The TGFβ type I receptor TGFβRI functions as an inhibitor of BMP signaling in cartilage.

The type I TGFβ receptor TGFβRI (encoded by Tgfbr1) was ablated in cartilage. The resulting Tgfbr1 Col2 mice exhibited lethal chondrodysplasia. Similar defects were not seen in mice lacking the type II TGFβ receptor or SMADs 2 and 3, the intracellular mediators of canonical TGFβ signaling. However, we detected elevated BMP activity in Tgfbr1 Col2 mice. As previous studies showed that TGFβRI can physically interact with ACVRL1, a type I BMP receptor, we generated cartilage-specific Acvrl1 (Acvrl1 Col2 ) and Acvrl1/Tgfbr1 (Acvrl1/Tgfbr1 Col2 ) knockouts. Loss of ACVRL1 alone had no effect, but Acvrl1/Tgfbr1 Col2 mice exhibited a striking reversal of the chondrodysplasia seen in Tgfbr1 Col2 mice. Loss of TGFβRI led to a redistribution of the type II receptor ACTRIIB into ACVRL1/ACTRIIB complexes, which have high affinity for BMP9. Although BMP9 is not produced in cartilage, we detected BMP9 in the growth plate, most likely derived from the circulation. These findings demonstrate that the major function of TGFβRI in cartilage is not to transduce TGFβ signaling, but rather to antagonize BMP signaling mediated by ACVRL1

Role of Hedgehog Signaling in Olfactory Epithelium Regeneration

The olfactory epithelium (OE) lines the nasal cavities and is responsible for smell (odorant) sensation. Specialized neurons called olfactory sensory neurons (OSNs) bind to odorant molecules and transduce the smell signal to brain circuits for olfactory perception. Due to the constant contact of the OE with the air, which contains environmental stressors and pathogens, the cells of the OE must be constantly replenished. This makes the OE one of the few sites of adult neurogenesis. Two presumed stem cell populations can proliferate and differentiate into OE cells – the mitotically active globose basal cells (GBCs) and relatively quiescent horizontal basal cells (HBCs). The signaling pathways that coordinate stem cell mediated turnover, homeostasis, and regeneration of the OE remain understudied. The Hedgehog (HH) signaling pathway is an attractive candidate for study in the context of OE regeneration. Previous work from our lab demonstrated that HBCs have functional primary cilia that are necessary for HBC-mediated regeneration of the OE. Importantly, primary cilia are also necessary for proper HH signaling and processing of downstream HH transcription factors called GLIs. Therefore, we hypothesized that GLI transcription factors could be functioning in the OE. In this dissertation I investigate 1) the expression of GLI transcription factors in the OE, 2) the consequence of driving GLI activator in HBCs, and 3) the consequence of depletion of GLIs in HBCs on OE regeneration. Furthermore, I describe a novel role for HH transcription factors GLI2 and GLI3 in OE regeneration. Specifically, I demonstrate that driving overactive GLI2 in HBCs results in aberrant cell identity and loss of OSN lineages following injury. Additionally, I show that loss of both GLI2 and GLI3 in HBCs results in defects in OE regeneration following injury. My data suggest that proper levels of both GLI2 and GLI3 in HBCs are necessary for the OE to be able to recover from acute injury. My work can be a springboard for future studies in HH signaling in the OE and further expand our understanding of this unique, neuroregenerative epithelium.PHDCell and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/175675/1/shiraza_1.pd

Smad2 and Smad3 Regulate Chondrocyte Proliferation and Differentiation in the Growth Plate.

TGFβs act through canonical and non-canonical pathways, and canonical signals are transduced via Smad2 and Smad3. However, the contribution of canonical vs. non-canonical pathways in cartilage is unknown because the role of Smad2 in chondrogenesis has not been investigated in vivo. Therefore, we analyzed mice in which Smad2 is deleted in cartilage (Smad2CKO), global Smad3-/- mutants, and crosses of these strains. Growth plates at birth from all mutant strains exhibited expanded columnar and hypertrophic zones, linked to increased proliferation in resting chondrocytes. Defects were more severe in Smad2CKO and Smad2CKO;Smad3-/- (Smad2/3) mutant mice than in Smad3-/- mice, demonstrating that Smad2 plays a role in chondrogenesis. Increased levels of Ihh RNA, a key regulator of chondrocyte proliferation and differentiation, were seen in prehypertrophic chondrocytes in the three mutant strains at birth. In accordance, TGFβ treatment decreased Ihh RNA levels in primary chondrocytes from control (Smad2fx/fx) mice, but inhibition was impaired in cells from mutants. Consistent with the skeletal phenotype, the impact on TGFβ-mediated inhibition of Ihh RNA expression was more severe in Smad2CKO than in Smad3-/- cells. Putative Smad2/3 binding elements (SBEs) were identified in the proximal Ihh promoter. Mutagenesis demonstrated a role for three of them. ChIP analysis suggested that Smad2 and Smad3 have different affinities for these SBEs, and that the repressors SnoN and Ski were differentially recruited by Smad2 and Smad3, respectively. Furthermore, nuclear localization of the repressor Hdac4 was decreased in growth plates of Smad2CKO and double mutant mice. TGFβ induced association of Hdac4 with Smad2, but not with Smad3, on the Ihh promoter. Overall, these studies revealed that Smad2 plays an essential role in the development of the growth plate, that both Smads 2 and 3 inhibit Ihh expression in the neonatal growth plate, and suggested they accomplish this by binding to distinct SBEs, mediating assembly of distinct repressive complexes

Smad2 and Smad3 Regulate Chondrocyte Proliferation and Differentiation in the Growth Plate.

TGFβs act through canonical and non-canonical pathways, and canonical signals are transduced via Smad2 and Smad3. However, the contribution of canonical vs. non-canonical pathways in cartilage is unknown because the role of Smad2 in chondrogenesis has not been investigated in vivo. Therefore, we analyzed mice in which Smad2 is deleted in cartilage (Smad2CKO), global Smad3-/- mutants, and crosses of these strains. Growth plates at birth from all mutant strains exhibited expanded columnar and hypertrophic zones, linked to increased proliferation in resting chondrocytes. Defects were more severe in Smad2CKO and Smad2CKO;Smad3-/- (Smad2/3) mutant mice than in Smad3-/- mice, demonstrating that Smad2 plays a role in chondrogenesis. Increased levels of Ihh RNA, a key regulator of chondrocyte proliferation and differentiation, were seen in prehypertrophic chondrocytes in the three mutant strains at birth. In accordance, TGFβ treatment decreased Ihh RNA levels in primary chondrocytes from control (Smad2fx/fx) mice, but inhibition was impaired in cells from mutants. Consistent with the skeletal phenotype, the impact on TGFβ-mediated inhibition of Ihh RNA expression was more severe in Smad2CKO than in Smad3-/- cells. Putative Smad2/3 binding elements (SBEs) were identified in the proximal Ihh promoter. Mutagenesis demonstrated a role for three of them. ChIP analysis suggested that Smad2 and Smad3 have different affinities for these SBEs, and that the repressors SnoN and Ski were differentially recruited by Smad2 and Smad3, respectively. Furthermore, nuclear localization of the repressor Hdac4 was decreased in growth plates of Smad2CKO and double mutant mice. TGFβ induced association of Hdac4 with Smad2, but not with Smad3, on the Ihh promoter. Overall, these studies revealed that Smad2 plays an essential role in the development of the growth plate, that both Smads 2 and 3 inhibit Ihh expression in the neonatal growth plate, and suggested they accomplish this by binding to distinct SBEs, mediating assembly of distinct repressive complexes

The TGFβ type I receptor TGFβRI functions as an inhibitor of BMP signaling in cartilage.

The type I TGFβ receptor TGFβRI (encoded by Tgfbr1) was ablated in cartilage. The resulting Tgfbr1 Col2 mice exhibited lethal chondrodysplasia. Similar defects were not seen in mice lacking the type II TGFβ receptor or SMADs 2 and 3, the intracellular mediators of canonical TGFβ signaling. However, we detected elevated BMP activity in Tgfbr1 Col2 mice. As previous studies showed that TGFβRI can physically interact with ACVRL1, a type I BMP receptor, we generated cartilage-specific Acvrl1 (Acvrl1 Col2 ) and Acvrl1/Tgfbr1 (Acvrl1/Tgfbr1 Col2 ) knockouts. Loss of ACVRL1 alone had no effect, but Acvrl1/Tgfbr1 Col2 mice exhibited a striking reversal of the chondrodysplasia seen in Tgfbr1 Col2 mice. Loss of TGFβRI led to a redistribution of the type II receptor ACTRIIB into ACVRL1/ACTRIIB complexes, which have high affinity for BMP9. Although BMP9 is not produced in cartilage, we detected BMP9 in the growth plate, most likely derived from the circulation. These findings demonstrate that the major function of TGFβRI in cartilage is not to transduce TGFβ signaling, but rather to antagonize BMP signaling mediated by ACVRL1

Hdac4, SnoN, and Ski protein distribution in the neonatal growth plate and binding to S1-3 in the <i>Ihh</i> promoter.

<p><b>(A)</b> Immunohistochemical staining of Hdac4 in E18.5 proximal tibias. Bottom panel shows magnifications of the boxed regions in top panel, representing the lower columnar and prehypertrophic zones. <b>(B)</b> ChIP analysis of Hdac4 binding to SBE 1–3 in ATDC5 cells. <b>(C and D)</b> Immunohistochemical staining of SnoN and Ski in E18.5 proximal tibias. Asterisks in (C and D) indicated SnoN or Ski present in columnar zone cells. <b>(E)</b> ChIP analysis of SnoN and Ski binding to SBE 1–3 in ATDC5 cells. Cells in (B and E) were matured to prehypertrophy and subsequently treated with TGFβ1 (5 ng/ml) or non-treated (Control) for 24 hrs. Asterisk in (B and E), p = or < 0.05. All experiments were performed in triplicate and repeated twice. <i>Smad2</i>^<i>CKO</i> = <i>Smad2</i>^<i>fx/fx</i><i>;Col2a1Cre</i>.</p

Increased <i>Ihh</i> mRNA and protein levels and activity in <i>Smad2</i> and <i>Smad3</i> mutant growth plates.

<p>All images are P0 proximal tibias <b>(A)</b> RNA <i>in situ</i> hybridization for <i>Ihh</i> showing increased expression in <i>Smad2</i>^<i>CKO</i>, <i>Smad3</i>^<i>-/-</i>, and double mutant growth plates. <b>(B)</b> Immunostaining for Ihh protein showing diffusion into the resting zones <i>of Smad2</i>^<i>CKO</i>, <i>Smad3</i>^<i>-/-</i>, and double mutant growth plates. <b>(C)</b> Magnifications of the boxed regions in (B). <b>(D)</b> Immunostaining for Ptch1, showing increased protein levels throughout the growth plate in mutants, particularly in the resting zone. <b>(E)</b> Immunostaining for phospho-Smad1/5/8 (PS158) protein showing no significant difference in levels of activated BMP signaling comparing <i>Smad2</i>^<i>CKO</i>, <i>Smad3</i>^<i>-/-</i>, or double mutant to control <i>Smad2</i>^<i>fx/fx</i> growth plates. <i>Smad2</i>^<i>CKO</i> = <i>Smad2</i>^<i>fx/fx</i><i>;Col2a1Cre</i>.</p

Smad2 and Smad3 Regulate Chondrocyte Proliferation and Differentiation in the Growth Plate

<div><p>TGFβs act through canonical and non-canonical pathways, and canonical signals are transduced via Smad2 and Smad3. However, the contribution of canonical vs. non-canonical pathways in cartilage is unknown because the role of Smad2 in chondrogenesis has not been investigated <i>in vivo</i>. Therefore, we analyzed mice in which Smad2 is deleted in cartilage (<i>Smad2</i>^<i>CKO</i>), global <i>Smad3</i>^<i>-/-</i> mutants, and crosses of these strains. Growth plates at birth from all mutant strains exhibited expanded columnar and hypertrophic zones, linked to increased proliferation in resting chondrocytes. Defects were more severe in <i>Smad2</i>^<i>CKO</i> and <i>Smad2</i>^<i>CKO</i><i>;Smad3</i>^-/- <i>(Smad2/3)</i> mutant mice than in <i>Smad3</i>^<i>-/-</i> mice, demonstrating that Smad2 plays a role in chondrogenesis. Increased levels of <i>Ihh</i> RNA, a key regulator of chondrocyte proliferation and differentiation, were seen in prehypertrophic chondrocytes in the three mutant strains at birth. In accordance, TGFβ treatment decreased <i>Ihh</i> RNA levels in primary chondrocytes from control (<i>Smad2</i>^<i>fx/fx</i>) mice, but inhibition was impaired in cells from mutants. Consistent with the skeletal phenotype, the impact on TGFβ-mediated inhibition of <i>Ihh</i> RNA expression was more severe in <i>Smad2</i>^<i>CKO</i> than in <i>Smad3</i>^<i>-/-</i> cells. Putative Smad2/3 binding elements (SBEs) were identified in the proximal <i>Ihh</i> promoter. Mutagenesis demonstrated a role for three of them. ChIP analysis suggested that Smad2 and Smad3 have different affinities for these SBEs, and that the repressors SnoN and Ski were differentially recruited by Smad2 and Smad3, respectively. Furthermore, nuclear localization of the repressor Hdac4 was decreased in growth plates of <i>Smad2</i>^<i>CKO</i> and double mutant mice. TGFβ induced association of Hdac4 with Smad2, but not with Smad3, on the <i>Ihh</i> promoter. Overall, these studies revealed that Smad2 plays an essential role in the development of the growth plate, that both Smads 2 and 3 inhibit <i>Ihh</i> expression in the neonatal growth plate, and suggested they accomplish this by binding to distinct SBEs, mediating assembly of distinct repressive complexes.</p></div

Elevated proliferation in <i>Smad2</i>^<i>CKO</i> and <i>Smad3</i>^<i>-/-</i> growth plates.

<p>All images are P0 proximal tibias. <b>(A)</b> Sections stained with safranin O. Heights of distinct growth plate zones are indicated by double arrows and demarcated by dashed lines. White dashed line is the demarcation between the columnar and prehypertrophic zones. All images are aligned to this boundary. Blue dashed lines demarcate the approximate boundary between the resting and columnar zones. Green dashed lines demarcate the boundary between the hypertrophic zone and zone of ossification. Blue double-headed arrows indicate the extent of the proliferative zone in the control <i>Smad2</i>^<i>fx/fx</i> growth plate. Green double-headed arrows demarcate the extent of the control <i>Smad2</i>^<i>fx/fx</i> hypertrophic zone. The double-headed arrows are superimposed on the images of <i>Smad2</i>^<i>CKO</i>, <i>Smad3</i>^<i>-/-</i> and <i>Smad2</i>^<i>CKO</i><i>;Smad3</i>^<i>-/-</i> mutants to clarify the differences in lengths of these zones compared with control <i>Smad2</i>^<i>fx/fx</i>. 3 litters containing mice of all of the genotypes shown in the figures were examined. At least 5 mice per genotype were examined. The images shown are from littermates. <b>(B)</b> PCNA immunostaining. All mice are littermates. <b>(C)</b> Quantitation of the percentage of PCNA-positive cells in distinct zones. The percentage of PCNA-positive cells in the resting zone (blue bars) and columnar zone (red bars) were quantified. Data are expressed as percent PCNA-positive + SE (n = 5). Asterisk, p < 0.05 compared with control <i>Smad2</i>^<i>fx/fx</i>. <b>(D)</b> Quantitation of the percentage of TUNEL-positive cells in chondro-osseous junctions. Data are expressed as percent TUNEL-positive + SE (n = 3). There are no significant differences between <i>Smad2</i>^<i>CKO</i>, <i>Smad3</i>^<i>-/-</i> and <i>Smad2</i>^<i>CKO</i><i>;Smad3</i>^<i>-/-</i> and control <i>Smad2</i>^<i>fx/fx</i> mice. <i>S2</i>^<i>fx/fx</i> = <i>Smad2</i>^<i>fx/fx</i>. <i>S2</i>^<i>CKO</i> = <i>Smad2</i>^<i>fx/fx</i><i>;Col2a1Cre</i>. <i>S3</i>^<i>-/-</i> = <i>Smad3</i>^<i>-/-</i>. <i>S2</i>^<i>CKO</i>;<i>S3</i>^<i>-/-</i> = <i>Smad2</i>^<i>fx/fx</i><i>;Col2a1Cre;Smad3</i>^<i>-/-</i>.</p

Smads 2 and 3 regulate <i>Ihh</i> levels in chondrocytes.

<p><b>(A)</b><i>Ihh</i> mRNA levels in primary rib chondrocytes isolated from P0 mice of the indicated genotypes. Chondrocytes were maintained in chondrogenic medium (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006352#sec011" target="_blank">Material and Methods</a>) to mature them to prehypertrophy. At this stage, they were treated with TGFβ1 (5 ng/ml) or TGFβ2 (5 ng/ml) for 24 hrs. <i>Ihh</i> RNA levels were assessed by quantitative real time PCR. Values were normalized using <i>Gapdh</i> and are plotted relative to control (<i>Smad2</i>^<i>fx/fx</i>) + SE. <b>(B)</b> 742 bp <i>Ihh</i>-Luc activity in ATDC5 chondrocytes matured to prehypertrophy. Cells were treated with siRNAs against <i>Smad2</i> and/or <i>Smad3</i>, and then treated with TGFβ1 (5 ng/ml) for 24 hrs. Scr, scrambled siRNA control. <b>(C)</b> P3TP-Luc activity in ATDC5 chondrocytes under conditions of <i>Smad2/Smad3</i> knockdown, verifying regulation of this control reporter by Smad2 and Smad3. All experiments were performed in triplicate and repeated twice. Asterisks, p < 0.05. <i>Smad2</i>^<i>CKO</i> = <i>Smad2</i>^<i>fx/fx</i><i>;Col2a1Cre</i>.</p