Shh Transcription Research Articles

Ectopic Activation of Fgf8 in Dental Mesenchyme Causes Incisor Agenesis and Molar Microdontia.

Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice (Osr2-creKI; Rosa26R-Fgf8) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-creKI; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-creKI; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-creKI; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin, Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-creKI; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-creKI; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia.

Abstract IA001: Cellular and molecular underpinnings of field cancerization and progression to invasive carcinoma in bladder cancer

Abstract Two major concerns in clinical management of bladder cancer are: (i) the high frequency of recurrence; and (ii) the morbidity and mortality associated with progression to invasive disease (invasive urothelial carcinoma, IUC). To gain a better understanding of these issues we have used the murine BBN (N-butyl-N-(4-hydroxylbutyl) nitrosamine) experimental model of bladder cancer, as well as tissue samples from normal human bladders and from bladders of patients at various stages of bladder cancer formation and progression. We have documented in the BBN model a CIS-like stage, prior to invasion, in which clonal expansion from a single cell occupies a significant fraction of the urothelium. With this experimental murine model alongside observations from patient samples and data from The Cancer Genome Atlas (TCGA), we have ascertained that a key event in progression of pre-invasive to invasive carcinoma is the silencing of the Sonic hedgehog (Shh) member of the Hedgehog gene family. Silencing-associated invasion appears due to the loss of SHH-induced differentiation signals, which when present slow growth and block invasion. Despite the consistent loss of SHH expression in invasive murine and human bladder cancers, however, TCGA data show few if any mutations in the SHH gene of IUC cells. Instead, our studies of patient samples and cancer-derived cell lines reveal that the loss of SHH expression is due to tighter chromosomal packaging and reduced accessibility of a bladder-specific enhancer located ~200 kb upstream of the SHH transcription start site. We have found that inhibitors of certain chromatin-modifying enzymes, including an FDA-approved drug, are capable of re-activating SHH expression, and are testing the possibility that these drugs may block or reverse invasion of urothelial carcinoma. The high frequency of bladder cancer recurrence is thought to result from field cancerization, that is, an increased probability of new cancer formation in apparent normal urothelium, even after successful resection of the primary tumor. To understand the basis for this field effect, we compared apparent normal urothelium from cancer patients to truly normal urothelium from bladders never affected by malignancy. We found that apparent normal samples display a dramatically increased basal character in transcriptomic assays, using as a metric of basalization an index constructed from single cell RNA-seq analysis of normal urothelium. We and others have demonstrated previously by lineage tracing and cell ablation studies in the BBN model that IUC arises from basal stem cells of the urothelium. The basalization of urothelium distant from the primary tumor that we observe thus may account for the increased probability of cancer formation in the apparent normal urothelium of bladder cancer patients. We have found that several FDA-approved antagonists of EGF signaling can reverse this basalization in the mouse BBN model as well as in organoid culture, and that these drugs dramatically reduce the formation of tumors following BBN exposure. Citation Format: Philip A. Beachy, Kris B. Prado, Aaron M. Kershner, Karim Mrouj, Bernard M. Kiss, Hua Dong, Ozgu Aydogdu, Ye Tian, Joseph C. Liao. Cellular and molecular underpinnings of field cancerization and progression to invasive carcinoma in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr IA001.

Characterization of Sonic Hedgehog transcripts in the adult mouse brain: co-expression with neuronal and oligodendroglial markers

In the adult mammalian brain, astrocytes are proposed to be the major Sonic Hedgehog (Shh)-responsive cells. However, the sources of the Shh molecule mediating activation of the pathway are still poorly characterized. The present work investigates the distribution and phenotype of cells expressing Shh mRNA in the adult mouse brain. Using single-molecule fluorescent in situ hybridization (smfISH), we report much broader expression of Shh transcripts in almost all brain regions than originally reported. We identify Shh mRNA in HuC/D+ neuronal populations, including GABAergic (glutamic acid decarboxylase 67, Gad67), cholinergic (choline acetyltransferase, ChAT), dopaminergic (tyrosine hydroxylase, TH), nitrergic (neuronal nitric oxide synthase, nNOS), and in a small population of oligodendroglial cells expressing Sox10 and Olig2 mRNA transcription factors. Further analysis of Shh mRNA in cerebral cortical and hypothalamic neurons suggests that Shh is also expressed by glutamatergic neurons. Interestingly, we did not observe substantial Desert Hedgehog and Indian Hedgehog mRNA signals, nor Shh signals in S100β+ astrocytes and Iba1+ microglial cells. Collectively, the present work provides the most robust central map of Shh-expressing cells to date and underscores the importance of nitrergic neurons in regulating Shh availability to brain cells. Thus, our study provides a framework for future experiments aimed at better understanding of the functions of Shh signaling in the brain in normal and pathological states, and the characterization of novel regulatory mechanisms of the signaling pathway.

Mirk/Dyrk1B controls ventral spinal cord development via Shh pathway

Cross-talk between Mirk/Dyrk1B kinase and Sonic hedgehog (Shh)/Gli pathway affects physiology and pathology. Here, we reveal a novel role for Dyrk1B in regulating ventral progenitor and neuron subtypes in the embryonic chick spinal cord (SC) via the Shh pathway. Using in ovo gain-and-loss-of-function approaches at E2, we report that Dyrk1B affects the proliferation and differentiation of neuronal progenitors at E4 and impacts on apoptosis specifically in the motor neuron (MN) domain. Especially, Dyrk1B overexpression decreases the numbers of ventral progenitors, MNs, and V2a interneurons, while the pharmacological inhibition of endogenous Dyrk1B kinase activity by AZ191 administration increases the numbers of ventral progenitors and MNs. Mechanistically, Dyrk1B overexpression suppresses Shh, Gli2 and Gli3 mRNA levels, while conversely, Shh, Gli2 and Gli3 transcription is increased in the presence of Dyrk1B inhibitor AZ191 or Smoothened agonist SAG. Most importantly, in phenotype rescue experiments, SAG restores the Dyrk1B-mediated dysregulation of ventral progenitors. Further at E6, Dyrk1B affects selectively the medial lateral motor neuron column (LMCm), consistent with the expression of Shh in this region. Collectively, these observations reveal a novel regulatory function of Dyrk1B kinase in suppressing the Shh/Gli pathway and thus affecting ventral subtypes in the developing spinal cord. These data render Dyrk1B a possible therapeutic target for motor neuron diseases.

Distinct expression patterns of Hedgehog signaling components in mouse gustatory system during postnatal tongue development and adult homeostasis.

The Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae. HH signaling also mediates taste organ maintenance and regeneration in adults. However, there are knowledge gaps in HH pathway component expression during postnatal taste organ differentiation and maturation. Importantly, the HH transcriptional effectors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; the HH receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, also remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Notably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stages. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development.

A Sonic Hedgehog-Gli-Bmi1 signaling pathway plays a critical role in p27 deficiency induced bone anabolism.

To explore the mechanism of the bone anabolic action of p27 deficiency, we first confirmed that osteoblast formation and osteogenesis were significantly increased in p27 deficient mice compared with their wild-type littermates. Microarray analysis of differential gene expression profiles, followed by real-time RT-PCR and Western blots revealed that p27 deletion significantly upregulated the expression of Sonic hedgehog (Shh), Gli1 and 2 and their target gene Bmi1 in bone tissue, and significantly down regulated the expression of the negative regulators of the Shh pathway Sufu, Patched 1 and Gli3 in bone tissue. The Shh antagonist KAAD-cyclopamine or vismodegib significantly reduced osteogenesis of bone marrow mesenchymal stem cells (BM-MSCs) in vitro and osteoblastic bone formation in vivo. The results of chromatin immunoprecipitation and double luciferase assay demonstrated that p27 inhibited Shh transcription mediated via E2F4. Bmi1 knockout blocked the increase of osteoblastic bone formation induced by p27 deficiency in vivo. In conclusion, the results of this study indicate that the signaling pathway Shh-Gli-Bmi1 plays a critical role in p27 deficiency induced bone anabolic action, suggesting that Bmi1 may be an important therapeutic target for osteoporosis induced by activation of p27 signaling or inactivation of sonic hedgehog signaling.

Tbx5 drives Aldh1a2 expression to regulate a RA-Hedgehog-Wnt gene regulatory network coordinating cardiopulmonary development.

The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. Here using Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA) signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary (CP) development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing CP evolution and birth defects.

Noggin Overexpression Impairs the Development of Muscles, Tendons, and Aponeurosis in Soft Palates by Disrupting BMP-Smad and Shh-Gli1 Signaling.

The roles of bone morphogenetic protein (BMP) signaling in palatogenesis were well documented in the developing hard palate; however, little is known about how BMP signaling regulates the development of soft palate. In this study, we overexpressed Noggin transgene via Osr2-creKI allele to suppress BMP signaling in the developing soft palate. We found that BMP-Smad signaling was detected in the palatal muscles and surrounding mesenchyme. When BMP-Smad signaling was suppressed by the overexpressed Noggin, the soft palatal shelves were reduced in size with the hypoplastic muscles and the extroversive hypophosphatasia (HPP). The downregulated cell proliferation and survival in the Osr2-creKI;pMes-Noggin soft palates were suggested to result from the repressed Shh transcription and Gli1 activity, implicating that the BMP-Shh-Gli1 network played a similar role in soft palate development as in the hard palate. The downregulated Sox9, Tenascin-C (TnC), and Col1 expression in Osr2-creKI;pMes-Noggin soft palate indicated the impaired differentiation of the aponeurosis and tendons, which was suggested to result in the hypoplasia of palatal muscles. Intriguingly, in the Myf5-creKI;pMes-Noggin and the Myf5-creKI;Rosa26R-DTA soft palates, the hypoplastic or abrogated muscles affected little the fusion of soft palate. Although the Scx, Tnc, and Co1 transcription was significantly repressed in the tenogenic mesenchyme of the Myf5-creKI;pMes-Noggin soft palate, the Sox9 expression, and the Tnc and Col1 transcription in aponeurosis mesenchyme were almost unaffected. It implicated that the fusion of soft palate was controlled by the mesenchymal clues at the tensor veli palatini (TVP) and levator veli palatini (LVP) levels, but by the myogenic components at the palatopharyngeus (PLP) level.

Interaction of tacrolimus through hedgehog signaling pathway: An in vitro evaluation using rat hepatocytes.

Tacrolimus (TAC) is the drug of choice in immunosuppressive therapy for organ transplantation; however, adverse effects are still a major concern. The current study aims to decipher the short-term exposure of TAC on rat hepatocytes in relation to activation of hedgehog (HH) signaling pathway. Time dependent study was conducted using primary rat hepatocytes treated with TAC (36 µM) for 6, 12, 24 and 48 h. Western blot analysis was performed using cell lysate in order to analyze the regulation of HH pathway proteins including HHIP, SMO, PTCH, IHH, SHH, and GLI transcription factors. The study revealed change in protein expression of HH signaling molecules with activation of HH pathway, due to downregulation of HHIP, and enrichment of HH ligands with activation of SMO and GLI transcription factors. It is therefore, concluded that short term TAC exposure leads to upregulation of HH pathway in liver, which may initially act to repair the liver damage but can worsen the condition in case of prolonged immunosuppressive therapy. This insight could lead to understand association of off target effects of immunosuppressive drugs and occurrence of other liver diseases in transplant patients when it comes to long term immunosuppressive therapy. These findings also illuminate a novel direction that use of HH inhibitor might provide a therapeutic strategy for immune suppression related liver disorders.

Upregulation of LINC01426 promotes the progression and stemness in lung adenocarcinoma by enhancing the level of SHH protein to activate the hedgehog pathway

Long noncoding RNAs (lncRNAs) play crucial roles in regulating a variety of biological processes in lung adenocarcinoma (LUAD). In our study, we mainly explored the functional roles of a novel lncRNA long intergenic non-protein coding RNA 1426 (LINC01426) in LUAD. We applied bioinformatics analysis to find the expression of LINC01426 was upregulated in LUAD tissue. Functionally, silencing of LINC01426 obviously suppressed the proliferation, migration, epithelial–mesenchymal transition (EMT), and stemness of LUAD cells. Then, we observed that LINC01426 functioned through the hedgehog pathway in LUAD. The effect of LINC01426 knockdown could be fully reversed by adding hedgehog pathway activator SAG. In addition, we proved that LINC01426 could not affect SHH transcription and its mRNA level. Pull-down sliver staining and RIP assay revealed that LINC01426 could interact with USP22. Ubiquitination assays manifested that LINC01426 and USP22 modulated SHH ubiquitination levels. Rescue assays verified that SHH overexpression rescued the cell growth, migration, and stemness suppressed by LINC01426 silencing. In conclusion, LINC01426 promotes LUAD progression by recruiting USP22 to stabilize SHH protein and thus activate the hedgehog pathway.

Sonic hedgehog signalling as a potential endobronchial biomarker in COPD

BackgroundThe hedgehog (HH) pathway has been associated with chronic obstructive pulmonary disease (COPD) in genome-wide association studies and recent studies suggest that HH signalling could be altered in COPD. We therefore used minimally invasive endobronchial procedures to assess activation of the HH pathway including the main transcription factor, Gli2, and the ligand, Sonic HH (Shh).MethodsThirty non-COPD patients and 28 COPD patients were included. Bronchial brushings, bronchoalveolar lavage fluid (BALF) and bronchial biopsies were obtained from fiberoptic bronchoscopy. Characterization of cell populations and subcellular localization were evaluated by immunostaining. ELISA and RNAseq analysis were performed to identify Shh proteins in BAL and transcripts on lung tissues from non-COPD and COPD patients with validation in an external and independent cohort.ResultsCompared to non-COPD patients, COPD patients exhibited a larger proportion of basal cells in bronchial brushings (26 ± 11% vs 13 ± 6%; p < 0.0001). Airway basal cells of COPD subjects presented less intense nuclear staining for Gli2 in bronchial brushings and biopsies (p < 0.05). Bronchial BALF from COPD patients contained lower Shh concentrations than non-COPD BALF (12.5 vs 40.9 pg/mL; p = 0.002); SHH transcripts were also reduced in COPD lungs in the validation cohort (p = 0.0001).ConclusionThis study demonstrates the feasibility of assessing HH pathway activation in respiratory samples collected by bronchoscopy and identifies impaired bronchial epithelial HH signalling in COPD.

Foxa1 and Foxa2 orchestrate development of the urethral tube and division of the embryonic cloaca through an autoregulatory loop with Shh

Congenital anomalies of external genitalia affect approximately 1 in 125 live male births. Development of the genital tubercle, the precursor of the penis and clitoris, is regulated by the urethral plate epithelium, an endodermal signaling center. Signaling activity of the urethral plate is mediated by Sonic hedgehog (SHH), which coordinates outgrowth and patterning of the genital tubercle by controlling cell cycle kinetics and expression of downstream genes. The mechanisms that govern Shh transcription in urethral plate cells are largely unknown. Here we show that deletion of Foxa1 and Foxa2 results in persistent cloaca, an incomplete separation of urinary, genital, and anorectal tracts, and severe hypospadias, a failure of urethral tubulogenesis. Loss of Foxa2 and only one copy of Foxa1 results in urethral fistula, an additional opening of the penile urethra. Foxa1/a2 participate in an autoregulatory feedback loop with Shh, in which FOXA1 and FOXA2 positively regulate transcription of Shh in the urethra, and SHH feeds back to negatively regulate Foxa1 and Foxa2 expression. These findings reveal novel roles for Foxa genes in development of the urethral tube and in division of the embryonic cloaca.

C9C5 positive mature oligodendrocytes are a source of Sonic Hedgehogin the mouse brain.

In the mature rodent brain, Sonic Hedgehog (Shh) signaling regulates stem and progenitor cell maintenance, neuronal and glial circuitry and brain repair. However, the sources and distribution of Shh mediating these effects are still poorly characterized. Here, we report in the adult mouse brain, a broad expression pattern of Shh recognized by the specific monoclonal C9C5 antibody in a subset (11–12%) of CC1+ mature oligodendrocytes that do not express carbonic anhydrase II. These cells express also Olig2 and Sox10, two oligodendrocyte lineage-specific markers, but not PDGFRα, a marker of oligodendrocyte progenitors. In agreement with oligodendroglial cells being a source of Shh in the adult mouse brain, we identify Shh transcripts by single molecule fluorescent in situ hybridization in a subset of cells expressing Olig2 and Sox10 mRNAs. These findings also reveal that Shh expression is more extensive than originally reported. The Shh-C9C5-associated signal labels the oligodendroglial cell body and decorates by intense puncta the processes. C9C5+ cells are distributed in a grid-like manner. They constitute small units that could deliver locally Shh to its receptor Patched expressed in GFAP+ and S100β+ astrocytes, and in HuC/D+ neurons as shown in PtcLacZ/+ reporter mice. Postnatally, C9C5 immunoreactivity overlaps the myelination peak that occurs between P10 and P20 and is down regulated during ageing. Thus, our data suggest that C9C5+CC1+ oligodendroglial cells are a source of Shh in the mouse postnatal brain.

Sonic hedgehog expression in the postnatal brain.

ABSTRACTBeyond its role in patterning the neural tube during embryogenesis, additional functions of Sonic hedgehog (Shh) in post-embryonic and mature brains have been coming into focus. However, the question of the abundance of endogenous Shh – the ligand of the signaling pathway – and its changes over time in post-embryonic and mature brains are less well understood. Here we find that while the amounts of Shh transcript and protein in rat brains are nearly undetectable at birth, they increase continuously during postnatal development and remain at readily detectable levels in young adults. This developmental age-associated increase in Shh levels is also seen in hippocampal neurons grown in culture, in which very young neurons produce minimal amounts of Shh protein but, as neurons grow and form synapses, the amounts of Shh increase significantly. Using immunolabeling with antibodies to different residues of Shh, we observed that the N-terminal fragment and the C-terminal fragment of Shh are present in hippocampal neurons, and that these two Shh forms co-exist in most compartments of the neuron. Our findings provide a better understanding of Shh expression in the brain, laying the groundwork for further comprehending the biogenesis of Shh protein in the young and mature brain and neurons.

Relationship between spinal Sonic Hedgehog signaling pathway and inflammatory responses in rats with neuropathic pain

Objective To evaluate the relationship between spinal Sonic hedgehog(Shh) signaling pathway and inflammatory responses in rats with neuropathic pain(NP). Methods Forty-eight clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 250-300 g, were divided into 4 groups(n=12 each) using a random number table method: sham operation group(group S), group NP, dimethyl sulfoxide(DMSO) group(group D) and specific Shh signaling pathway inhibitor cyclopamine group(group CP). Spared nerve injury was produced by exposing the sciatic nerve and branches followed by ligation and transection of tibial and common fibular nerves in anesthetized rats. The mechanical paw withdrawal threshold(MWT) was measured at 1 day before establishing the model and 1 and 7 days after establishing the model., The animals were sacrificed after measurement of pain threshold at 7 days after operation, and the lumbar segment L4-6 of the spinal cord was obtained for determination of the expression of Shh, Patched homolog(Ptch), Smoothened(Smo) and zinc finger-containing transcription factors 1(Gli1)(by Western blot) and contents of tumor necrosis factor-alpha(TNF-α) and interleukin-1beta(IL-1β)(by enzyme-linked immunosorbent assay). Results Compared with group S, the MWT was significantly decreased, the expression of Shh, Patched, Smo and Gli1 was up-regulated, and the contents of TNF-α and IL-1β were increased in NP, D and CP groups(P<0.05). Compared with group NP, the MWT was significantly increased, the expression of Shh, Patched, Smo and Gli1 was down-regulated, and the contents of TNF-α and IL-1β were decreased in group CP(P<0.05), and no significant change was found in the parameters mentioned above in group D(P<0.05). Conclusion The mechanism by which Shh signaling pathway is involved in the development and maintenance of inflammatory responses is related to inhibiting inflammatory responses of the spinal cord in rats with NP. Key words: Neuralgia; Hedgehog proteins; Signal transduction; Inflammation; Spinal cord

Hedgehog and Wnt Signaling Pathways Regulate Tail Regeneration.

Urodele amphibians have a tremendous capacity for the regeneration of appendages, including limb and tail, following injury. While studies have focused on the cellular and morphological changes during appendicular regeneration, the signaling mechanisms that govern these cytoarchitectural changes during the regenerative response are unclear. In this study, we describe the essential role of hedgehog (Hh) and Wnt signaling pathways following tail amputation in the newt. Quantitative PCR studies revealed that members of both the Hh and Wnt signaling pathways, including the following: shh, ihh, ptc-1, wnt-3a, β-catenin, axin2, frizzled (frzd)-1, and frzd-2 transcripts, were induced following injury. Continuous pharmacological-mediated inhibition of Hh signaling resulted in spike-like regenerates with no evidence of tissue patterning, whereas activation of Hh signaling enhanced the regenerative process. Pharmacological-mediated temporal inhibition experiments demonstrated that the Hh-mediated patterning of the regenerating tail occurs early during regeneration and Hh signals are continuously required for proliferation of the blastemal progenitors. BrdU incorporation and PCNA immunohistochemical studies demonstrated that Hh signaling regulates the cellular proliferation of the blastemal cells following amputation. Similarly, Wnt inhibition resulted in perturbed regeneration, whereas its activation promoted tail regeneration. Using an inhibitor-activator strategy, we demonstrated that the Wnt pathway is likely to be upstream of the Hh pathway and together these signaling pathways function in a coordinated manner to facilitate tail regeneration. Mechanistically, the Wnt signaling pathway activated the Hh signaling pathway that included ihh and ptc-1 during the tail regenerative process. Collectively, our results demonstrate the absolute requirement of signaling pathways that are essential in the regulation of tail regeneration.

Bivalency of Shh Modulates Cell Fate Decision of Dental Epithelium Stem Cells

Rodent incisor grows throughout whole life of the animal and owns every stage of dental epithelium development, thus it provides an ideal model to study the fate decision of dental epithelial stem cells (DESCs) as well as to understand the mechanisms of tooth regeneration. Bivalency, characterized as concurrence of H3K27me3 and H3K4me3, manages key developmental genes transcript in some organs, however no evidences answer whether it works on enamel organ development. Here using mouse incisor models, we first of time reveal the dynamic alterations of bivalency during DESCs differentiation, which is dominated via H3K27me3 rather than H3K4me3. Ezh2 is identified to catalyze this H3K27me3 change, then determining cell fate of DESCs. Ezh2 defecient DESCs show impaired stemness but activated amelogenic gene amelogenin (Amelx). Conversely, elevating Ezh2 maintains pluripotency and represses Amelx, which is rescued by deleting SET trimethylase activity domain in Ezh2. Mechanistically, Ezh2 is validated to catalyze H3K27me3 within promoter of Shh in a methytransferase activity dependent manner, thus determining Shh transcript. In vitro silencing Ezh2 results in activation of Shh expression, increasing the recruitment of GLI1 within Amelx promoter, however, Ezh2 overexpression represses such Shh signaling. In vivo bivalency related Shh activation occurs in pre-ameloblasts (PAB), which directly triggers Ptch1/Gli1 signaling to enhance the enrichment of Gli1 within promoter of Amelx, finally opens the door of differentiation. These results bring light to brand new insights on role of bivalency in enamel organ formation and cell fate choices of stem cell. Funding Sources: This work was supported by National Natural Science Foundation of China (NSFC grant 81470711&81771033 to LWZ), preeminent youth fund of Sichuan province (2016JQ0054 to LWZ), Sichuan Province Science and Technology Innovation Team Program (2015TD0011 to YL, 2017TD0016 to Y.Q). Conflicts of Interest: The authors declare no potential conflicts of interest with respect to the authorship and publication of this article. Ethics Approval Statement: All mouse experiments were conducted in accordance with protocols approved by ethical committees of the West China School of Stomatology, Sichuan University and State Key Laboratory of Oral Diseases.

Upstream Enhancer Elements of Shh Regulate Oral and Dental Patterning

Sonic hedgehog (Shh) is important in pattern formation during development. Shh transcription is modulated by a long-range regulatory mechanism containing a number of enhancers, which are spread over nearly 850 kb in the mouse genome. Shh enhancers in the nervous system have been found between intron and 430 kb upstream of Shh. Enhancers in the oral cavity, pharynx, lung, gut, and limbs have been discovered between 610 kb and 850 kb upstream of Shh. However, the intergenic region ranging from 430 to 610 kb upstream of Shh remains to be elucidated. In the present study, we found a novel long-range enhancer located 558 kb upstream of Shh. The enhancer showed in vivo activity in oral cavity and whiskers. A targeted deletion from the novel enhancer to mammal reptile conserved sequence 1 (MRCS1), which is a known enhancer of Shh in oral cavity, resulted in supernumerary molar formation, confirming the essential role of this intergenic region for Shh transcription in teeth. Furthermore, we clarified the binding of Lef1/Tcfs to the new enhancer and MRCS1, suggesting that Wnt/β-catenin signaling regulates Shh signaling in the oral cavity via these enhancers.

Transcriptional changes in chick wing bud polarization induced by retinoic acid.

Background: Retinoic acid is implicated in the induction of the gene encoding Sonic hedgehog (Shh) that specifies anteroposterior positional values and promotes growth of the developing limb bud. However, because retinoic acid is involved in limb initiation, it has been difficult to determine if it could have additional roles in anteroposterior patterning. To investigate this, we implanted retinoic acid–soaked beads to the anterior margin of the chick wing bud and performed microarray analyses prior to onset of Shh expression. Results: Retinoic acid up‐regulates expression of Hoxd11‐13 that encode transcription factors implicated in inducing Shh transcription and that are involved in digit development. In our assay, retinoic acid induces Shh transcription and, consequently, a new pattern of digits at a much later stage than anticipated. Retinoic acid represses many anteriorly expressed genes, including Bmp4, Lhx9, Msx2, and Alx4. We provide evidence that retinoic acid influences transcription via induction of dHAND and inhibition of Gli3 to establish a new anteroposterior pre‐pattern. We show that transient exposure to retinoic acid can suppress distal development and expedite cells to transcriptionally respond to Shh. Conclusions: Our findings reveal how retinoic acid and Shh signaling could cooperate in anteroposterior patterning of the limb. Developmental Dynamics 246:682–690, 2017. © 2017 Wiley Periodicals, Inc.

The transcription factor Gli3 promotes B cell development in fetal liver through repression of Shh.

Before birth, B cells develop in the fetal liver (FL). In this study, we show that Gli3 activity in the FL stroma is required for B cell development. In the Gli3-deficient FL, B cell development was reduced at multiple stages, whereas the Sonic hedgehog (Hh [Shh])-deficient FL showed increased B cell development, and Gli3 functioned to repress Shh transcription. Use of a transgenic Hh-reporter mouse showed that Shh signals directly to developing B cells and that Hh pathway activation was increased in developing B cells from Gli3-deficient FLs. RNA sequencing confirmed that Hh-mediated transcription is increased in B-lineage cells from Gli3-deficient FL and showed that these cells expressed reduced levels of B-lineage transcription factors and B cell receptor (BCR)/pre-BCR-signaling genes. Expression of the master regulators of B cell development Ebf1 and Pax5 was reduced in developing B cells from Gli3-deficient FL but increased in Shh-deficient FL, and in vitro Shh treatment or neutralization reduced or increased their expression, respectively.