Relationship: 2734: Antagonism Smoothened leads to Decrease, SMO relocation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The relationship between antagonism of SMO and a decrease in SMO relocation and activation has been shown repeatedly in mice models as detailed in the empirical evidence section. The relationship is biologically plausible in human, but to date no specific experiments have addressed this question. The SHH pathway is well understood to be fundamental to proper embryonic development and that aberrant SHH signaling during embryonic development can cause birth defects indculding orofacial clefts (OFCs). For this reason, this KER is applicable to the embryonic stage with a high level of confidence.  

Key Event Relationship Description

The Smoothened (SMO) receptor is Class F G protein coupled receptor involved in signal transduction of the Sonic Hedgehog (SHH) pathway. It includes distinct functional groups including ligand binding pockets, cysteine rich domain (CRD), transmembrane helix (TM), extracellular loop (ECL), intracellular loop (ICL), and a carboxyl-terminal tail (C-term tail) (Arensdorf, Marada et al. 2016).  SMO signaling is dependent upon its relocation to a subcellular location. This relocation occurs in the primary cilium (PC) in vertebrates (Huangfu and Anderson 2005). Relocation of SMO to the PC typically occurs within ~20 minutes of agonist stimulation (Arensdorf, Marada et al. 2016).

In the absence of SHH ligand, the Patched (PTCH) receptor suppresses the activation of SMO. When HH ligand binds to PTCH, suppression on SMO is released and SMO can relocate, accumulate, and signal to intracellular effectors (Denef, Neubüser et al. 2000, Rohatgi and Scott 2007). It has been shown that SMO localization to the tip of the primary cilia is essential for the SHH signaling cascade in vertebrates (Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Rohatgi, Milenkovic et al. 2009). This relocation then leads to signaling to effectors resulting in the activation of the GLI transcription factors and the subsequent induction of HH target gene expression (Alexandre, Jacinto et al. 1996, Von Ohlen and Hooper 1997). The exact mechanism through which PTCH and SMO interact is not known.

Evidence Supporting this KER

SMO signaling is dependent upon its relocation to a subcellular location. This relocation occurs in the primary cilium (PC) in vertebrates (Huangfu and Anderson 2005). It has been shown that SMO localization to the tip of the primary cilia is essential for the SHH signaling cascade in vertebrates (Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Rohatgi, Milenkovic et al. 2009)

• In vitro
  • NIH 3t3 (murine fibroblast) were used to study the effects of three SHH pathway antagonists, SANT 1, SANT2, and cyclopamine on SMO localization using fluorescent microscopy. Cells were treated with increasing concentrations of the antagonists in the presence of SHH ligand. SANT1 and SANT2 both blocked SMO localization in the cilia with IC50 values of 5 and 13nM respectively. Cyclopamine did not inhibit the accumulation of SMO in the cilia even when dosed at 5-10um (>10 fold above kd). All three antagonists inhibited SHH pathway transduction and target gene expression (Rohatgi, Milenkovic et al. 2009).  
  • A small molecule screen of 10,000 compounds identified six inhibitors of SHH signaling, four of which bind directly to SMO (SANT1-4). Screening was conducted using NIH 3T3 SHH LightII cells cultured in media conditioned from HEK 293 transfected to stably express Shh-N. Cells were dosed with the compound library at 0.714ug/ml and SHH activity was quantified at 30h using Renilla luciferase activity.  A fluorescent binding assay using BODIPY-cyclopamine was used to verify binding to SMO for the SANT compounds. Dose response reported as IC50 for the inhibition of SHH signaling was conducting in NIH 3T3 SHH light2, NIH 3T3 SmoA1-Light2, P2 Ptch1-/- (mouse embryonic fibroblasts) (Chen, Taipale et al. 2002).

• • Direct binding of cyclopamine to SMO was verified using a photoaffinity form of cyclopamine (PA-cyclopamine). PA-cyclopamine had previously been shown to inhibit SHH signaling in NIH 3T3 Shh-LightII cells with similar IC50 values to cyclopamine (300nm and 150nm respectively) (Taipale, Chen et al. 2000). Binding to SMO was verified using a COS-1 (fibroblast, monkey) line transfected to over express SMO. The location of cyclopamine binding was further investigated using BODIPY- cyclopamine and COS-1 cells modified to lack either a N-terminal, extracellular cysteine-rich domain, or the cytoplasmic C terminal of SMO. The findings support that cyclopamine does not require these domains and instead binds directly to the heptahelical domain  (Chen, Taipale et al. 2002).
  • To investigate whether SMO localization is regulated by SHH, a renal epithelial MDCK (Madin-Darby canine kidney) line was engineered to express Myc-tagged SMO. Following culture for 1hr in SHH conditioned media SMO presence in the primary cilium is upregulated while cells cultured in the presence of cyclopamine see a downregulation of SMO in the primary cilia (Corbit, Aanstad et al. 2005)
  • To determine whether PTCH1 regulates localization of SMO MEFs from PTCH1^-/- mice were used. These showed SHH activity and SMO localization in the primary cilium in the absence of SHH ligand or SAG. Reintroduction of PTCH1 via a retrovirus suppressed SHH activity and prevented SMO accumulation in primary cilia (Rohatgi and Scott 2007)
  • A high content assay to detect compounds that block SMO accumulation to the primary cilia in the presence of SHH was used to screen a library of ~5600 compounds. This screen identified 26 hits with DY131 and its analog GSK4716 further investigated as potent hits. These compounds inhibited SHH induced accumulation of SMO::EGFP with IC50s of 0.8um and 2um respectively. DY131 and GSK4716 both inhibited the activation of a Glireporter with IC50s of 2um and 10um respectively (Wang, Arvanites et al. 2012).  
• In vivo         
  • The presence of critical periods for disruption of SHH was investigated using C57BL/6J mice. Vismodegib was suspended at 3mg/ml in 0.5% methyl cellulose and 0.2% tween. Pregnant dams were administered 40mg/kg vismodegib at GD7.0, 7.25, 7.5, 7.75, 8.0, 8.25,8.5, 8.625, 8.75, 8.875, 9.0, 9.25, 9.5, 9.75, and 10.0. Cyclopamine was dosed at 120mg/kg/d via subcutaneous infusion between GD8.25-9.375. Pregnant dams were euthanized at GD17 and fetal specimens were collected and fixed for imaging. The control group consisted of fetuses exposed to 0.5% methyl cellulose and 0.2% tween at GD7.75, 8.875, or 9.5.  Acute exposure to vismodegib resulted in a peak incidence of lateral cleft lip and palate at GD8.875 (13%). Exposure at GD9.0 and 10.0 resulted in clefts of the secondary palate only (34%).  A higher penetrance (81%) was found for cyclopamine exposure (Heyne, Melberg et al. 2015).
  • Two-week-old mice were dosed with 40mg/kg vismodegib (GDC-0449) via ip injection twice a day for 3 consecutive days. Quantification of immunofluorescence and ciliary length showed that like SMO^fl/+ mice, ciliary M71/M72 OR was reduced while cilia lengths were not changed. To determine if SMO regulates ciliary localization an OMP-CRE mouse line was used. It was found that immunofluorescence of M71/M72 was reduced in both SMO^fl/+, SMO^fl/fl, as compared to SMO^+/+ control (Maurya, Bohm et al. 2017).
  • To explore how a conditional loss of primary cilia on neural crest cells Kif3a^f/f Wnt1-Cre mice were used to explore the molecular basis of aglossia. Aglossia was found to be due to a lack of mesoderm derived muscle precursor migration. RNA-seq was used on E11.5 embryos on the mandibular prominces of wildtype and knock mice. The key SHH readout, GLI1 was downregulated two-fold in mutants (Millington, Elliott et al. 2017).
  • Cyclopamine was found to inhibit SHH signaling in White leghorn neural plate explants. Explants were dissected from stage 9-10 embryo chicks and cultured in collagen gels. Tissues were cultured in Shh-N media from COS-1 cells. Cyclopamine was dissolved in ethanol and added to test tissues. Tissues were fixed at 24-29hr and processed for immunofluorescence. 120nm cyclopamine was found to repress SHH induction as determined by Pax7 repression and the blockage of floor plate and motor neuron induction (Incardona, Gaffield et al. 1998).
  • • Multiple ciliopathies associated with clefting in humans including Meckel-Gruber syndrome (OMIM 249000) and Ellis-van Creveld syndrome (OMIM 225500)(Brugmann, Cordero et al. 2010)

While we know that entry to the cilia is tightly controlled, the exact mechanism of SMO ciliary trafficking is not fully understood. The PC is separated from the plasma membrane by the ciliary pockets and the transition zone which function together to regulate the movement of lipids and proteins in and out of the organelle (Goetz, Ocbina et al. 2009, Rohatgi and Snell 2010). The SHH receptor PTCH contains a ciliary localization sequence in its’ carboxy tail. Localization of PTCH to the PC is essential for inhibition of SMO as deletion of the CLS in PTCH prevents PTCH localization as well as inhibition of SMO (Kim, Hsia et al. 2015) (53). SMO also contains a CLS, but only accumulates in the PC upon ligand binding (Corbit, Aanstad et al. 2005). The entry of SMO into the PC is thought to occur either laterally through the ciliary pockets or internally via recycling endosomes (Milenkovic, Scott et al. 2009). Once inside the PC, SMO can diffuse freely, however it will usually accumulate in specific locations depending upon its’ activation state. Inactive SMO will accumulate more at the base of the PC while active SMO will accumulate in the tip of the PC (Milenkovic, Weiss et al. 2015).

An endogenous ligand for SMO has not been discovered although evidence for one exists and that PTCH controls SMO by controlling its’ availability or accessibility. To support this, it has been shown that PTCH and SMO do not physically interact (Chen and Struhl 1998). PTCH acts catalytically with SMO with one PTCH receptor capable of controlling many (~50) SMO receptors (Taipale, Cooper et al. 2002). Since PTCH includes a sterol sensing domain and shares characteristics of ancient bacterial transporters, a model of PTCH functioning by pumping a sterol-like MSO regulator has been proposed (Mukhopadhyay and Rohatgi 2014).  SMO is constitutively active in the absence of PTCH suggesting that the elusive molecule is an agonist (Rohatgi and Scott 2007). Conversely, the discovery that oxysterols bind to the CRD binding domain acting as positive modulators suggest that the molecule could be an agonist with PTCH functioning to sequester away or limit cellular concentration (Corcoran and Scott 2006, Nachtergaele, Mydock et al. 2012)

The activity of SMO is controlled by ligand binding (Kobilka 2007). Two separate binding pockets, one in the groove of the extracellular CRD and the other in the helices of the TMD have been identified (Nachtergaele, Mydock et al. 2012, Rana, Carroll et al. 2013, Wang, Wu et al. 2013, Byrne, Sircar et al. 2016, Huang, Zheng et al. 2018). These two binding pockets have been shown to interact in an allosteric manner (Nachtergaele, Mydock et al. 2012). The binding pocket in the helices of the TMD binds several SMO agonists including SAG as well as antagonists Vismodegib and Sonidegib. The CRD binding pocket binds cholesterol and its’ oxidized derivates (Byrne, Luchetti et al. 2018). The antagonist cyclopamine binds to the TMD binding pocket and inhibits SHH signal transduction. However, in mSMO carrying the mutations D477G/E552K that disable the TMD binding pocket, cyclopamine binds to the CRD pocket and activates the pathway (Huang, Nedelcu et al. 2016). To date several oxysterols including 20(S)-hydroxylcholesterol, 22(S)-hydroxylcholesterol, 7-keto-25-hydroxylcholesterol and 7-keto-27-hydroxylcholesterol have been identified as activators of SMO (Dwyer, Sever et al. 2007, Nachtergaele, Mydock et al. 2012, Myers, Sever et al. 2013). A binding site for 24(S),25-epoxycholesterol has been identified in the TMD pocket using cryo-EM of SMO in complex with 24(S),25-epoxycholesterol (Qi, Liu et al. 2019).      

While it is well understood that cyclopamine is an antagonist of SMO, contradictory in vivo data was found regarding whether cyclopamine blocks SMO relocation to the primary cilia. Rohatgi et al used NIH 3T3s cell and found that cyclopamine did not inhibit the accumulation of SMO in the cilia even when dosed at 5-10um (>10 fold above kd). All three antagonists inhibited SHH pathway transduction and target gene expression (Rohatgi, Milenkovic et al. 2009).  Corbit et al used a renal epithelial MDCK (Madin-Darby canine kidney) line was engineered to express Myc-tagged SMO. Following culture for 1hr in SHH conditioned media SMO presence in the primary cilium is upregulated while cells cultured in the presence of cyclopamine see a downregulation of SMO in the primary cilia (Corbit, Aanstad et al. 2005). Further work is required to determine if SMO antagonism via cyclopamine results in decrease in SMO relocation.

Quantitative Understanding of the Linkage

Relocation of SMO to the PC typically occurs within ~20 minutes of agonist stimulation (Arensdorf, Marada et al. 2016). No data was found on how fast antagonism of SMO will stop its’ relocation to the primary cilia.

References

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Brugmann, S. A., D. R. Cordero and J. A. Helms (2010). "Craniofacial ciliopathies: A new classification for craniofacial disorders." Am J Med Genet A 152A(12): 2995-3006.

Byrne, E. F. X., G. Luchetti, R. Rohatgi and C. Siebold (2018). "Multiple ligand binding sites regulate the Hedgehog signal transducer Smoothened in vertebrates." Current Opinion in Cell Biology 51: 81-88.

Byrne, E. F. X., R. Sircar, P. S. Miller, G. Hedger, G. Luchetti, S. Nachtergaele, M. D. Tully, L. Mydock-McGrane, D. F. Covey, R. P. Rambo, M. S. P. Sansom, S. Newstead, R. Rohatgi and C. Siebold (2016). "Structural basis of Smoothened regulation by its extracellular domains." Nature 535(7613): 517-522.

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Relationship: 2735: Decrease, SMO relocation leads to Decrease, GLI1/2 translocation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The relationship between a decrease in translocation of SMO and a decrease in GLI1/2 translocation to the nucleus has been shown repeatedly in mice models as detailed in the empirical evidence section. The relationship is biologically plausible in human, but to date no specific experiments have addressed this question. The SHH pathway is well understood to be fundamental to proper embryonic development and that aberrant SHH signaling during embryonic development can cause birth defects including orofacial clefts (OFCs). For this reason, this KER is applicable to the embryonic stage with a high level of confidence. 

Key Event Relationship Description

The Smoothened (SMO) receptor is Class F G protein coupled receptor involved in signal transduction of the Sonic Hedgehog (SHH) pathway. It includes distinct functional groups including ligand binding pockets, cysteine rich domain (CRD), transmembrane helix (TM), extracellular loop (ECL), intracellular loop (ICL), and a carboxyl-terminal tail (C-term tail) (Arensdorf, Marada et al. 2016).  SMO signaling is dependent upon its relocation to a subcellular location. This relocation occurs in the primary cilium (PC) in vertebrates (Huangfu and Anderson 2005). Relocation of SMO to the PC typically occurs within ~20 minutes of agonist stimulation (Arensdorf, Marada et al. 2016).

The Glioma-associated onocogene (Gli) family of zinc finger transcription factors (Gli1, Gli2, Gli3) are the primarily downstream effectors of the Hedgehog (HH) signaling cascade. When HH ligand binds to Patched (PTCH), its’ inhibition on SMO is relieved. SMO this then able to accumulate to the tip of primary cilium in its’ active form (Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Kim, Kato et al. 2009). SMO causes the GLI family to become dislodged from their complex with the negative regulator of HH signaling, Suppressor of Fused (Sufu) (Kogerman, Grimm et al. 1999, Pearse, Collier et al. 1999, Stone, Murone et al. 1999, Tukachinsky, Lopez et al. 2010). The GLI-Sufu complex maintains retention of Gli in the cytosol allowing for exposure to phosphorylation via protein kinase A (PKA) which inhibits downstream signal transduction  (Tuson, He et al. 2011). When SMO is activated, the GLI2/3-Sufu complex is dismantled allowing for retrograde transport of GLI back into the nucleus (Kim, Kato et al. 2009). Multiple ciliopathies are associated with clefting in humans including Meckel-Gruber syndrome (OMIM 249000) and Ellis-van Creveld syndrome (OMIM 225500)(Brugmann, Cordero et al. 2010).

The GLI family is found in both a long activator form (GliA) or a proteolytically cleaved repressor form (GliR). Current understanding is that Gli3 functions primarily as a repressor while Gli1 and Gli2 function mainly as activators of the pathway and that recruitment of SMO to the cilium leads to an increase in the ratio of GliA:GliR (Hui and Angers 2011, Liu 2016). Downstream transcription is primarily activated by Gli2 and repressed by Gli3 (Wang, Fallon et al. 2000, Bai, Auerbach et al. 2002, Persson, Stamataki et al. 2002). Gli1 serves primarily as an activator of transcription and works through amplification of the activated state (Park, Bai et al. 2000).

Evidence Supporting this KER

SMO signaling is dependent upon its relocation to a subcellular location. This relocation occurs in the primary cilium (PC) in vertebrates (Huangfu and Anderson 2005). It has been shown that SMO localization to the tip of the primary cilia is essential for the SHH signaling cascade via the GLI transcription factors(Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Rohatgi, Milenkovic et al. 2009)

• In vitro
  • NIH 3T3 clones with stable HA-Gli2 expression were created and a line with low HA-Gli2 expression was selected for further study. The reporter activity was induced by ShhN and fully inhibited by cyclopamine. When stimulated with ShhN, antibody staining was used to verify that Gli2 accumulates at the tip of the primary cilia. Immunostaining was also used to find that Gli2 accumulated in the nucleus of cells treated with ShhN. Using nuclear extracts of unstimulated cells HA-Gli2R was predominantly localized in the nucleus while in stimulated cells HA-Gli2 increased and HA-Gli2 decreased. Cells treated with Shh agonist SAG also had SMO accumulation in the primary cilia and increased HA-Gli2A in the nucleus (Kim, Kato et al. 2009).
  • NIH 3T3 cells were used to study whether the oxysterols and/or cholesterol are required for SHH signaling. Cells were depleted of sterols via incubation with methyl-β-cyclodextrin (MCD). Fluorinated sterols were added back as soluble components and the cells were stimulated with Shh ligand. Assays were performed for recruitment of endogenous SMO to the primary cilia and for pathway activation using a transcriptional reporter assay. Sterol depletion blocked relocation of SMO to the cilia and SHH activation. Cholesterol and 25-fluorocholesterol both rescued sterol depleted cells and restored SHH pathway activation (Huang, Nedelcu et al. 2016).
  • MMS1 (human myeloma) cells were used to study whether activation of Gli1 is required for its’ translocation to the nucleus. Forskolin (FSK) which acts by blocking GLI1 access to PKA was added to culture for 24h at 10µm. The nuclear localization of GLI1 was significantly decreased in the Prescence of FSK (Blotta, Jakubikova et al. 2012).
• In vivo
  • To explore how a conditional loss of primary cilia on neural crest cells, Kif3a^f/f Wnt1-Cre mice were used to explore the molecular basis of aglossia (congenital absence of tongue). Aglossia was found to be due to a lack of mesoderm derived muscle precursor migration. RNA-seq was used on E11.5 embryos on the mandibular prominces of wildtype and knock mice. The key SHH readout, GLI1 was downregulated two-fold in mutants (Millington, Elliott et al. 2017).
  • To test whether vertebrate SUFU, in situ hybridization was used to analyze SUFU expression in mouse embryos at 8.5-15.5 days post-coitum. The expression was found to partially overlap with expression of PTCH and GLI1-3 supporting a role of SUFU in SHH signaling. Radioactive in situ hybridization was used to analyze expression of SUFUH and PTCH1 in a 12-week-old human embryo. SUFUH expression was to be preferentially expressed in cells that receive SHH signaling (Kogerman, Grimm et al. 1999)
  • Multiple ciliopathies associated with clefting in humans including Meckel-Gruber syndrome (OMIM 249000) and Ellis-van Creveld syndrome (OMIM 225500)(Brugmann, Cordero et al. 2010).

 While we know that entry to the cilia is tightly controlled, the exact mechanism of SMO ciliary trafficking is not fully understood. The PC is separated from the plasma membrane by the ciliary pockets and the transition zone which function together to regulate the movement of lipids and proteins in and out of the organelle (Goetz, Ocbina et al. 2009, Rohatgi and Snell 2010). The SHH receptor PTCH contains a ciliary localization sequence in its’ carboxy tail. Localization of PTCH to the PC is essential for inhibition of SMO as deletion of the CLS in PTCH prevents PTCH localization as well as inhibition of SMO (Kim, Hsia et al. 2015) (53). SMO also contains a CLS, but only accumulates in the PC upon ligand binding (Corbit, Aanstad et al. 2005). The entry of SMO into the PC is thought to occur either laterally through the ciliary pockets or internally via recycling endosomes (Milenkovic, Scott et al. 2009). Once inside the PC, SMO can diffuse freely, however it will usually accumulate in specific locations depending upon its’ activation state. Inactive SMO will accumulate more at the base of the PC while active SMO will accumulate in the tip of the PC (Milenkovic, Weiss et al. 2015).

Quantitative Understanding of the Linkage

The data presented in support of this KER includes both in vitro and in vivo studies. The in vitro work offers data that SMO relocates to the tip of the primary cilium and that this plays a role in the translocation of the GLI transcription factors to the nucleus. The in vivo work shows that loss of cilia in embryonic mice leads to a significant decrease in GLI1 in the nucleus. SUFU expression was found to overlap with PTCH1 expression in both mice and human embryos supporting that SUFU plays a role in SHH signal transduction.  

Relocation of SMO to the PC typically occurs within ~20 minutes of agonist stimulation (Arensdorf, Marada et al. 2016). No data was found with regards to GLI1/2 translocation.  

References

Arensdorf, A. M., S. Marada and S. K. Ogden (2016). "Smoothened Regulation: A Tale of Two Signals." Trends Pharmacol Sci 37(1): 62-72.

Bai, C. B., W. Auerbach, J. S. Lee, D. Stephen and A. L. Joyner (2002). "Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway." Development 129(20): 4753-4761.

Blotta, S., J. Jakubikova, T. Calimeri, A. M. Roccaro, N. Amodio, A. K. Azab, U. Foresta, C. S. Mitsiades, M. Rossi, K. Todoerti, S. Molica, F. Morabito, A. Neri, P. Tagliaferri, P. Tassone, K. C. Anderson and N. C. Munshi (2012). "Canonical and noncanonical Hedgehog pathway in the pathogenesis of multiple myeloma." Blood 120(25): 5002-5013.

Brugmann, S. A., D. R. Cordero and J. A. Helms (2010). "Craniofacial ciliopathies: A new classification for craniofacial disorders." Am J Med Genet A 152A(12): 2995-3006.

Corbit, K. C., P. Aanstad, V. Singla, A. R. Norman, D. Y. R. Stainier and J. F. Reiter (2005). "Vertebrate Smoothened functions at the primary cilium." Nature 437(7061): 1018-1021.

Goetz, S. C., P. J. Ocbina and K. V. Anderson (2009). "The primary cilium as a Hedgehog signal transduction machine." Methods Cell Biol 94: 199-222.

Huang, P., D. Nedelcu, M. Watanabe, C. Jao, Y. Kim, J. Liu and A. Salic (2016). "Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling." Cell 166(5): 1176-1187.e1114.

Huangfu, D. and K. V. Anderson (2005). "Cilia and Hedgehog responsiveness in the mouse." Proc Natl Acad Sci U S A 102(32): 11325-11330.

Hui, C. C. and S. Angers (2011). "Gli proteins in development and disease." Annu Rev Cell Dev Biol 27: 513-537.

Kim, J., E. Y. Hsia, A. Brigui, A. Plessis, P. A. Beachy and X. Zheng (2015). "The role of ciliary trafficking in Hedgehog receptor signaling." Sci Signal 8(379): ra55.

Kim, J., M. Kato and P. A. Beachy (2009). "Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus." Proc Natl Acad Sci U S A 106(51): 21666-21671.

Kogerman, P., T. Grimm, L. Kogerman, D. Krause, A. B. Undén, B. Sandstedt, R. Toftgård and P. G. Zaphiropoulos (1999). "Mammalian suppressor-of-fused modulates nuclear-cytoplasmic shuttling of Gli-1." Nat Cell Biol 1(5): 312-319.

Liu, K. J. (2016). "Craniofacial Ciliopathies and the Interpretation of Hedgehog Signal Transduction." PLoS Genet 12(12): e1006460.

Milenkovic, L., M. P. Scott and R. Rohatgi (2009). "Lateral transport of Smoothened from the plasma membrane to the membrane of the cilium." J Cell Biol 187(3): 365-374.

Milenkovic, L., L. E. Weiss, J. Yoon, T. L. Roth, Y. S. Su, S. J. Sahl, M. P. Scott and W. E. Moerner (2015). "Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1." Proc Natl Acad Sci U S A 112(27): 8320-8325.

Millington, G., K. H. Elliott, Y. T. Chang, C. F. Chang, A. Dlugosz and S. A. Brugmann (2017). "Cilia-dependent GLI processing in neural crest cells is required for tongue development." Dev Biol 424(2): 124-137.

Park, H. L., C. Bai, K. A. Platt, M. P. Matise, A. Beeghly, C. C. Hui, M. Nakashima and A. L. Joyner (2000). "Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation." Development 127(8): 1593-1605.

Pearse, R. V., 2nd, L. S. Collier, M. P. Scott and C. J. Tabin (1999). "Vertebrate homologs of Drosophila suppressor of fused interact with the gli family of transcriptional regulators." Dev Biol 212(2): 323-336.

Persson, M., D. Stamataki, P. te Welscher, E. Andersson, J. Böse, U. Rüther, J. Ericson and J. Briscoe (2002). "Dorsal-ventral patterning of the spinal cord requires Gli3 transcriptional repressor activity." Genes Dev 16(22): 2865-2878.

Rohatgi, R., L. Milenkovic, R. B. Corcoran and M. P. Scott (2009). "Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process." Proc Natl Acad Sci U S A 106(9): 3196-3201.

Rohatgi, R., L. Milenkovic and M. P. Scott (2007). "Patched1 regulates hedgehog signaling at the primary cilium." Science 317(5836): 372-376.

Rohatgi, R. and W. J. Snell (2010). "The ciliary membrane." Curr Opin Cell Biol 22(4): 541-546.

Stone, D. M., M. Murone, S. Luoh, W. Ye, M. P. Armanini, A. Gurney, H. Phillips, J. Brush, A. Goddard, F. J. de Sauvage and A. Rosenthal (1999). "Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli." J Cell Sci 112 ( Pt 23): 4437-4448.

Tukachinsky, H., L. V. Lopez and A. Salic (2010). "A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu-Gli protein complexes." J Cell Biol 191(2): 415-428.

Tuson, M., M. He and K. V. Anderson (2011). "Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube." Development 138(22): 4921-4930.

Wang, B., J. F. Fallon and P. A. Beachy (2000). "Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb." Cell 100(4): 423-434.

Relationship: 2721: Decrease, GLI1/2 translocation leads to Decrease, GLI1/2 target gene expression

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

All presented evidence for the relationship is performed in mice. The relationship is biologically plausible in human, but to date no specific experiments have addressed this question. The SHH pathway is well understood to be fundamental to proper embryonic development and that aberrant SHH signaling during embryonic development can cause birth defects including orofacial clefts (OFCs). For this reason, this KER is applicable to the embryonic stage with a high level of confidence.  

Key Event Relationship Description

The Glioma-associated onocogene (Gli) family of zinc finger transcription factors (Gli1, Gli2, Gli3) are the primarily downstream effectors of the Hedgehog (HH) signaling cascade. When HH ligand binds to Patched (PTCH), its’ inhibition on SMO is relieved. SMO this then able to accumulate to the tip of primary cilium in its’ active form (Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Kim, Kato et al. 2009). SMO causes the GLI family to become dislodged from their complex with the negative regulator of HH signaling, Suppressor of Fused (Sufu) (Kogerman, Grimm et al. 1999, Pearse, Collier et al. 1999, Stone, Murone et al. 1999, Tukachinsky, Lopez et al. 2010). The GLI-Sufu complex maintains retention of Gli in the cytosol allowing for exposure to phosphorylation via protein kinase A (PKA) which inhibits downstream signal transduction  (Tuson, He et al. 2011). When SMO is activated, the GLI2/3-Sufu complex is dismantled allowing for retrograde transport of GLI back into the nucleus (Kim, Kato et al. 2009). This relocation then leads to signaling to effectors resulting in the activation of the GLI transcription factors and the subsequent induction of SHH target gene expression (Alexandre, Jacinto et al. 1996, Von Ohlen and Hooper 1997).

The GLI family is found in both a long activator form (GliA) or a proteolytically cleaved repressor form (GliR). Current understanding is that Gli3 functions primarily as a repressor while Gli1 and Gli2 function mainly as activators of the pathway and that recruitment of SMO to the cilium leads to an increase in the ratio of GliA:GliR (Hui and Angers 2011, Liu 2016). Downstream transcription is primarily activated by Gli2 and repressed by Gli3 (Wang, Fallon et al. 2000, Bai, Auerbach et al. 2002, Persson, Stamataki et al. 2002). Gli1 serves primarily as an activator of transcription and works through amplification of the activated state (Park, Bai et al. 2000).

Evidence Supporting this KER

The evidence presented for this KER is low.The relationship between GLI1/2 translocation and a decrease in GLI1/2 target gene expression relocation has been shown indirectly in multiple mouse models through disruption of SHH signaling at the level of SMO. From our understanding of the SHH pathway, we can infer that disruption of the SHH signaling pathway at the level of SMO is causing a decrease in GLI1/2 translocation and it is this that is causing the altered gene expression While clear evidence that disruption of SHH signaling leads to altered gene expression especially those of the Fox family, insufficient evidence exists for the direct relationship between GLI1/2 translocation and SHH target gene expression. The evidence also lacks direct human applicability as all presented work was performed in vitro on murine models or in vitro on murine cell lines.

SHH signaling is well established to be essential for proper embryonic development in vertebrates including mice and humans. Activation of the pathway results in a downstream signaling cascade resulting in the relocation of GLI to the nucleus and subsequent gene transcription (Carballo, Honorato et al. 2018).

• In vitro
  • A mouse cNCC line (09-1) with the expression signature (AP-2alpha (Tfap2a, Twist1, Sox9, Cd44) was used to study whether foxf2 is a target of SHH signalling. Addition of SHH ligand (0.4µg/ml) was found to upregulate both GLI1 and Foxf2. This upregulation was completely blocked by the addition of vismodegib (120nm)(Everson, Fink et al. 2017).
  • To determine if SHH pathway inhibition was downstream for GANT 61 and GANT 58, a Sufu-null MEF cell line was used. Treatment of cells with either GANT at 10µm led to a significant reduction of SHH target genes GLI1 and Hip1 as determined by qPCR. As expected, cyclopamine was unable to inhibit signalling in this system as activation occurs downstream of SMO. GANT 61 is believed to act through addition of the modification to GLI1 that compromises its’ ability to properly bind DNA (Lauth, Bergström et al. 2007).
  • GLI activators bind to the GACCACCCA motif to promote transcription of  GLI1, PTCH1, PTCH2, HHIP1, MYCN, CCND1, CCND2, BCL2, CFLAR, FOXF1, FOXL1, PRDM1 (BLIMP1), JAG2, GREM1, and Follistatin (Katoh and Katoh 2009)
• In vivo
  • In situ hybridization was used to determine expression of GLI1 in C57BL/6J mice to better understanding temporal SHH signalling. At GD 9.0 no difference was found between control and embryos exposed to cyclopamine (120mg/kg/day). GLI1 was downregulated in the ventral frontonasal prominence (FNP) of clomipramine exposed embryos by GD 9.25. FNP tissue was micro dissected and cDNA microarray analysis was performed. 210 genes were found to be dysregulated including a significant enrichment to the forkhead box (Fox) family. RT-PCR confirmed significant down regulation of the SHH target genes GLI1 and PTCH1 as well as nine Fox members: Foxa2, Foxb2, Foxc1, Foxc2, Foxd1, Foxe1, Foxf1, Foxf2, Foxl1. Two members of the fox family, Foxm1 and Foxo1 were not found to differentially expressed in either the cDNA microarray or RT-PCR (Everson, Fink et al. 2017).
  • Using mutant Osr2-IresCre;Smo^c/c mice Foxf2 and Foxf1were found to be positively regulated by SHH-SMO signalling. Expression of Osr2 was found to be reduced by E13.5 in the mutants. Expression of Osr1, Pax9, Tbx22 were not found to be altered (Lan and Jiang 2009).
  • To study whether SHH signalling regulates the developmental fate of the ecto-mesenchyme via regulation of gene activity in the facial primoridia, Wnt1-Cre;Smo^n/c, (removal of SHH signalling) and Wnt1-Cre;R26SmoM2 (activation of SHH signalling). Positive regulation from SHH activity was found for Foxc2, Foxd1, Foxd2, Foxf1, and Foxf2. The Fox genes were found to be dissimilar in expression pattern with spatial activation even with uniform activation of the SHH pathway. Foxc2 and Foxd1 were found to be expressed ubiquitously in the MNA except at the midline, while Foxf1 is expressed at the lateral ends. Foxd2 and Foxf2 are both expressed along the mediolateral axis with Foxd2 having an increasing gradient from medial to lateral and Foxf2 having an opposing gradient (Jeong, Mao et al. 2004).

Quantitative Understanding of the Linkage

The quantitative understanding for this KER is low. Studies to investigate response-response relationship as well as time scale have not been conducted or were not found in the literature review. The empirical evidence presented establishes that disruption of SHH signaling results in the altered gene expression of SHH target genes.

Positive feedback loop of gene expression from GLI1 and negative feedback loop for PTCH1, PTCH2, HHIP1 (Katoh and Katoh 2009)

References

Alexandre, C., A. Jacinto and P. W. Ingham (1996). "Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins." Genes Dev 10(16): 2003-2013.

Bai, C. B., W. Auerbach, J. S. Lee, D. Stephen and A. L. Joyner (2002). "Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway." Development 129(20): 4753-4761.

Carballo, G. B., J. R. Honorato, G. P. F. de Lopes and T. C. L. d. S. e. Spohr (2018). "A highlight on Sonic hedgehog pathway." Cell Communication and Signaling 16(1): 11.

Corbit, K. C., P. Aanstad, V. Singla, A. R. Norman, D. Y. R. Stainier and J. F. Reiter (2005). "Vertebrate Smoothened functions at the primary cilium." Nature 437(7061): 1018-1021.

Everson, J. L., D. M. Fink, J. W. Yoon, E. J. Leslie, H. W. Kietzman, L. J. Ansen-Wilson, H. M. Chung, D. O. Walterhouse, M. L. Marazita and R. J. Lipinski (2017). "Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis." Development 144(11): 2082-2091.

Hui, C. C. and S. Angers (2011). "Gli proteins in development and disease." Annu Rev Cell Dev Biol 27: 513-537.

Jeong, J., J. Mao, T. Tenzen, A. H. Kottmann and A. P. McMahon (2004). "Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia." Genes Dev 18(8): 937-951.

Katoh, Y. and M. Katoh (2009). "Hedgehog target genes: mechanisms of carcinogenesis induced by aberrant hedgehog signaling activation." Curr Mol Med 9(7): 873-886.

Kim, J., M. Kato and P. A. Beachy (2009). "Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus." Proc Natl Acad Sci U S A 106(51): 21666-21671.

Kogerman, P., T. Grimm, L. Kogerman, D. Krause, A. B. Undén, B. Sandstedt, R. Toftgård and P. G. Zaphiropoulos (1999). "Mammalian suppressor-of-fused modulates nuclear-cytoplasmic shuttling of Gli-1." Nat Cell Biol 1(5): 312-319.

Lan, Y. and R. Jiang (2009). "Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth." Development 136(8): 1387-1396.

Lauth, M., A. Bergström, T. Shimokawa and R. Toftgård (2007). "Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists." Proc Natl Acad Sci U S A 104(20): 8455-8460.

Liu, K. J. (2016). "Craniofacial Ciliopathies and the Interpretation of Hedgehog Signal Transduction." PLoS Genet 12(12): e1006460.

Park, H. L., C. Bai, K. A. Platt, M. P. Matise, A. Beeghly, C. C. Hui, M. Nakashima and A. L. Joyner (2000). "Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation." Development 127(8): 1593-1605.

Pearse, R. V., 2nd, L. S. Collier, M. P. Scott and C. J. Tabin (1999). "Vertebrate homologs of Drosophila suppressor of fused interact with the gli family of transcriptional regulators." Dev Biol 212(2): 323-336.

Persson, M., D. Stamataki, P. te Welscher, E. Andersson, J. Böse, U. Rüther, J. Ericson and J. Briscoe (2002). "Dorsal-ventral patterning of the spinal cord requires Gli3 transcriptional repressor activity." Genes Dev 16(22): 2865-2878.

Rohatgi, R., L. Milenkovic and M. P. Scott (2007). "Patched1 regulates hedgehog signaling at the primary cilium." Science 317(5836): 372-376.

Stone, D. M., M. Murone, S. Luoh, W. Ye, M. P. Armanini, A. Gurney, H. Phillips, J. Brush, A. Goddard, F. J. de Sauvage and A. Rosenthal (1999). "Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli." J Cell Sci 112 ( Pt 23): 4437-4448.

Tukachinsky, H., L. V. Lopez and A. Salic (2010). "A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu-Gli protein complexes." J Cell Biol 191(2): 415-428.

Tuson, M., M. He and K. V. Anderson (2011). "Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube." Development 138(22): 4921-4930.

Von Ohlen, T. and J. E. Hooper (1997). "Hedgehog signaling regulates transcription through Gli/Ci binding sites in the wingless enhancer." Mech Dev 68(1-2): 149-156.

Wang, B., J. F. Fallon and P. A. Beachy (2000). "Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb." Cell 100(4): 423-434.

Relationship: 2731: Decrease, GLI1/2 target gene expression leads to Decrease, SHH second messenger production

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The relationship between a decrease in shh target gene expression and a decrease in secondary messenger production has been shown in mouse models. The relationship is biologically plausible in human, but to date no specific experiments have addressed this question. The SHH pathway is well understood to be fundamental to proper embryonic development and that aberrant SHH signaling during embryonic development can cause birth defects including orofacial clefts (OFCs). For this reason, this KER is applicable to the embryonic stage with a high level of confidence. 

Key Event Relationship Description

A network of reciprocal growth factor signaling between the epithelium and mesenchyme is required for proper growth and patterning of the early palatal shelves. This signaling is largely comprised of a network between bone morphogenic protein (BMP), Fibroblast growth factor (Fgf), and Sonic Hedgehog (SHH) (Zhang, Song et al. 2002, Rice, Spencer-Dene et al. 2004). Activation of the SHH pathway results in a downstream signaling cascade resulting in the relocation of GLI to the nucleus and subsequent gene transcription (Carballo, Honorato et al. 2018). This gene expression drives secondary messenger signaling for the pathway. Proper Msx1 activity in the mesenchyme is required for the expression of SHH in the overlying epithelium (Zhang, Song et al. 2002). Maintenance of SHH expression in the epithelium is believed to be dependent on Fgf10 expression in the mesenchyme and its’ signaling through Fgfr2b in the epithelium (Rice, Spencer-Dene et al. 2004).  

Evidence Supporting this KER

• • In Osr2-IresCre;Smo^c/c (SHH pathway inactive) mutant mice Fgf10 mRNA was found to be significantly reduced in the anterior palatal mesenchyme. The expression of Fgf10 correlated with a downregulation of PTCH1 (Lan and Jiang 2009).
  • To determine if SHH can induce Fgf10, SHH overexpressing cells were implanted in the anterior region of the wing bud of chick embryos. By 27 hours, the expression of Fgf10 had significantly increased and expanded from the anterior mesenchyme to the bifurcating wing bud (Ohuchi, Nakagawa et al. 1997).
  • To investigate whether MSX-1 is in the same pathway as Fgf10, MSX-1 expression was examined in Fgf10-/- mice and Fgf10 expression was examined in Msx-1-/- mice. No change in expression was found and it is concluded that MSX-1 is not a downstream target of Fgf10 (Alappat, Zhang et al. 2005).
  • SHH expression is reduced in the palatal epithelium of both Fgf10-/- and Fgfr2b -/- mutants. Exogenous Fgf10 induced SHH in WT palatal epithelium  (Rice, Spencer-Dene et al. 2004).
  • BMP2 and BMP4 is downregulated in the anterior palate of Osr2-IresCre;Smo^c/c (SHH pathway inactive) mutant mice (Lan and Jiang 2009).
  • Upregulation of mesenchymal BMP4 by SHH via Foxf1 or Foxl1 (Katoh and Katoh 2009).

The relationships and feedback/feedforward loops that exist between SHH and its’ secondary messengers primary Fgf10 and BMP4 is not well understood. Some evidence exists that expression of both Fgf10 and BMP4 correlates with that of SHH. The state of evidence is lacking and no dose response data was found.

Relationship: 2732: Decrease, SHH second messenger production leads to Decrease, Cell proliferation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The relationship between a decrease in SHH secondary messengers and a decrease in cellular proliferation translocation has been demonstrated in both mouse and chick models. The relationship is biologically plausible in human, but to date no specific experiments have addressed this question. The SHH pathway is well understood to be fundamental to proper embryonic development and that aberrant SHH signaling during embryonic development can cause birth defects including orofacial clefts (OFCs). For this reason, this KER is applicable to the embryonic stage with a high level of confidence. 

Key Event Relationship Description

SHH is a mitogen that regulates cell proliferation during development. SHH regulation of proliferation works at least in part through regulation of cyclin D1 (ccnd 1) and cyclin D2 (Ccnd 2) (Kenney and Rowitch 2000, Ishibashi and McMahon 2002, Lobjois, Benazeraf et al. 2004, Mill, Mo et al. 2005, Hu, Mo et al. 2006). The regulation of ccnd 1 and ccnd 2 by SHH is not fully understood but is believed to be in part by regulation via SHH signaling and its signaling to SHH secondary messengers, namely the fibroblast growth factor family. A network of reciprocal growth factor signaling between the epithelium and mesenchyme is required for proper growth and patterning of the early palatal shelves. This signaling is largely comprised of a network between bone morphogenic protein (BMP), Fibroblast growth factor (Fgf), and Sonic Hedgehog (SHH) (Zhang, Song et al. 2002, Rice, Spencer-Dene et al. 2004). Activation of the SHH pathway results in a downstream signaling cascade resulting in the relocation of GLI to the nucleus and subsequent gene transcription (Carballo, Honorato et al. 2018). This gene expression drives secondary messenger signaling for the pathway. Proper Msx1 activity in the mesenchyme is required for the expression of SHH in the overlying epithelium (Zhang, Song et al. 2002). Maintenance of SHH expression in the epithelium is believed to be dependent on Fgf10 expression in the mesenchyme and its’ signaling through Fgfr2b in the epithelium (Rice, Spencer-Dene et al. 2004). 

Evidence Supporting this KER

The SHH pathway is well known to be associated with cellular proliferation. There is a high biological probability that this proliferation results through regulation of SHH secondary messengers.

Empirical evidence

• In vitro
  • Mouse cerebellar granule cells exposed to cycloheximide and SHH did not promote upregulation of ccnd 1, ccnd 2, or ccn3 mRNA. This supports that there is a protein intermediate between the SHH pathway and regulation of the G1 cyclins(Kenney and Rowitch 2000).
• In vivo
  • In mouse palate explants application of SHH was found to induce proliferation in the palatal mesenchyme as measured by BrdU (Rice, Spencer-Dene et al. 2004).
  • In CD-1 WT and MSX-1-/-, SHH soaked beads were able to induce proliferation in palatal mesenchyme explants at 24hr but not after 8hr suggesting the induction of proliferation is through an indirect mechanism (Zhang, Song et al. 2002).
  • IHC staining for Ccnd-1 and Ccnd-2 in Osr2-IresCre Smo^c/c (SHH inactive) and control embryos was used to determine if expression patterns differed between the mesenchyme and epithelium in mutants. Expression for both Ccnd-1 and Ccnd-2 was found to be reduced in the mesenchyme for mutants. mRNA was found to be reduced for both Ccnd-1 and Ccnd-2 in the palatal mesenchyme (Lan and Jiang 2009).
  • In Osr2-IresCre;Smo^c/c (SHH pathway inactive) mutant mice Fgf10 mRNA was found to be significantly reduced in the anterior palatal mesenchyme. The expression of Fgf10 correlated with a downregulation of PTCH1 (Lan and Jiang 2009).
  • To determine if SHH can induce Fgf10, SHH overexpressing cells were implanted in the anterior region of the wing bud of chick embryos. By 27 hours, the expression of Fgf10 had significantly increased and expanded from the anterior mesenchyme to the bifurcating wing bud (Ohuchi, Nakagawa et al. 1997).
  • To investigate whether MSX-1 is in the same pathway as Fgf10, MSX-1 expression was examined in Fgf10-/- mice and Fgf10 expression was examined in Msx-1-/- mice. No change in expression was found and it is concluded that MSX-1 is not a downstream target of Fgf10 (Alappat, Zhang et al. 2005).
  • SHH expression is reduced in the palatal epithelium of both Fgf10-/- and Fgfr2b -/- mutants. Exogenous Fgf10 induced SHH in WT palatal epithelium  (Rice, Spencer-Dene et al. 2004).
  • Fgf8 activity was found to sustain ccnd 2 expression in the neural groove and that the attenuation of fgf signalling is necessary for the up regulation of ccnd 1. This was conducted using chick embryos and replacing a small piece of the rostral presomitic mesoderm with an Fgf8 soaked bead. To test the necessity of the Fgf pathway, SU5402 treatment was used (Lobjois, Benazeraf et al. 2004).
  • Cyclopamine treatment of stage 9-10 chick embryos in the neural tube and neural grove resulted in a strong down regulation of ccnd 1 transcripts as well as SHH target genes (e.g. Gli1). Toxicity was assessed using sox2 and effects due to non-specific toxicity were not found. Ccnd 2 expression was not affected by cyclopamine treatment. This suggests that the iniation of ccnd 1 in the neural groove is SHH dependent while ccnd 2 is not (Lobjois, Benazeraf et al. 2004).

 The regulation of proliferation by SHH has been shown but questions to the exact mechanism of regulation remain. Evidence exists that there is likely an intermediate between SHH and regulation of ccnd 1 and ccnd 2. Some evidence exists that the intermediate could be a member(s) of the Fgf family. The relationship between a decrease is SHH secondary messenger production and a decrease in cellular proliferation is plausible and data is shown that supports a decrease in ccnd 1 and 2 in correlation with the Fgf and SHH pathways.  Further studies are needed to further out understanding of the regulation of proliferation by shh.

References

Alappat, S. R., Z. Zhang, K. Suzuki, X. Zhang, H. Liu, R. Jiang, G. Yamada and Y. Chen (2005). "The cellular and molecular etiology of the cleft secondary palate in Fgf10 mutant mice." Dev Biol 277(1): 102-113.

Carballo, G. B., J. R. Honorato, G. P. F. de Lopes and T. C. L. d. S. e. Spohr (2018). "A highlight on Sonic hedgehog pathway." Cell Communication and Signaling 16(1): 11.

Hu, M. C., R. Mo, S. Bhella, C. W. Wilson, P. T. Chuang, C. C. Hui and N. D. Rosenblum (2006). "GLI3-dependent transcriptional repression of Gli1, Gli2 and kidney patterning genes disrupts renal morphogenesis." Development 133(3): 569-578.

Ishibashi, M. and A. P. McMahon (2002). "A sonic hedgehog-dependent signaling relay regulates growth of diencephalic and mesencephalic primordia in the early mouse embryo." Development 129(20): 4807-4819.

Kenney, A. M. and D. H. Rowitch (2000). "Sonic hedgehog promotes G(1) cyclin expression and sustained cell cycle progression in mammalian neuronal precursors." Mol Cell Biol 20(23): 9055-9067.

Lan, Y. and R. Jiang (2009). "Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth." Development 136(8): 1387-1396.

Lobjois, V., B. Benazeraf, N. Bertrand, F. Medevielle and F. Pituello (2004). "Specific regulation of cyclins D1 and D2 by FGF and Shh signaling coordinates cell cycle progression, patterning, and differentiation during early steps of spinal cord development." Dev Biol 273(2): 195-209.

Mill, P., R. Mo, M. C. Hu, L. Dagnino, N. D. Rosenblum and C. C. Hui (2005). "Shh controls epithelial proliferation via independent pathways that converge on N-Myc." Dev Cell 9(2): 293-303.

Ohuchi, H., T. Nakagawa, A. Yamamoto, A. Araga, T. Ohata, Y. Ishimaru, H. Yoshioka, T. Kuwana, T. Nohno, M. Yamasaki, N. Itoh and S. Noji (1997). "The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF8, an apical ectodermal factor." Development 124(11): 2235-2244.

Rice, R., B. Spencer-Dene, E. C. Connor, A. Gritli-Linde, A. P. McMahon, C. Dickson, I. Thesleff and D. P. Rice (2004). "Disruption of Fgf10/Fgfr2b-coordinated epithelial-mesenchymal interactions causes cleft palate." J Clin Invest 113(12): 1692-1700.

Zhang, Z., Y. Song, X. Zhao, X. Zhang, C. Fermin and Y. Chen (2002). "Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis." Development 129(17): 4135-4146.

Relationship: 2724: Decrease, Cell proliferation leads to Decrease, outgrowth

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The relationship between a decrease in cellular proliferation and a decrease in outgrowth has been demonstrated in both mouse and chick models. The relationship is biologically plausible in human, but to date no specific experiments have addressed this question. The SHH pathway is well understood to be fundamental to proper embryonic development and that aberrant SHH signaling during embryonic development can cause birth defects including orofacial clefts (OFCs). For this reason, this KER is applicable to the embryonic stage with a high level of confidence. 

Key Event Relationship Description

SHH is a mitogen that regulates cell proliferation during development. SHH regulation of proliferation works at least in part through regulation of cyclin D1 (ccnd 1) and cyclin D2 (Ccnd 2) (Kenney and Rowitch 2000, Ishibashi and McMahon 2002, Lobjois, Benazeraf et al. 2004, Mill, Mo et al. 2005, Hu, Mo et al. 2006). The regulation of ccnd 1 and ccnd 2 by SHH is not fully understood but is believed to be in part by regulation via SHH signaling and its signaling to SHH secondary messengers, namely the fibroblast growth factor family. A network of reciprocal growth factor signaling between the epithelium and mesenchyme is required for proper growth and patterning of the early palatal shelves.

The development of the face occurs early in embryogenesis and involves precise coordination of multiple tissues. The oropharyngeal membrane appears early in the 4^th week of gestation and gives rise to the frontonasal process and the 1^st pharyngeal arch. The frontonasal process is derived from the neural crest and in turn gives rise to two medial nasal process and two lateral nasal processes that later fuse and form the intermaxillary process. The pharyngeal arch is derived from mesoderm and the neural crest. It gives rise to two mandibular process and two maxillary processes (Som and Naidich 2013). These processes are comprised of mesenchymal cells from neural crest migration and the craniopharyngeal ectoderm and are coated in an epithelium (Ferguson 1988). The upper lip is formed during weeks 5-7 when the maxillary processes grow towards the midline and fuse intermaxillary process that have formed the philtrum and columella (Warbrick 1960, Kim, Park et al. 2004). The palate develops between week 6-12 from a median palatine process and a pair of lateral palatine processes. The primary palate is formed from the posterior extension of the intermaxillary process. The lateral palatine processes arise as medial mesenchymal processes from both maxillary processes. These processes initially grow inferiorly until the tongue is pulled downwards by the elongation of the maxilla and mandible. Once above the tongue, the lateral processes grow medially until they make contact and fuse (Som and Naidich 2014). For normal facial development and growth coordinated multivariate signaling is required. For example, retinoic acid, BMP, FGF, and SHH signal together to control facial growth (Liu, Rooker et al. 2010). SHH is an important modulator of epithelial-mesenchyme interaction (EMi) during development. SHH has been shown to regulate growth and formation of the palatal shelves prior to elevation and fusion (Rice, Connor et al. 2006). During development, SHH ligand is secreted by the epithelium into the underlying mesenchyme. This causes a gradient of signaling where mesenchyme proximal to the epithelium is exposed to higher concentrations of SHH than more distal cells (Cohen, Kicheva et al. 2015). Disruption of SHH during critical windows of development is believed to work in an EMi dependent, but epithelial-mesenchyme transition (Emt) independent manner. OFCs caused by disruption to SHH are believed to be due to a reduction in epithelial induced proliferation and the subsequent decrease in tissue outgrowth and the failure of the facial processes to meet and fuse (Lipinski, Song et al. 2010, Heyne, Melberg et al. 2015). 

Evidence Supporting this KER

The SHH pathway is well known to be associated with cellular proliferation and growth of the facial prominences. There is a high biological probability that disruption to proliferation of the facial prominences disrupts outgrowth.

• In vitro
  •  
• In vivo
  • To investigate how SHH might regulate early pharyngeal arch (PA1) development SHH-/- embryos were generated. At E9.5, the mutant embryos were thinner with hypoplasia on PA1. Morphometrics of PA1 of mutant vs. control showed a significant decrease in size in the mutant (P<0.05) for both the dorsal-ventral and the anteroposterior axis. Hypoplasia was quantified using a Pax3-Cre/R26R transgenic mouse line marked with LacZ and stained with X-gal (Yamagishi, Yamagishi et al. 2006).
  • SHH expressed in thickened palatal epithelium prior to palatal shelf outgrowth (E13.0-14.5) (Rice, Connor et al. 2006)
  • Using Wnt1-Cre;Smon/c embryos, a significant decrease in the growth of the mandibular arch in both the proximodistal and dorsoventral (D-V) axes. This supports that observation that the wild type, but not the mutants undergo rapid growth in the D-V axis around E11.5 (Jeong, Mao et al. 2004).
  • SHH is expressed in oral epithelium and shown as a key signal for palatal shelf outgrowth in explant culture (Lan and Jiang 2009)

 The regulation of proliferation by SHH has been shown but questions to the exact mechanism of regulation remain. Evidence exists that there is likely an intermediate between SHH and regulation of ccnd 1 and ccnd 2. Some evidence exists that the intermediate could be a member(s) of the Fgf family. The relationship between a decrease in cellular proliferation and a decrease in outgrowth is plausible and data is shown that supports that disruption of the SHH pathway leads to decrease in palatal outgrowth.  Further studies are needed to further out understanding of the regulation of proliferation by SHH and its subsequent impact on outgrowth of the facial prominences.

References

en, M., A. Kicheva, A. Ribeiro, R. Blassberg, K. M. Page, C. P. Barnes and J. Briscoe (2015). "Ptch1 and Gli regulate Shh signalling dynamics via multiple mechanisms." Nature Communications 6(1): 6709.

Ferguson, M. W. (1988). "Palate development." Development 103 Suppl: 41-60.

Heyne, G. W., C. G. Melberg, P. Doroodchi, K. F. Parins, H. W. Kietzman, J. L. Everson, L. J. Ansen-Wilson and R. J. Lipinski (2015). "Definition of critical periods for Hedgehog pathway antagonist-induced holoprosencephaly, cleft lip, and cleft palate." PLoS One 10(3): e0120517.

Hu, M. C., R. Mo, S. Bhella, C. W. Wilson, P. T. Chuang, C. C. Hui and N. D. Rosenblum (2006). "GLI3-dependent transcriptional repression of Gli1, Gli2 and kidney patterning genes disrupts renal morphogenesis." Development 133(3): 569-578.

Ishibashi, M. and A. P. McMahon (2002). "A sonic hedgehog-dependent signaling relay regulates growth of diencephalic and mesencephalic primordia in the early mouse embryo." Development 129(20): 4807-4819.

Jeong, J., J. Mao, T. Tenzen, A. H. Kottmann and A. P. McMahon (2004). "Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia." Genes Dev 18(8): 937-951.

Kenney, A. M. and D. H. Rowitch (2000). "Sonic hedgehog promotes G(1) cyclin expression and sustained cell cycle progression in mammalian neuronal precursors." Mol Cell Biol 20(23): 9055-9067.

Kim, C. H., H. W. Park, K. Kim and J. H. Yoon (2004). "Early development of the nose in human embryos: a stereomicroscopic and histologic analysis." Laryngoscope 114(10): 1791-1800.

Lan, Y. and R. Jiang (2009). "Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth." Development 136(8): 1387-1396.

Lipinski, R. J., C. Song, K. K. Sulik, J. L. Everson, J. J. Gipp, D. Yan, W. Bushman and I. J. Rowland (2010). "Cleft lip and palate results from Hedgehog signaling antagonism in the mouse: Phenotypic characterization and clinical implications." Birth Defects Res A Clin Mol Teratol 88(4): 232-240.

Liu, B., S. M. Rooker and J. A. Helms (2010). "Molecular control of facial morphology." Semin Cell Dev Biol 21(3): 309-313.

Lobjois, V., B. Benazeraf, N. Bertrand, F. Medevielle and F. Pituello (2004). "Specific regulation of cyclins D1 and D2 by FGF and Shh signaling coordinates cell cycle progression, patterning, and differentiation during early steps of spinal cord development." Dev Biol 273(2): 195-209.

Mill, P., R. Mo, M. C. Hu, L. Dagnino, N. D. Rosenblum and C. C. Hui (2005). "Shh controls epithelial proliferation via independent pathways that converge on N-Myc." Dev Cell 9(2): 293-303.

Rice, R., E. Connor and D. P. C. Rice (2006). "Expression patterns of Hedgehog signalling pathway members during mouse palate development." Gene Expression Patterns 6(2): 206-212.

Som, P. M. and T. P. Naidich (2013). "Illustrated review of the embryology and development of the facial region, part 1: Early face and lateral nasal cavities." AJNR Am J Neuroradiol 34(12): 2233-2240.

Som, P. M. and T. P. Naidich (2014). "Illustrated review of the embryology and development of the facial region, part 2: Late development of the fetal face and changes in the face from the newborn to adulthood." AJNR Am J Neuroradiol 35(1): 10-18.

Warbrick, J. G. (1960). "The early development of the nasal cavity and upper lip in the human embryo." J Anat 94(Pt 3): 351-362.

Yamagishi, C., H. Yamagishi, J. Maeda, T. Tsuchihashi, K. Ivey, T. Hu and D. Srivastava (2006). "Sonic Hedgehog Is Essential for First Pharyngeal Arch Development." Pediatric Research 59(3): 349-354.

Relationship: 2726: Decrease, outgrowth leads to OFC

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The relationship is biologically plausible in human, but to date no specific experiments have addressed this question. The SHH pathway is well understood to be fundamental to proper embryonic development and that aberrant SHH signaling during embryonic development can cause birth defects including orofacial clefts (OFCs). For this reason, this KER is applicable to the embryonic stage with a high level of confidence. 

Key Event Relationship Description

Orofacial clefts (OFCs) are one of the most common human birth defects and occur in approximately 1 in 700 live births (Mossey, Little et al. 2009, Dixon, Marazita et al. 2011). Formation of the upper lip and palate requires the orchestrated proliferation and fusion of embryonic facial growth centers and is dependent on paracrine intercellular signaling through multiple pathways. Genetic and chemical disruption of the Sonic Hedgehog (SHH), Transforming growth factor-beta (Tgf-β), bone morphogenic protein (BMP), epidermal growth factor (EGF) etc. pathways have been shown to cause OFCs (Jiang, Bush et al. 2006, Bush and Jiang 2012, Lan, Xu et al. 2015).  Early orofacial development involves epithelial ectoderm derived SHH ligand driving tissue outgrowth through an induced gradient of SHH dependent transcription in the underlying mesenchyme, which is thought to drive mesenchymal proliferation (Lan and Jiang 2009, Kurosaka 2015).

The development of the face occurs early in embryogenesis and involves precise coordination of multiple tissues. The oropharyngeal membrane appears early in the 4^th week of gestation and gives rise to the frontonasal process and the 1^st pharyngeal arch. The frontonasal process is derived from the neural crest and in turn gives rise to two medial nasal process and two lateral nasal processes that later fuse and form the intermaxillary process. The pharyngeal arch is derived from mesoderm and the neural crest. It gives rise to two mandibular process and two maxillary processes (Som and Naidich 2013). These processes are comprised of mesenchymal cells from neural crest migration and the craniopharyngeal ectoderm and are coated in an epithelium (Ferguson 1988). The upper lip is formed during weeks 5-7 when the maxillary processes grow towards the midline and fuse intermaxillary process that have formed the philtrum and columella (Warbrick 1960, Kim, Park et al. 2004). The palate develops between week 6-12 from a median palatine process and a pair of lateral palatine processes. The primary palate is formed from the posterior extension of the intermaxillary process. The lateral palatine processes arise as medial mesenchymal processes from both maxillary processes. These processes initially grow inferiorly until the tongue is pulled downwards by the elongation of the maxilla and mandible. Once above the tongue, the lateral processes grow medially until they make contact and fuse (Som and Naidich 2014). For normal facial development and growth coordinated multivariate signaling is required. For example, retinoic acid, BMP, FGF, and SHH signal together to control facial growth (Liu, Rooker et al. 2010). SHH is an important modulator of epithelial-mesenchyme interaction (EMi) during development. SHH has been shown to regulate growth and formation of the palatal shelves prior to elevation and fusion (Rice, Connor et al. 2006). During development, SHH ligand is secreted by the epithelium into the underlying mesenchyme. This causes a gradient of signaling where mesenchyme proximal to the epithelium is exposed to higher concentrations of SHH than more distal cells (Cohen, Kicheva et al. 2015). Disruption of SHH during critical windows of development is believed to work in an EMi dependent, but epithelial-mesenchyme transition (Emt) independent manner. OFCs caused by disruption to SHH are believed to be due to a reduction in epithelial induced proliferation and the subsequent decrease in tissue outgrowth and the failure of the facial processes to meet and fuse (Lipinski, Song et al. 2010, Heyne, Melberg et al. 2015).

Evidence Supporting this KER

The SHH pathway is well known to be associated with development of the face including the lip and palatal. Disruption of SHH at critical periods of development has been shown to cause OFCs.  

• In vitro
  •  
• In vivo
  • ~85% of K14-Cre;Shh^c/n mice had cleft palate with rudimentary palatal shelves spaced apart without contact suggesting that the cleft is due to insufficient outgrowth of the shelves (Rice, Spencer-Dene et al. 2004).
  • 100% (n=22) Osr2-IresCre;Smo^c/c had a cleft palate. At E14.5 the palatal shelves were underdeveloped and had not grown out to make contact compared to control littermates that had met and initiated fusion. This supports that disruption of SHH signalling impairs palatal shelf outgrowth and can lead to cleft palate (Lan and Jiang 2009)

The quantitative understanding of this relationship is low. No studies were found to exist to address dose response or time-scale data. Further work is needed to address these questions and create a better understanding of this relationship.

References

Bush, J. O. and R. Jiang (2012). "Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development." Development 139(2): 231-243.

Cohen, M., A. Kicheva, A. Ribeiro, R. Blassberg, K. M. Page, C. P. Barnes and J. Briscoe (2015). "Ptch1 and Gli regulate Shh signalling dynamics via multiple mechanisms." Nature Communications 6(1): 6709.

Dixon, M. J., M. L. Marazita, T. H. Beaty and J. C. Murray (2011). "Cleft lip and palate: understanding genetic and environmental influences." Nature Reviews Genetics 12(3): 167-178.

Ferguson, M. W. (1988). "Palate development." Development 103 Suppl: 41-60.

Heyne, G. W., C. G. Melberg, P. Doroodchi, K. F. Parins, H. W. Kietzman, J. L. Everson, L. J. Ansen-Wilson and R. J. Lipinski (2015). "Definition of critical periods for Hedgehog pathway antagonist-induced holoprosencephaly, cleft lip, and cleft palate." PLoS One 10(3): e0120517.

Jiang, R., J. O. Bush and A. C. Lidral (2006). "Development of the upper lip: morphogenetic and molecular mechanisms." Dev Dyn 235(5): 1152-1166.

Kim, C. H., H. W. Park, K. Kim and J. H. Yoon (2004). "Early development of the nose in human embryos: a stereomicroscopic and histologic analysis." Laryngoscope 114(10): 1791-1800.

Kurosaka, H. (2015). "The Roles of Hedgehog Signaling in Upper Lip Formation." Biomed Res Int 2015: 901041.

Lan, Y. and R. Jiang (2009). "Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth." Development 136(8): 1387-1396.

Lan, Y., J. Xu and R. Jiang (2015). "Cellular and Molecular Mechanisms of Palatogenesis." Curr Top Dev Biol 115: 59-84.

Lipinski, R. J., C. Song, K. K. Sulik, J. L. Everson, J. J. Gipp, D. Yan, W. Bushman and I. J. Rowland (2010). "Cleft lip and palate results from Hedgehog signaling antagonism in the mouse: Phenotypic characterization and clinical implications." Birth Defects Res A Clin Mol Teratol 88(4): 232-240.

Liu, B., S. M. Rooker and J. A. Helms (2010). "Molecular control of facial morphology." Semin Cell Dev Biol 21(3): 309-313.

Mossey, P. A., J. Little, R. G. Munger, M. J. Dixon and W. C. Shaw (2009). "Cleft lip and palate." The Lancet 374(9703): 1773-1785.

Rice, R., E. Connor and D. P. C. Rice (2006). "Expression patterns of Hedgehog signalling pathway members during mouse palate development." Gene Expression Patterns 6(2): 206-212.

Rice, R., B. Spencer-Dene, E. C. Connor, A. Gritli-Linde, A. P. McMahon, C. Dickson, I. Thesleff and D. P. Rice (2004). "Disruption of Fgf10/Fgfr2b-coordinated epithelial-mesenchymal interactions causes cleft palate." J Clin Invest 113(12): 1692-1700.

Som, P. M. and T. P. Naidich (2013). "Illustrated review of the embryology and development of the facial region, part 1: Early face and lateral nasal cavities." AJNR Am J Neuroradiol 34(12): 2233-2240.

Som, P. M. and T. P. Naidich (2014). "Illustrated review of the embryology and development of the facial region, part 2: Late development of the fetal face and changes in the face from the newborn to adulthood." AJNR Am J Neuroradiol 35(1): 10-18.

Warbrick, J. G. (1960). "The early development of the nasal cavity and upper lip in the human embryo." J Anat 94(Pt 3): 351-362.