFP signal was acquired using a 488 nm line of an Argon laser and was detected at 510 nm. A transmitted light image was acquired during scanning for visualization of cell outline by grouping of the transmitted detector with the argon laser. For confirmation of localization, cells were treated with 100 µM MitoView Blue  for 10 min. Blue fluorescence was acquired using the DAPI detection channel and overlapped with eGFP fluorescence.Fluorescence images were acquired using a Nikon A1R confocal microscope. Transformed SRZ strains were grown overnight at 28 °C in PD broth to an ODU. maydis Aox1 was used in a BLASTp search against the nuclear genome of SRZ. The resulting sequence of 409 amino acids was then used for MUSCLE analysis against known alternative oxidases in closely and distantly related species  and were consistent with the sizes of their predicted polypeptides . Greater size discrepancies were seen in U. maydis (448 amino acids), U. bromivora (417 amino acids) and, more notably, in the distantly related P. hubeiensis (323 amino acids). Amino acid conservation was strongest in the carboxyl end of the aligned polypeptide sequences, coinciding with a ferritin-like diiron-binding domain  common in alternative oxidases of plants, fungi and protists. This preliminary bioinformatic evidence strongly suggests the presence of an aox gene in SRZ, encoding a polypeptide of approximately 45 kDa and rich in alanine (13.45%).The amino acid sequence of the  species . ProteinTo determine whether the SRZ Aox ortholog identified bioinformatically corresponds to a mitochondrial protein, a fluorescent Aox-eGFP fusion protein was designed. The SRZ transformants generated with this construct were then analyzed using confocal microscopy. Fluorescence was detected as thin threads that spread out throughout the cell, and even extended to budding daughter cells . To further confirm that the fluorescence detected originated from mitochondrial structures, cells were treated with the mitochondrial fluorogenic dye MitoView Blue. This chemical has light absorbance and emission at 398 nm and 440 nm, respectively, and can permeate membranes. Upon accumulation in the mitochondrial membrane, the chemical becomes brightly fluorescent. Treatment of cells with this mitochondrial fluorogenic dye confirmed that the green fluorescence coincided with mitochondrial structures .To functionally confirm the presence of an alternative oxidase in the ETC of SRZ, cells were grown in the presence of specific respiratory inhibitors. Cells were treated with antimycin A (AMA), salicylhydroxamic acid (SHAM), or both to infer the predominant respiratory pathway as a function of cell survival, with AMA targeting complex III and SHAM targeting Aox. In addition to an untreated control group, cells were treated with the respective solvents used to prepare the respiratory inhibitors to account for their basal toxicity on SRZ cells. Treatment with AMA and/or SHAM resulted in reduced survival rate in WT strains, although it did not eliminate growth . A signiaox deletion strains. Addition of AMA and of AMA and SHAM, but not of SHAM alone had a severe growth inhibitory effect on the deletion strains  and are illustrated in aox deletion strains displayed a reduction in serious symptoms  compared to plants inoculated with the WT strains. This last finding was discernable in the overall disease incidence analysis, in which individual symptoms were taken into consideration to ascribe single ratings to the different symptomatic profiles generated. Along these lines, mating combinations of the WT strains were responsible for significantly higher disease incidence indexes than those caused by combinations between the aox mutants. The mutants’ reduced disease incidence without fully disrupting the fungal life cycle may indicate that Aox is acting synergistically with other cellular components during the pathogenic stage of SRZ.To determine whether Aox is a crucial component of SRZ during its pathogenic stage, maize seedlings were inoculated with compatible combinations of both WT and aox transcriptional expression was compared via qRT-PCR during the teliospore (diploid), mated (beginning of dikaryon phase) and sporidium (haploid) stages of SRZ. Relative expression analysis of aox in teliospores was performed separately in reference to SRZ1  using AMA. AMA is a bacterial secondary metabolite that acts by binding to the Qi site of complex III, thereby competing with coenzyme Q to arrest the movement of electrons. Ultimately, this blockage leads to electron efflux, with the potential to become harmful as electrons leak into mitochondrial spaces to form highly toxic products, such as reactive oxygen species (ROS).Functional verification of the putative Aox was based on the effect of mitochondrial inhibitors on overall cell growth. This assay proved to be uncomplicated and cost-effective, as it permitted the direct assessment of the effects of specific mitochondrial inhibitors on cell survival. The classical cytochrome c pathway  was blocThe presence of an alternative oxidase provides an alternate route for electron transport, receiving electrons directly from reduced ubiquinol to reduce oxygen into water. This one-step process only depends on NADH oxidoreductase (complex I) as the sole contributor to the proton motive force in the intermembrane space of the mitochondrion. An organism equipped with this diverging route for electron transport can dissipate the build-up of electrons caused by AMA and survive the inhibition of the classical cytochrome c pathway, albeit with a significant decrease in ATP synthesis.aox resulted in overall weaker symptoms. Deletion of aox in U. maydis did not result in weaker virulence. However, U. maydis only produces local infections, and symptoms are scored as early as 2 wpi. SRZ infection provides the ability for long-term evaluation of the phytopathogen, as disease evaluation requires fully mature maize plants. Accordingly, Aox may be involved in the endurance of the fungus once inside of the host plant. The controlled nature of the infection experiments possibly contributed to the survival of the deletion mutants in the plant tissue, as a natural population of maize is constantly challenged by environmental insults. Further experimentation is required and should take into consideration environmental factors that may affect both the fungus and the host plant, such as extreme temperatures, to uncover additional aspects of the involvement of Aox in pathogenicity.A loss-of-function approach suggested notable differences during the pathogenic stage of SRZ. Although WT and mutant crosses of SRZ successfully colonized, invaded and induced symptoms in the plant, they did so at significantly different frequencies and deletion of aox is upregulated in teliospores, in contrast with haploid sporidia and even mated cells. The analysis was performed in reference to different haploid strains, as these behaved differently in the conducted qRT-PCR experiments. However, aox transcript levels of teliospores remained significantly higher regardless of the reference strain used. This finding is consistent with what was found in other organisms, in which specific life cycle stages favor secondary respiration in response to different metabolic demands. This finding suggests that Aox may play an important role in the teliospore stage of SRZ. Additional experimentation is needed to confirm if these specialized developmental structures, which remain relatively quiescent until they disseminate and germinate, do in fact have increased respiration rates mediated by Aox. In contrast, given that haploid sporidia are in constant division and must grow by budding or, after successful mating, filamentously for successful pathogenic development, ATP requirements are significantly higher. The higher metabolic demand imposed by sporidia can thus be fulfilled by the canonical cytochrome c pathway of the ETC, with three of the four classical protein complexes contributing to the mitochondrial proton motive force.Expression analysis of different stages of the life cycle of SRZ revealed that Zea mays  that was identified and extensively characterized early, was linked to thermotolerance and osmotic and oxidative regulation [The presence of Aox in plants contributes additional complexities to studies of this nature, as the evolutionary history of host-pathogen interactions should not be ignored. Quite surprisingly, the Aox of gulation ,38,39. A


This study examined the effects of ovo injection of clenbuterol on fat deposition and growth performance in chickens, which is prejudicial to poultry consumers and muscle growth-related genes, egg hatchability, and fertility. The achieved result showed a definite effect of clenbuterol on body gain and hatchability. It decreased fat deposition and upregulation of muscle growth-related gene expressions accompanied by modulation of fatty and amino acid composition, reflecting a new insight into the intracellular pathways of clenbuterol supplementation on chicks.p < 0.01). Clenbuterol also modulated saturated fatty acid levels in the breast muscles and improved essential amino acids when administered at 10 and 15 ppm. Additionally, clenbuterol at 15 ppm significantly decreased myostatin gene expression (p < 0.01) and considerably increased IGF1r and IGF-binding protein (IGFBP) expression. Clenbuterol administration led to a significant upregulation of hepatic PPARα, growth hormone receptor, and Lipoprotein lipase (LPL) mRNA expression with a marked decrease in fatty acid synthase (FAS) and sterol regulatory element-binding protein 1 (SREBP-1c) expression. In conclusion, the current study revealed that in ovo injection of clenbuterol showed positive effects on the growth of hatched chicks through reduced abdominal fat deposition, improved intestinal morphology, and modulation of hepatic gene expressions in myogenesis, lipogenesis, and lipolysis.The present study aimed to assess the in ovo administration of clenbuterol on chick fertility, growth performance, muscle growth, myogenic gene expression, fatty acid, amino acid profile, intestinal morphology, and hepatic lipid-related gene expressions. In this study, 750 healthy fertile eggs from the local chicken breed Dokki-4 strain were analyzed. Fertile eggs were randomly divided into five experimental groups (150 eggs/3 replicates for each group). On day 14 of incubation, in addition to the control group, four other groups were established where 0.5 mL of worm saline (30 °C) was injected into the second group of eggs. In the third, fourth, and fifth groups, 0.5 mL of worm saline (30 °C), 0.9% of NaCl, and 10, 15, and 20 ppm of clenbuterol were injected into the eggs. Results suggested that clenbuterol increased growth efficiency up to 12 weeks of age, especially at 15 ppm, followed by 10 ppm, decreased abdominal body fat mass, and improved hatchability ( A significant proportion of high-quality meat supply is poultry. Recently, poultry meat consumption has increased; therefore, poultry meat prices are considerably more competitive than those of other species, especially chicken broilers . In ovo In ovo feeding of such substances influenced the pre- and post-hatching physiological status of broiler embryos, leading to improved hatchability, the superior nutritional quality of hatchlings, greater vigor, and higher post-hatch growth . This prClenbuterol is widely used in farm animals for respiratory disorders and enhances protein creation . These cAlthough poultry signifies a critical source of protein worldwide, with comparatively minimal costs and a high processing ratio for feed , it contAccording to the Egyptian code of ethics, this experiment was performed and approved by the animal ethics committee at the Kafrelsheikh University, Egypt.Clenbuterol was provided by Sigma . A total of 900 incubating eggs from local chicken breeds, the Dokki-4 strain were obtained from the Research Station, Sakha, Institute for Animal Development, Agricultural Research Centre, and Agriculture Ministry of Egypt. All eggs were placed large end up in an automatic incubator. In this study, 750 healthy fertile eggs that contained embryos were weighed and selected at seven days of incubation. Eggs were instantaneously cleaned, disinfected, and dried with soft tissue paper. The eggs were incubated at 36 °C–38 °C with a relative humidity of 55%. All eggs were placed large end up in an automatic turner. On day 14 of incubation, fertile eggs were randomly divided into five experimental groups of three replicates (50 eggs/replicate). An auto-needle hole using a 23-G needle with a depth of 25 mm was created at the broad end of each egg and 0.5 mL was injected in ovo-food solutions. In addition, a control group was used where 0.5 mL of worm saline (30 °C) was injected into the second group’s eggs. In the third, fourth, and fifth groups, eggs were injected with 0.5 mL of worm saline (30 °C), 0.9% NaCl, and clenbuterol at 10, 15, and 20 ppm/egg, respectively. After feeding, holes were filled with a nontoxic glue stick. At the end of 18 days of incubation, eggs were sprayed with TH4 disinfectant solution (2 mL/1000 mL of water)  and tran2) were housed at the Kafrelsheikh University, Egypt, in an environmentally managed space. The house was kept at a temperature that was dependent on the bird’s age. The temperature was regulated through an air conditioner. During the experiment, air humidity was maintained at almost 70% [A total of 200 healthy hatched chicks  were selected and used for the feeding trial. The chicks grew for 12 weeks, and standard feed was provided for local chicken strains. Chicks were maintained in brooder pens for two weeks after hatching and transferred into separate locations. The chicks  divided by the body production. The slaughtering technique was performed following the Malaysian institute’s method . The carg/15 min by centrifuge of the clotted blood at 4 °C and stored at −20 °C for additional biochemical analysis.At 12 weeks of age, two blood samples were obtained by wing vein puncture from five birds from each replicate, randomly selected from each group, under gentle restraint. One sample was obtained for hematological analysis. Non-heparinized syringes were used in one sample for serum collection, separated at 3000× Blood samples were used to assess the hemoglobin content (g/dL) with Drabkin’s technique utilizing the colorimetric form of cyanmethemoglobin after centrifugation . Blood wThe spectrophotometric analysis was conducted to evaluate global protein (g/dL), globulin, and albumin (g/dL) concentrations . Biodiagg at 4 °C for 20 min, and the supernatant was added for further evaluation. The manufacturer’s protocol was used to calculate malondialdehyde concentration with a biodiagnostic kit . The biodiagnostic package  was tested following the protocol for GSH-Px. The biodiagnostic kit  protocol was used to test SOD. MDA, GSH-Px, and SOD contents were measured at 534 nm, 340 nm, and 560 nm, respectively, using a UV–VIS spectrophotometer  using the software Excel 2016 . Breast muscle samples in 100 mM cold potassium phosphate buffer, pH 7.2, were homogenized. Homogenate muscles were spun at 1500× Polymorphonuclear cell phagocytosis using Candida albicans was achieved according to . The folg/15 min, filtered through a 0.5 μm membrane, and dried. The derivatized samples and AA standards were inserted into the column for separation by high-performance liquid chromatography using a Nova-PakTM C18 column .Five birds from each replicate per group (20 birds/group) were selected to estimate muscle fatty acids and amino acid patterns. Extraction of fat was performed in the breast muscle using the chloroform–methanol (2:1) mixture to extract lipids, centrifuged for 10 min at 3000 rpm. The esterification procedure was achieved by adding the supernatant to 2 mL of methanol–sulfuric acid mix (95:5). The free fatty acids were provided by Sigma-Aldrich . Fatty acid quantity was assessed using Agilent gas chromatography techniques (7890A GC). The flow rate conditions through the GC column and the splitless feeding mode were applied . The amoTotal RNA was extracted from the tissue samples using the manufacturer’s easy-RED Total RNA Extraction Kits . The RNA integrity was tested by agarose gel electrophoresis, and NanoDrop’s spectrophotometer was used to analyze the sample quantities. The first-strand cDNA was achieved by the HiSenScript cDNA package . The selected genes, with GAPDH as a standard gene, were amplified with specific primers and stable in the sample groups . The mRNTissue samples from both ascending and descending limbs of the duodenum and the bursa of Fabricius and spleen were collected from five chickens from each treated group. The samples were fixed in 10% formaldehyde solution and then dehydrated in graded ethanol. The dehydrated samples were cleared in xylene and then embedded in paraffin. Moreover, 5-µm-thick paraffin-tissue sections were stained with hematoxylin and eosin. The stained sections were examined under a light microscope (Leica). The obtained images were subjected to morphometric analysis, including intestinal villi length of duodenum, total cell count of splenic parenchyma, and lymphoid follicles of the bursa of Fabricius using image analysis software . A total of 5 images from each bird were selected and the average was calculated (Mean ± SE) .p-value  <  0.05. Before conducting this test, Shapiro–Wilk and Levene’s experiments assessed normality. Polynomial contrasts were applied to find linear and quadratic impacts of different clenbuterol levels on the different parameters [Data analysis was performed using SPSS version 23  . A one-wrameters .p = 0.023) weight gain and final body weight (p = 0.01) compared to other treated groups. Moreover, clenbuterol at 15 ppm improved the hatchability (p < 0.01) concerning normal saline and control groups and recovered the hatchability and fertility percentage of injected eggs. Additionally, there was a significant improvement in carcass yield (p = 0.041). Furthermore, there was a significantly decreased abdominal fat weight percentage at 10 and 15 ppm dose of clenbuterol concerning other treated groups.The effects of in ovo feeding on growth and carcass traits are shown in ss yield . Moreovep > 0.05) in RBC count, Hb level, and H/L ratio in different treated groups  compared to other treated groups, with a noteworthy increase in HDL level (p = 0.011). Clenbuterol feeding resulted in considerably increased total protein and albumin levels compared to other treated groups. Moreover, clenbuterol had no considerable effect on AST and ALT levels. Additionally, clenbuterol groups showed a nonsignificant alteration in the total antioxidant capacity in other treated groups.There were no significant differences (d groups . In ovo p > 0.05)  . Lysosomp < 0.05) low saturated fatty acids  contents of the breast muscles compared to the other treated groups. Meanwhile, the current study revealed no significant variations of the in ovo feeding of clenbuterol to modulate the polyunsaturated FA (PUFA) contents in the breast muscles in other treated groups, including α-linolenic acid linoleic acid, docosahexaenoic acid, and eicosapentaenoic acid. As shown in p < 0.01) in the control and normal saline groups. Clenbuterol at 15 ppm showed significant upregulation of IGF1r, and clenbuterol significantly upregulated IGFBP expression at 15 ppm concentration in other treated groups; in the same context, clenbuterol feeding showed significant upregulation of hepatic PPARα in other treated groups  for clenbuterol at 10 ppm, 15 ppm, and 20 ppm, respectively. Clenbuterol feeding showed significant upregulation in LPL mRNA expression with a marked decrease in FAS mRNA expression. Moreover, there was significant downregulation in SREBP-1c expression in clenbuterol groups in other treated groups with marked upregulation in GHr expression, as shown in p < 0.001) in the clenbuterol 10 ppm group compared with the control and clenbuterol 15 ppm groups  lower saturated fatty acid  content of the breast muscles compared to other treated groups.Lipid markers decreased with in ovo clenbuterol feeding relative to saline feeding and control, as shown in M. longissimus Dorsi steers treated with beta-adrenergic agonist and showed that stearic acid might be negatively cholesterolemic, which decreased the cholesterol level. No previous studies have examined the effect of in ovo clenbuterol feeding on the amino acid contour of chicken muscle. In this context, the current study suggested that, as shown in Meanwhile, the current study revealed no significant variations of in ovo feeding of clenbuterol to modulate the PUFA contents in the breast muscles concerning other treated groups: α-linolenic acid, linoleic acid, and docosahexaenoic acid. These data were consistent with , in whicMoreover, in ovo feeding of clenbuterol significantly increased the nonessential amino acid contents in chicken breast muscles, such as serine, alanine, arginine, proline, and aspartic acid at 15 ppm in other treated groups. These data may be accredited to improving the protein content of muscles compared to the control group. Kheiri and Alibeyghi  revealedp < 0.01) in the control and normal saline groups. Similarly, Ijiri et al. [IGF-1 had a positive regulatory impact on muscle growth. These reports supported our finding in which clenbuterol at 15 ppm had significant upregulation of IGF1r. These findings were consistent with [IGF-1 upregulation. Moreover, Abo et al. [IGF-1 receptor level. These data were in harmony with [IGF-1 expression. These data were also supported by IGFBP expression in which clenbuterol significantly upregulates IGFBP expression at 15 ppm concentration in other treated groups. These data were in line with [IGFBP content.Additionally, it is crucial to investigate the transcriptomic pathway of clenbuterol concerning muscle growth. It was found that in ovo feeding of clenbuterol led to marked downregulation and myostatin gene expression , (p < 0.01), (p < 0.05) for clenbuterol at 10 ppm, 15 ppm, and 20 ppm, respectively. PPARα is highly expressed in the liver and plays a key role in lipid metabolism-boosting fatty acid oxidation [LPL mRNA expression with markedly decreased FAS mRNA expression, and this was consistent with [LPL gene. Clenbuterol modulates mRNA expression levels through attenuation of lipogenic activity (downregulated levels of the FAS in the liver) and fatty acid oxidation (increased levels of LPL in the liver gene expression), which facilitate lipid catabolism in agreement with our results. Kim et al. [l growth . Clenbutthose of , who repactivity . In the xidation . This suxidation . Clenbutm et al.  reportedSREBP-1c expression in clenbuterol groups than in other treated groups. Sterol regulatory element-binding proteins- 1 and -2 (SREBP-1 and -2) are key transcript components implicated in cholesterol and fatty acid biosynthesis. Our data were in harmony with [SREBP-1c expression, which supports our earlier result.Moreover, there was a significant downregulation in ony with . They reIn ovo feeding of clenbuterol improved hatchability, fertility, and growth efficiency, promoted lipolysis, modulated lipid markers, and decreased abdominal fat. Moreover, clenbuterol enhanced poultry body gain via upregulation of insulin growth factor 1 receptor and insulin-like growth factor-binding protein 2 expression, downregulated myostatin gene, and increased protein synthesis. In addition, clenbuterol increased the intestinal villi without significant alterations in the histopathology of the bursa of Fabricius and spleen. The in ovo clenbuterol feeding led to higher oxidation of fatty acids and increased growth weight.


Human T-lymphotropic virus type-1 (HTLV-1) is a neglected infection most often associated with an indolent process. However, a subset of HTLV-1 seropositive patients face the risk to develop life-threatening T-cell lymphoma/leukemia, or the highly disabling and incurable HTLV1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Over the years, other complications to HTLV-1 have been proposed and debated intensely. One of these, although rare, associations include cerebellar ataxia occurring most often in Japanese patients with manifest HAM/TSP. Here we present a HTLV-1 seropositive patient from the Middle East featuring a slowly progressive cerebellar syndrome with cerebellar atrophy but not evidence of spastic paraparesis. In addition, this patient suffered from autoimmune conditions such as Sjögren’s syndrome and vitiligo which are putatively associated with HTLV-1. Infection with human T cell lymphotropic virus type 1 (HTLV-1) is in most cases indolent; however, some patients develop adult T cell leukemia, associated with poor prognosis, or the highly disabling and incurable HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) , normal Achilles reflexes, and absence of Babinski’s sign. In addition, her muscle tone, strength, and sensation to touch and pin prick were normal. Vibration sense in her malleoli and proprioception were impaired with subsequent abnormal Romberg’s test; the retropulsion test was normal. Her altering hand movements were irregular; bradykinesia was absent. The patient also displayed hypermetric saccades. Her Scale for the Assessment and Rating of Ataxia score rose from 7.5 to 9.5 and Instituto de Pesquisa Clinica Evandro Chagas HAM disability scale, from 8 to 10, over 5 years.Neuroimaging demonstrated cerebellar atrophy Fig.  but not The most common genetic ataxias, syphilis, Lyme’s and Whipple’s disease, and autoimmune ataxias were ruled out (Supplementary document). Thus, onconeuronal antibodies, antibodies for celiac disease, and against GAD were absent. PVL in blood was initially 0.54% and 1.4% when repeated whereas serum β2M increased from 2.5 to 4.4 mg/L (ref < 2.0) over 4 years. In the CSF, white cell count, albumin concentration, and neurofilament light protein (NfL) concentration were normal, with no malignant cells, protein 14-3-3 or oligoclonal bands. However, CSF PVL (4 HTLV DNA copies/15 cells), neopterin 3.7 pg/mL (reference 2–3 pg/mL), and CXCL10 249.1 pg/mL (reference 115–160.8 pg/mL) were high.Computed tomography of her chest and abdomen showed an enlarged thyroid gland but the patient was euthyroid; a biopsy aspirate demonstrated colloid cells only. Mammography was initially normal but at age 68, she was diagnosed with a breast tumor treated with surgery and chemotherapy. Oral steroids were given with chemotherapy, but this did not alleviate the patient’s motor symptoms.So far, 20 HTLV-1 seropositive patients displaying ataxia have been reported, mostly in Japan. All but two had manifest HAM/TSP, which suggests that pyramidal signs may overshadow subtle cerebellar signs and nystagmus . In this case, β2M and PVL increased during follow-up and PVL in the CSF was high. Of note, determining PVL in the CSF is challenging due to the low number of leukocytes in CSF.Importantly, neurological complications are associated with high PVL . β2M is a component of the major histocompatibility complex class 1 and a key element in immediate immune response. As a consequence of the inflammatory reaction, chronic inflammation of the nervous system may occur in predisposed individuals, leading to permanent neurological dysfunction. The pattern of interleukin elevation in the CSF has been reported in HAM/TSP patients with very slow progression  Supplementary file2 (DOC 59 KB)Below is the link to the electronic supplementary material.


The canonical Wnt/β-catenin signaling plays a fundamental role in regulating embryonic development, injury repair and the pathogenesis of human diseases. In vertebrates, low density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6), the single-pass transmembrane proteins, act as coreceptors of Wnt ligands and are indispensable for Wnt signal transduction. LRP5 and LRP6 are highly homologous and widely co-expressed in embryonic and adult tissues, and they share similar function in mediating Wnt signaling. However, they also exhibit distinct characteristics by interacting with different protein partners. As such, each of them possesses its own unique functions. In this review, we systematically discuss the similarity and divergence of LRP5 and LRP6 in mediating Wnt and other signaling in the context of kidney diseases. A better understanding of the precise role of LRP5 and LRP6 may afford us to identify and refine therapeutic targets for the treatment of a variety of human diseases. Drosophila segmental polarity gene “Wingless” and mouse protooncogene “Int-1” , dishevelled (Dvl), casein kinase 1 (CK1) and glycogen synthase kinase-3 (GSK-3) . CK1 andIn vertebrates, there are 19 different Wnt ligands and 10 Fzd receptors, but only two coreceptors (LRP5/6) in Wnt signaling . LRP5/6 There are several structurally unrelated, transmembrane receptor proteins that mediate different Wnt signaling . Both caIn humans, there are 10 Fzd proteins, named Fzd1-10 . Fzd is The current model for Wnt signaling is that the binding of Wnt to Fzd and LRP5/6 leads to the dimerization or clustering of these two receptors, resulting in the formation of ternary complexes together with different downstream components . ActivatLow-density lipoprotein receptors (LDLR) are involved in a variety of cellular functions. The LDLR family members include LDL receptor, LRP , megalin (LRP2), VLDL receptor, apoER2 (LRP8), SorLA/LR11, LRP1b, LRP3, MEGF7 (LRP4), and LRP5/6 . However+-adenosine triphosphatase (V-ATPase) activity. (Pro)renin receptor, as a specific adaptor between LRP6 and V-ATPase, is an essential component of the Wnt receptor complex and obligatory for its signal transduction in a (pro)renin-independent manner (in vitro and in vivo ((Pro)renin receptor (PRR) is a single-pass transmembrane protein at the plasma membrane that transmits renin and prorenin signals . It has t manner . Meanwhi in vivo . As such in vivo .2+ pathway. The Wnt/PCP signaling can be initiated through the interaction between Wnt and Fzd receptors and their co-receptors, the receptor-like tyrosine kinase (RYK) and receptor tyrosine kinase-like orphan receptor (ROR), to recruit and activate Dvl, which then serves as a scaffold and activator for RhoA and Ras. Activated RhoA and Ras then regulate the activities of Rho-associated kinase (ROCK) and c-Jun N-terminal kinase (JNK), thereby participating in polarized cell orientation and asymmetric cell movement  and calcineurin. Activated calcineurin via dephosphorylation activates the nuclear factor of activated T cells (NFAT), which translocates to the nucleus and regulates the expression of target genes, thereby regulating cell fate  pathway and Wnt/Camovement . In the ell fate .Lrp5 cDNAs are 95% identical, indicating that the protein is highly conserved during evolution  < Lrp6+/– < Lrp5–/– < Lrp5+/–/Lrp6+/– < Lrp5–/–/Lrp6+/– < Lrp6–/– < Lrp5+/–/Lrp6–/– < Lrp5–/–/Lrp6–/–, indicating that loss of Lrp6 alleles consistently produces a more severe phenotype than loss of Lrp5. This difference of LRP5 and LRP6 in organ development may be contributed by their affinity with Wnt ligands or signaling efficacy  family proteins act as potent activators of Wnt/β-catenin signaling by binding to Fzd8 and LRP6 receptors . It has Norrin, a cystine-knot like growth factor that is unrelated to Wnt, has been shown to bind to Fzd4 and LRP5/6 to form a ternary complex, thus activating Wnt/β-catenin signaling . In addiper se, but regulate the activity of kinases used to phosphorylate LRP6, thereby activating β-catenin signaling. For example, hepatocyte growth factor (HGF) can stimulate GSK3-dependent and Wnt-independent LRP6 phosphorylation, thereby stabilizing β-catenin and activating Wnt signaling  and transforming growth factor-β (TGF-β) receptor 1 (TβR1) on the cell membrane . PDGF phIt has also been found that the CTGF domain 4 exhibits low-affinity binding to LRP6, rapidly stimulating LRP6 phosphorylation as well as downstream effectors JNK and p42/p44 activation, which can be inhibited by DKK1 or by silencing LRP6 . MoreoveThe Hippo pathway is an evolutionarily conserved signaling cascade that regulates organ size and tissue homeostasis by governing cell proliferation and apoptosis . There aIt is reported that in the absence of Wnt, YAP, and TAZ are components of the β-catenin destruction complex, and YAP/TAZ are associated with the destruction complex by binding to Axin . In thisLRP5 mutants lacking the extracellular domain act as constitutive active forms that bind Axin and induce LEF-1 activation by destabilizing Axin and stabilizing β-catenin . IntriguOur own data suggest that overexpression of LRP6 alone can activate not only β-catenin but also YAP/TAZ, whereas LRP5 merely activate β-catenin . These results could be explained by two possible mechanisms. One is that overexpression of LRP6 alone leads to homo-oligomerization of LRP6 and conformational changes to form activated forms, thus recruiting Axin and resulting the activation of β-catenin and YAP/TAZ. Alternatively, due to the direct binding of LRP6 to Axin, overexpression of LRP6 alone could compete with YAP/TAZ to bind Axin, resulting in the simultaneous release of YAP/TAZ and β-catenin from the destruction complex. As shown in Lrp5 gene may play a role in sporadic autosomal dominant polycystic kidney disease (ADPKD) , macroscopic small cystic kidneys are visible, suggesting a PKD phenotype (It has been shown that the  (ADPKD) . Earlierignaling . Furtherhenotype . These fWnt/β-catenin signaling is reactivated after kidney injury , and susThe expression of LRP6 is also up-regulated in the streptozotocin (STZ)-induced diabetic rat kidneys . In anotin vivo in response to kidney injury caused by UUO and unilateral renal ischemia-reperfusion injury (UIRI), thus alleviating renal fibrosis, capillary rarefaction and inflammation , characterized by mineral metabolism disorders, vascular calcification (VC), and renal osteodystrophy, is a severe complication in patients with end-stage renal disease (ESRD) . MountinOver the last two decades, significant progress has been made in our understanding the role of LRP5/6 in mediating Wnt signaling and other signal pathways. It becomes clear that although LRP5 and LRP6 are highly homologous, they are expressed differentially in diverse tissues and organs throughout embryonic and adult stages and possess similar yet divergent actions. In general, LRP6 appears more potent than LRP5 in transmitting Wnt signaling. In addition, LRP6 retains many unique actions by acting as a common coreceptor for numerous extracellular cues and by coupling Wnt with Hippo signaling. In short, LRP6 possesses many unique functions that extend beyond Wnt signaling.in vivo for therapeutic intervention remains in its infant stage. Given the complexity of these coreceptors, there will be many obstacles to overcome before any effective remedies can be developed and used in treating various human diseases.Despite these advances, our current knowledge on the commonality and uniqueness of LRP5 and LRP6 is limited. Up to date, we still know little about the structural basis that accounts for the functional divergence of LRP5 and LRP6. Furthermore, how to target LRP5 and LRP6 by exploiting their commonality and uniqueness QR reviewed the literature and wrote the manuscript. JC prepared the figures. YL planned the study and revised the manuscript. All authors gave the final approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aimed to explore the precise temporospatial distributions of bone morphogenetic protein (BMP) and Wnt signaling pathways during postnatal development of mammalian tooth roots after the termination of crown morphogenesis. A total of two transgenic mouse lines, BRE-LacZ mice and BAT-gal mice, were undertaken. The mice were sacrificed on every postnatal (PN) day from PN 3d up to PN 21d. Then, the first lower molars were extracted, and the dissected mandibles were stained with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside  and fixed. Serial sections at 10 µm were prepared after decalcification, dehydration, and embedding in paraffin. We observed BMP/Smads and Wnt/β-catenin signaling activities in the dental sac, dental pulp, and apical papilla with a certain degree of variation. The position of activation of the BMP/Smad signaling pathway was located more coronally in the early stage, which then gradually expanded as root elongation proceeded and was associated with blood vessels in the pulp and developing complex apical tissues in the later stage. However, Wnt/β-catenin signaling was highly concentrated in the mesenchyme below the cusps in the early stage, gradually expanded to regions around the root in the transition/root stage, and then disappeared entirely in the later stage. These results further confirmed the participation of both BMP and Wnt canonical signaling pathways in tooth root development, as well as formed the basis for future studies on how precisely integrated signaling pathways regulate root morphogenesis and regeneration. The HERS is known to induce the differentiation of odontoblasts and cementoblasts, to guide root growth, and to determine the number of tooth roots. HERS disintegration orchestrates the deposition of root dentin, which subsequently initiates dental sac cell cementogenesis onto the dentin surface.5 Moreover, it may also participate in cementum formation via epithelial-mesenchymal transition.7 Thus, HERS plays a vital role in root development.Analogous to the ectodermal organogenesis, a series of reciprocal and sequential epithelial-mesenchymal interactions are essential for tooth root development, which occurs after crown formation.8 Moreover, the ablation of Smad4, the key intracellular mediator of the TGF-β/BMP signaling pathway, in odontoblasts disrupts odontoblast differentiation and causes abnormal root odontogenesis.10 Previous studies also revealed the essential role of Wnt signaling during root formation; we detected the canonical Wnt activity in the mesenchyme and odontoblasts in BAT-gal/ TOP-gal reporter mice.12 Furthermore, recent reports showed that the disruption of β-catenin in immature odontoblasts generates molars without roots, whereas the constitutive stabilization of β-catenin can also lead to aberrant short roots with excessive cementum.14BMP and Wnt signaling pathways are crucial for root formation, including cell fate, growth, and patterning. The BMP–Smad4–Shh–Gli1–Sox2 signaling cascade worked in root development, where the disruption of BMP signaling in the dental epithelium causes delays in HERS formation and affects the stem cell niche environment.16 However, the journey toward the functional tooth starts postnatally. Furthermore, a comprehensive study on BMP and Wnt signaling activity during postnatal mouse M1 morphogenesis remains elusive.The cervical loop structure of mouse molars disappears after crown formation, and then HERS is formed to guide root development postnatally, which is similar to human tooth organogenesis. Thus, the first lower mouse molar (M1) is an ideal model for studying tooth root development. In the past decades, the molecular regulatory network of early tooth morphogenesis was studied extensively, with research mainly concentrating on prenatal stages.BRE-LacZ reporter contains a BMP-response element (BRE) labeled by β-galactosidase, in which phosphorylated BMP Smad DNA-binding sites are originated with the mouse Id1 promoter.17 Similarly, BAT-gal mouse is a Wnt-reporter transgenic strain that expresses β-galactosidase when the intracellular β-catenin is activated .18 These BRE-LacZ or BAT-gal reporter mouse lines can identify sites where the BMP-Smads or Wnt/β-catenin signaling pathway is activated. They represent an opportunity to monitor BMP/Smad-dependent or Wnt/β-catenin-dependent signaling during M1 development. Therefore, we used BRE-LacZ and BAT-gal mice to monitor their activities in postnatal stages of tooth development to explore the potential roles of BMP and canonical Wnt/β-catenin signaling in late tooth morphogenesis. Indeed, the findings indicated that BMP and Wnt signaling pathways orchestrated an active network that regulated postnatal tooth development.This study aimed to investigate postnatal M1 morphogenesis before eruption associated with active BMP and Wnt signaling distribution, matching with root development and odontogenesis. The Animal care and all experiments were conducted under the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health, approved by the Institutional Animal Care and Use Committee of Tulane University (Protocol #: 0184R5)BRE-LacZ mice17 and BAT-gal mice18. All studies involved CD-1 background mice. The mice were kept in a 12-hour light/dark period with a temperature of 22 ± 20C and humidity of 45% ± 15% of the rooms.The day of birth was designated as “postnatal day (PN) 0.” Before the sacrifice, the tail biopsies from each mouse were determined by PCR-based genotyping. The generation and genotyping protocols of transgenic mice used in our study were described previously: Pups from mice of both sexes were sacrificed on every PN day from day 3 (PN3) up to PN21 (n: 5 pups). Extracted first lower molars and dissected mandibles were stained with X-gal, fixed, decalcified in 10% ethylene diaminetetraacetic acid/phosphate-buffered saline (PBS) solution (pH 7.4), dehydrated through a graded alcohol series, and embedded in paraffin. Serial sections of 10 µm were prepared. Counterstaining for sectioned samples was done with eosin. For general histological observations, the specimens were examined under a light microscope .LacZ expression was determined with X-gal staining, as described earlier.19 Briefly, the mandibular samples were fixed with 4% paraformaldehyde (PFA) in 0.2 mM glutaraldehyde in PBS for 10 min, as well as were processed for X-gal staining in the buffer  for 20 h at 37°C in the dark. For general observations, the specimens were examined under a stereomicroscope .The results showed that M1 crown development was completed on postnatal day 3 (PN3) , 2A and BRE-LacZ reporter mouse line to identify BMP activity in tooth development. β-galactosidase reporter gene expression identified the broad and dynamic BMP signaling activities during postnatal root development. For the early postnatal days, we harvested the M1 with the whole dental sac. Since the newly formed cusp was fragile and soft, it was difficult to separate the dental sac or epithelium from the crown until PN14  in the initial stages. During the following stages of M1 morphogenesis (PN6–PN8), the teeth rapidly increased in size and length. BRE-LacZ activity became lower in the crown pulp but intensified in the root pulp, dental sac, and apical papilla  to assess the histomorphology. BRE-LacZ and BAT-gal mice. Both strains were associated with β-galactosidase. X-gal staining in BRE-LacZ reporter correlated with the nuclear accumulation of phosphorylated SMAD 1/5/8 (BMP activation), whereas BAT-gal transgenic strain in the presence of activated β-catenin  exhibited β-gal activity. This study revealed the spatiotemporal activities of both BMP/Smad and Wnt/β-catenin signaling pathways in the developing molar root.Tooth morphogenesis is a well-orchestrated process governed by many signaling pathways between resident/migratory cells and the extracellular matrix. Multiple signaling pathways and numerous transcription factors have been illustrated in the molar root development; however, few concerns regarding their dynamic expression postnatally remain. Contrary to incisor development, mouse molar development is similar to that in humans, including coronal morphogenesis followed by the initiation of root development and later root elongation. In mice, tooth root development begins after birth and lasts for almost 21 days. Therefore, mouse M1 is an ideal animal model in mammalian odontogenesis research. Currently, the underlying mechanisms that control root formation during odontogenesis are yet to be determined. Many studies showed that BMP and Wnt ligands persist throughout tooth root formation. However, the dynamic pattern of active BMP and Wnt signaling is still not well understood. To allow screening for the strength of the two signals, we chose to derive two transgenic reporter strains, 20 We observed that Bmp2 signaling is indispensable for proper enamel development and mineralization in vivo.21 The blockage of BMP signaling in the dental mesenchyme on PN3.5 resulted in impaired root development, whereas inhibiting BMP signaling in the dental epithelium before root growth certified that postnatal epithelial BMP signaling was dispensable in the regulation of root development.22 Similar to many other developmental processes, epithelial−mesenchymal interactions were previously demonstrated to be essential for tooth root development.4 Here, we showed that activated BMP signaling  was specifically present in dental follicles and HERS at the early postnatal stage; then, the HERS became perforated, contributing to cementum regeneration and root formation , the Axin2 expression was downregulated in the crown odontoblast lines.11 The canonical Wnt signaling may also be indispensable to the differentiation and maturation of odontoblasts, whereas the inactivation of canonical Wnt activity probably act a pivotal role in terminal differentiation. Specifically, the tissue-specific ablation of β-catenin in developing odontoblasts led to rootless molars, whereas the constitutive stabilization of β-catenin in the dental mesenchyme produced molars with short roots and thick cementum.14 Further insight of the regulatory mechanism of Wnt/β-catenin in cellular differentiation and matrix secretion will be needed during many phases of postnatal root development in future studies.In mouse M1, rapid tooth growth and root elongation begin on PN6.Within the constraints and limitations of our study, a few tissues in the M1, where BMP and/or Wnt activity was expected, were not stained. This might be because (1) the expression of BMP and/or Wnt signaling are so transient that it is hard to upregulate β-galactosidase expression, (2) the window of signaling activation is missed, or (3) BMP and/or Wnt signaling in these tissues worked in a noncanonical signaling cascade way.In short, the results of this study showed that BMP and Wnt signaling activities exhibited different and dynamic patterns during mouse M1 root development. The position of activation of the BMP/Smad signal pathway was located more coronally in the early stage, which then gradually expanded as root elongation proceeded and was associated with blood vessels in the pulp and DAC tissues in the later stage. However, Wnt/β-catenin signaling was highly concentrated in the mesenchyme below the cusps in the early stage, gradually expanded to regions around the root at the transition/root stage, and then disappeared nearly the later stage. These findings emphasized the importance of spatial and temporal epithelial-mesenchymal signaling, such as BMP and Wnt signaling pathways, for postnatal dentinogenesis, as well as provided some clues for future studies to precisely explore the cellular and molecular mechanisms that regulate tooth root development.