scholarly journals Thm2 interacts with paralog, Thm1, and sensitizes to Hedgehog signaling in postnatal skeletogenesis

2020 ◽  
Author(s):  
Bailey A Allard ◽  
Wei Wang ◽  
Tana S Pottorf ◽  
Hammad Mumtaz ◽  
Luciane M Silva ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Genetic Interaction ◽  
Skeletal Development ◽  
Intraflagellar Transport ◽  
Genetic Syndromes ◽  
Mineral Density ◽  
Proliferation Zone ◽  
Trabecular Bone Mineral Density ◽  
Redundant Functions

AbstractCiliopathies are genetic syndromes that link osteochondrodysplasias to dysfunction of primary cilia. Primary cilia extend from the surface of bone and cartilage cells, to receive extracellular cues and mediate signaling pathways. Mutations in several genes that encode components of the intraflagellar transport-A ciliary protein complex have been identified in skeletal ciliopathies, including THM1. Here, we report a role for genetic interaction between Thm1 and its paralog, Thm2, in skeletogenesis. THM2 localizes to the ciliary axoneme, but unlike its paralog, Thm2 deficiency does not affect ciliogenesis and Thm2-null mice survive into adulthood. Since paralogs often have redundant functions, we crossed a Thm1 null (aln) allele into the Thm2 colony. After 5 generations of backcrossing the colony onto a C57BL6/J background, we observed that by postnatal day 14, Thm2-/-; Thm1aln/+ mice are smaller than control littermates. Thm2-/-; Thm1aln/+ mice exhibit shortened long bones, narrow ribcage, shortened cranium and mandibular defects. Mutant mice also show aberrant architecture of the tibial growth plate, with an expanded proliferation zone and diminished hypertrophic zone, indicating impaired chondrocyte differentiation. Using microcomputed tomography, Thm2-/-; Thm1aln/+ tibia were revealed to have reduced cortical and trabecular bone mineral density. Deletion of one allele of Gli2, a major transcriptional activator of the Hedgehog (Hh) pathway, exacerbated the small phenotype of Thm2-/-; Thm1aln/+ mice and caused small stature in Thm2-null mice. Together, these data reveal Thm2 as a novel locus that sensitizes to Hh signaling in skeletal development. Further, Thm2-/-; Thm1aln/+ mice present a new postnatal ciliopathy model of osteochondrodysplasia.

scholarly journals Genetic interaction of mammalian IFT-A paralogs regulates cilia disassembly, ciliary protein trafficking, Hedgehog signaling and embryogenesis

2019 ◽  
Author(s):  
Wei Wang ◽  
Bailey A. Allard ◽  
Tana S. Pottorf ◽  
Jay L. Vivian ◽  
Pamela V. Tran
Keyword(s):  
Protein Trafficking ◽  
Hedgehog Signaling ◽  
Double Mutant ◽  
Primary Cilia ◽  
Genetic Interaction ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Ciliary Protein ◽  
Transport Defect

AbstractPrimary cilia are sensory organelles that are essential for eukaryotic development and health. These antenna-like structures are synthesized by intraflagellar transport protein complexes, IFT-B and IFT-A, which mediate bi-directional protein trafficking along the ciliary axoneme. Here using mouse embryonic fibroblasts (MEF), we investigate the ciliary roles of two mammalian orthologues of Chlamydomonas IFT-A gene, IFT139, namely Thm1 (also known as Ttc21b) and Thm2 (Ttc21a). Thm1 loss causes perinatal lethality, and Thm2 loss allows survival into adulthood. At E14.5, the number of Thm1;Thm2 double mutant embryos is lower than that for a Mendelian ratio, indicating deletion of Thm1 and Thm2 causes mid-gestational lethality. We examined the ciliary phenotypes of mutant MEF. Thm1-mutant MEF show decreased cilia assembly, shortened primary cilia, a retrograde IFT defect for IFT and BBS proteins, and reduced ciliary entry of membrane-associated proteins. Thm1-mutant cilia also show a retrograde transport defect for the Hedgehog transducer, Smoothened, and an impaired response to Smoothened agonist, SAG. Thm2-null MEF show normal ciliary dynamics and Hedgehog signaling, but additional loss of a Thm1 allele impairs response to SAG. Further, Thm1;Thm2 double mutant MEF show enhanced cilia disassembly, and relative to Thm1-null MEF, increased impairment of IFT81 retrograde transport and of INPP5E ciliary import. Thus, Thm1 and Thm2 have unique and redundant roles in MEF. Thm1 regulates cilia assembly, and together with Thm2, cilia disassembly. Moreover, Thm1 alone and together with Thm2, regulates ciliary protein trafficking, Hedgehog signaling, and embryogenesis. These findings shed light on mechanisms underlying Thm1-, Thm2- or IFT-A-mediated ciliopathies.

scholarly journals IFT20 is critical for early chondrogenesis during endochondral ossification

2021 ◽  
Author(s):  
Hiroyuki Yamaguchi ◽  
Megumi Kitami ◽  
Karin H Uchima Koecklin ◽  
Li He ◽  
Jianbo Wang ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Endochondral Ossification ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Sox9 Expression ◽  
Skeletal Defects ◽  
The Family ◽  
Limb Outgrowth ◽  
Mutant Cells ◽  
Critical Process

Ciliogenic components, such as the family of intraflagellar transport (IFT) proteins, are recognized to play key roles in endochondral ossification, a critical process to form most bones. However, it remains unclear how each IFT protein performs its unique function to regulate endochondral ossification. Here, we show that intraflagellar transport 20 (IFT20) is required for early chondrogenesis. Utilizing three osteo-chondrocyte lineage-specific Cre mice (Prx1-Cre, Col2-Cre and Aggrecan-CreERT2), we deleted Ift20 to examine its function. While chondrocyte-specific Ift20 deletion with Col2-Cre or Aggrecan-CreERT2 drivers did not cause overt skeletal defects, mesoderm-specific Ift20 deletion using Prx1-Cre (Ift20:Prx1-Cre) resulted in shortened limb outgrowth. Although primary cilia were not formed in Ift20:Prx1-Cre mice, ciliary Hedgehog signaling was only moderately affected. Interestingly, loss of Ift20 lead to upregulation of Fgf18 expression resulting in ERK1/2 activation and sustained Sox9 expression, thus preventing endochondral ossification. Inhibition of enhanced phospho-ERK1/2 activation partially rescued defective chondrogenesis in Ift20 mutant cells, supporting an important role for FGF signaling. Our findings demonstrate a novel mechanism of IFT20 in early chondrogenesis during endochondral ossification.

scholarly journals Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling

2018 ◽  
Author(s):  
Shi-Rong Hong ◽  
Cuei-Ling Wang ◽  
Yao-Shen Huang ◽  
Yu-Chen Chang ◽  
Ya-Chu Chang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Living Cells ◽  
Comprehensive Understanding ◽  
Cellular Functions ◽  
Post Translational Modifications ◽  
Dynamic Distribution ◽  
Wide Range

AbstractTubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here we develop a method to rapidly deplete tubulin glutamlyation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.

scholarly journals DYRK2 is a ciliary kinase involved in vertebrate Hedgehog signal transduction

2020 ◽  
Author(s):  
Nicholas Morante ◽  
Monika Abedin Sigg ◽  
Luke Strauskulage ◽  
David R. Raleigh ◽  
Jeremy F. Reiter
Keyword(s):  
Signal Transduction ◽  
Transcription Factors ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Skeletal Development ◽  
Skeletal Defects ◽  
Gli Transcription Factors ◽  
Hedgehog Signal Transduction ◽  
Hedgehog Signal

ABSTRACTPrimary cilia are organelles specialized for signaling. We previously defined the proteomes of sea urchin and sea anemone cilia to identify ciliary proteins that predate the origin of bilateria. This evolutionary perspective on cilia identified DYRK2, a kinase not been previously implicated in ciliary biology. We found that DYRK2 localizes to cilia and that loss of DYRK2 disrupts ciliary morphology. We also found that DYRK2 participates in ciliary Hh signal transduction, communicating between SMO and GLI transcription factors. Mutation of mouse Dyrk2 resulted in skeletal defects reminiscent of those caused by loss of Indian hedgehog (Ihh). Like Dyrk2 mutations, pharmacological inhibition of DYRK2 dysregulates ciliary length control and attenuates Hedgehog signaling. Thus, DYRK2 is required for ciliary morphology, for Hedgehog signaling in vitro, and for skeletal development. We propose that DYRK2 is part of the mechanism that transduces SMO to activate GLI transcription factors within cilia.

scholarly journals Intraflagellar transport is deeply integrated in hedgehog signaling

2018 ◽  
Vol 29 (10) ◽  
pp. 1178-1189 ◽  
Author(s):  
Thibaut Eguether ◽  
Fabrice P. Cordelieres ◽  
Gregory J. Pazour
Keyword(s):  
Transcription Factors ◽  
Membrane Protein ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Hedgehog Pathway ◽  
Gli Transcription Factors ◽  
Cilia Formation ◽  
Mutant Cells

The vertebrate hedgehog pathway is organized in primary cilia, and hedgehog components relocate into or out of cilia during signaling. Defects in intraflagellar transport (IFT) typically disrupt ciliary assembly and attenuate hedgehog signaling. Determining whether IFT drives the movement of hedgehog components is difficult due to the requirement of IFT for building cilia. Unlike most IFT proteins, IFT27 is dispensable for cilia formation but affects hedgehog signaling similarly to other IFTs, allowing us to examine its role in the dynamics of signaling. Activating signaling at points along the pathway in Ift27 mutant cells showed that IFT is extensively involved in the pathway. Similar analysis of Bbs mutant cells showed that BBS proteins participate at many levels of signaling but are not needed to concentrate Gli transcription factors at the ciliary tip. Our analysis showed that smoothened delivery to cilia does not require IFT27, but the role of other IFTs is not known. Using a rapamycin-induced dimerization system to sequester IFT-B proteins at the mitochondria in cells with fully formed cilia did not affect the delivery of Smo to cilia, suggesting that this membrane protein may not require IFT-B for delivery.

scholarly journals Uncoupling Intraflagellar Transport and Primary Cilia Formation Demonstrates Deep Integration of IFT in Hedgehog Signaling

2017 ◽  
Author(s):  
Thibaut Eguether ◽  
Fabrice P Cordelieres ◽  
Gregory J Pazour
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Embryonic Fibroblasts ◽  
Fk506 Binding Protein ◽  
Gli Transcription Factors ◽  
Cilia Formation ◽  
Deep Integration ◽  
Mutant Cells

AbstractThe vertebrate hedgehog pathway is organized in primary cilia and hedgehog components relocate into or out of cilia during signaling. Defects in intraflagellar transport (IFT) typically disrupt ciliary assembly and attenuate hedgehog signaling. Determining if IFT drives the movement of hedgehog components is difficult due to the requirement of IFT for building cilia. Unlike most IFT proteins, IFT27 is dispensable for cilia formation but affects hedgehog signaling similar to other IFTs allowing us to examine its role in the dynamics of signaling. Activating signaling at points along the pathway inIft27mutant cells showed that IFT is extensively involved in the pathway. Similar analysis ofBbsmutant cells showed that BBS proteins participate at many levels of signaling but are not needed to concentrate Gli transcription factors at the ciliary tip. Our analysis showed that smoothened delivery to cilia does not require IFT27, but the role of other IFTs is not known. Using a rapamycin-induced dimerization system to stop IFT after ciliary assembly was complete we show that smoothened delivery to cilia is IFT independent.AbbreviationsMEFsmouse embryonic fibroblastsSAGsmoothen agonistIFTintraflagellar transportFKBPFK506 Binding Protein 12FRBFKBP12-rapamycin binding

Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development

Development ◽  
1997 ◽  
Vol 124 (1) ◽  
pp. 113-123 ◽  
Author(s):  
R. Mo ◽  
A.M. Freer ◽  
D.L. Zinyk ◽  
M.A. Crackower ◽  
J. Michaud ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Sonic Hedgehog ◽  
Limb Development ◽  
Hedgehog Signaling ◽  
Skeletal Development ◽  
Mutant Mice ◽  
Indian Hedgehog ◽  
Lateral Plate ◽  
Skeletal Defects ◽  
Redundant Functions

The correct patterning of vertebrate skeletal elements is controlled by inductive interactions. Two vertebrate hedgehog proteins, Sonic hedgehog and Indian hedgehog, have been implicated in skeletal development. During somite differentiation and limb development, Sonic hedgehog functions as an inductive signal from the notochord, floor plate and zone of polarizing activity. Later in skeletogenesis, Indian hedgehog functions as a regulator of chondrogenesis during endochondral ossification. The vertebrate Gli zinc finger proteins are putative transcription factors that respond to Hedgehog signaling. In Drosophila, the Gli homolog cubitus interruptus is required for the activation of hedgehog targets and also functions as a repressor of hedgehog expression. We show here that Gli2 mutant mice exhibit severe skeletal abnormalities including cleft palate, tooth defects, absence of vertebral body and intervertebral discs, and shortened limbs and sternum. Interestingly, Gli2 and Gli3 (C.-c. Hui and A. L. Joyner (1993). Nature Genet. 3, 241–246) mutant mice exhibit different subsets of skeletal defects indicating that they implement specific functions in the development of the neural crest, somite and lateral plate mesoderm derivatives. Although Gli2 and Gli3 are not functionally equivalent, double mutant analysis indicates that, in addition to their specific roles, they also serve redundant functions during skeletal development. The role of Gli2 and Gli3 in Hedgehog signaling during skeletal development is discussed.

scholarly journals Canonical and noncanonical intraflagellar transport regulates craniofacial skeletal development

2016 ◽  
Vol 113 (19) ◽  
pp. E2589-E2597 ◽  
Author(s):  
Kazuo Noda ◽  
Megumi Kitami ◽  
Kohei Kitami ◽  
Masaru Kaku ◽  
Yoshihiro Komatsu
Keyword(s):  
Neural Crest ◽  
Intracellular Transport ◽  
Primary Cilia ◽  
Type I Collagen ◽  
Skeletal Development ◽  
Growth Factor Receptor ◽  
Intraflagellar Transport ◽  
Type I ◽  
Multipotent Stem Cells ◽  
Facial Region

The primary cilium is a cellular organelle that coordinates signaling pathways critical for cell proliferation, differentiation, survival, and homeostasis. Intraflagellar transport (IFT) plays a pivotal role in assembling primary cilia. Disruption and/or dysfunction of IFT components can cause multiple diseases, including skeletal dysplasia. However, the mechanism by which IFT regulates skeletogenesis remains elusive. Here, we show that a neural crest-specific deletion of intraflagellar transport 20 (Ift20) in mice compromises ciliogenesis and intracellular transport of collagen, which leads to osteopenia in the facial region. Whereas platelet-derived growth factor receptor alpha (PDGFRα) was present on the surface of primary cilia in wild-type osteoblasts, disruption ofIft20down-regulated PDGFRα production, which caused suppression of PDGF-Akt signaling, resulting in decreased osteogenic proliferation and increased cell death. Although osteogenic differentiation in cranial neural crest (CNC)-derived cells occurred normally inIft20-mutant cells, the process of mineralization was severely attenuated due to delayed secretion of type I collagen. In control osteoblasts, procollagen was easily transported from the endoplasmic reticulum (ER) to the Golgi apparatus. By contrast, despite having similar levels of collagen type 1 alpha 1 (Col1a1) expression,Ift20mutants did not secrete procollagen because of dysfunctional ER-to-Golgi trafficking. These data suggest that in the multipotent stem cells of CNCs, IFT20 is indispensable for regulating not only ciliogenesis but also collagen intracellular trafficking. Our study introduces a unique perspective on the canonical and noncanonical functions of IFT20 in craniofacial skeletal development.

scholarly journals ERICH3 in Primary Cilia Regulates Cilium Formation and the Localisations of Ciliary Transport and Sonic Hedgehog Signaling Proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mona Alsolami ◽  
Stefanie Kuhns ◽  
Manal Alsulami ◽  
Oliver E. Blacque
Keyword(s):  
Sonic Hedgehog ◽  
Primary Cilium ◽  
Molecular Motors ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Signaling Proteins ◽  
Sonic Hedgehog Signaling ◽  
And Function

Abstract Intraflagellar transport (IFT) is essential for the formation and function of the microtubule-based primary cilium, which acts as a sensory and signalling device at the cell surface. Consisting of IFT-A/B and BBSome cargo adaptors that associate with molecular motors, IFT transports protein into (anterograde IFT) and out of (retrograde IFT) the cilium. In this study, we identify the mostly uncharacterised ERICH3 protein as a component of the mammalian primary cilium. Loss of ERICH3 causes abnormally short cilia and results in the accumulation of IFT-A/B proteins at the ciliary tip, together with reduced ciliary levels of retrograde transport regulators, ARL13B, INPP5E and BBS5. We also show that ERICH3 ciliary localisations require ARL13B and BBSome components. Finally, ERICH3 loss causes positive (Smoothened) and negative (GPR161) regulators of sonic hedgehog signaling (Shh) to accumulate at abnormally high levels in the cilia of pathway-stimulated cells. Together, these findings identify ERICH3 as a novel component of the primary cilium that regulates cilium length and the ciliary levels of Shh signaling molecules. We propose that ERICH3 functions within retrograde IFT-associated pathways to remove signaling proteins from cilia.

Sign in / Sign up

Export Citation Format

Share Document