Relationship of the ciliary GTPase, ARL13B, and the GPCR, SMOOTHENED Open Access

Abstract

In vertebrates, the neurons of the brain and spinal cord are organized and specified during embryonic development. Cell fate decisions are influenced by extracellular ligands interacting with receptive progenitor cells. The progenitors of the neural tube are highly responsive to Sonic Hedgehog (Shh) ligand. In each progenitor, the core components of the Shh pathway dynamically localize to the membrane of the primary cilium. Shh ligand binds to its receptor Patched1 (PTCH1), causing PTCH1 export from cilia and removing PTCH1 mediated suppression over the G-protein coupled receptor SMOOTHENED (SMO). Activated SMO promotes the formation of activating GLI transcription factors which induce Shh-dependent genes. The mechanism by which PTCH1 suppresses SMO and the endogenous substrate that activates SMO are unknown, but they are linked to cilia. The ciliary GTPase ARL13B regulates Shh signaling at multiple levels, influencing the production of activating GLI transcription factor and SMO trafficking to cilia. ARL13B function on SMO is elusive because it is masked by ARL13B’s downstream function. My central hypothesis is that ARL13B regulates the ciliary processes that activate SMO as its loss disrupts both SMO trafficking and Shh pathway activation.

           This dissertation describes my work on the role of ARL13B in regulating the ciliary processes that traffic and activate SMO. I show that SMO activation of high level Shh signaling is dependent on its enrichment in cilia. I show that ARL13B regulates Shh signaling and the ciliary organization of the pathway’s components from outside cilia, effectively uncoupling ARL13B’s function from its location. I demonstrate new models of cystic kidney disease, and obesity and diabetes caused by excluding ARL13B from the ciliary environment. I present genetic evidence consistent with ARL13B function upstream of SMO, regulating a still unknown ciliary process. The core of this dissertation demonstrates that ARL13B plays an essential role in the regulation of SMO and the Shh pathway, and that it can fulfill this role from outside the cilium. Together, my results fill fundamental gaps in our knowledge of SMO activation and cilia and show that ARL13B has tissue specific ciliary and non-ciliary roles that still need to be deciphered.

Table of Contents

Table of Contents

CHAPTER 1 CILIA-DEPENDENT HEDGEHOG SIGNALING AND ITS REGULATION BY CILIA................................................................... 1

1.1 Introduction of cilia structure and function........................................... 2

1.2 The role of Intraflagellar transport (IFT)................................................................... 4

1.3 Vertebrate Hedgehog signaling requires the cilium................................................................... 5

1.4 Vertebrate Hedgehog components dynamically localize to cilia................................................................... 9

1.4.1 Other ciliary regulators of the Hedgehog Pathway................................................................. 11

1.5 The ciliary membrane................................................................. 12

1.6 Cilia protein and GTPase ARL13B................................................................. 14

1.6.1 The ciliary and non-ciliary functions of ARL13B................................................................. 15

1.6.2 The evolution of ARL13B................................................................. 17

1.6.3 ARL13B is one of 35 cilia genes that cause Joubert Syndrome..................................................... 17

1.6.4 ARL13B regulates Sonic Hedgehog signaling and ciliary trafficking of Hedgehog components..................................................... 19

1.7 G-protein coupled receptor SMOOTHENED................................................................. 22

1.7.1 Smoothened has the structural hallmarks of a GPCR................................................................. 23

1.7.2 Exogenous ligands inform our understanding of Smoothened mechanisms................................................................. 25

1.7.3 Additional ciliary GPCRs................................................................. 27

1.8 GPCR localization to cilia is a dynamic and highly regulated process................................................................. 28

1.8.1 Cilia are a specialized environment for GPCR signaling cascades................................................................. 30

1.8.2 Ciliary trafficking of GPCRs................................................................. 32

1.8.3 Ciliary trafficking and retention of SMOOTHENED................................................................. 33

1.9 Models of endogenous SMOOTHENED activation................................................................. 35

1.10 The evolution of Hedgehog Signaling................................................................. 37

1.11 Summary and Dissertation Roadmap ................................................................. 39

 

CHAPTER 2 MATERIALS AND METHODS................................................................. 41

2.1 Mice................................................................. 42

2.1.1 Mapping and identification of the cabbie mutation................................................................. 42

2.1.2 Arl13b^V358A mouse allele generation and identification................................................................. 43

2.2 Phenotypic analysis of embryos................................................................. 44

2.2.1 Neural tube patterning................................................................. 44

2.2.2 Embedding and sectioning................................................................. 44

2.2.3 Antibody staining of neural tube cell fates................................................................. 45

2.3 Western blot of ARL13B protein in whole embryo lysates................................................................. 46

2.4 Derivation of mouse embryonic fibroblasts (MEFs)................................................................. 48

2.4.1 Isolating primary MEFs................................................................. 48

2.4.2 Immortalizing MEFs................................................................. 49

2.4.3 Working with MEFs................................................................. 49

2.4.4 Transfecting MEFs................................................................. 50

2.4.5 MEF antibody staining of cilia................................................................. 50

2.4.6 Ciliobrevin-D treatment................................................................. 51

2.4.7 Image quantification of MEF cilia................................................................. 51

2.4.8 RT-qPCR analysis of Shh transcriptional targets in MEFs................................................................. 53

2.5 Protein purification and ALR3 GEF assay of ARL13B................................................................. 54

2.6 Structural prediction of SMO................................................................. 55

2.7 Cell-based, cAMP-sensitive GloSensor assay for Smoothened activation................................................................. 56

2.7.1 Working with HEK-293FT cells................................................................. 56

2.7.2 The cAMP-sensitive GloSensor assay for SMO activation................................................................. 57

2.7.3 Plasmid combinations for the Myers assay................................................................. 58

2.8 Analysis of diabesity phenotypes in Arl13b^V358A/V358A mice................................................................. 59

2.8.1 Weight curve................................................................. 59

2.8.2 Glucose and insulin tolerance tests................................................................. 59

2.8.3 C-Fos staining of hypothalamic neurons in ARL13B^V358A mice................................................................. 60

2.9 Recipes................................................................. 62

2.10 Primers, PCR, and antibodies................................................................. 65

 

CHAPTER 3 Smoothened is required in cilia for the highest levels of Sonic Hedgehog response................................................................. 72

3.1 Summary................................................................. 73

3.2 Introduction................................................................. 74

3.3 Results................................................................. 78

3.3.1 cabbie is a novel Smoothened allele................................................................. 78

3.3.2 Smo^cbb causes craniofacial and skeletal defects................................................................. 80

3.3.3 Smo^cbb mutant embryos display abnormal neural tube patterning................................................................. 82

3.3.4 In vitro analysis of Shh-dependent transcriptional targets in cultured mutant fibroblasts................................................................. 84

3.3.5 N223K disrupts the Smo ligand-binding pocket and SAG binding to SMO................................................................. 86

3.3.6 Smoothened localization defect in Smo^cbb in vivo and in vitro after Shh and SAG activation................................................................. 88

3.4 Discussion................................................................. 91

 

CHAPTER 4 Arl13b is not required in cilia for normal Sonic Hedgehog..................................................................... signaling................................................................. 96

4.1 Summary................................................................. 97

4.2 Introduction................................................................. 97

4.3 Results............................................................... 102

4.3.1 ARL13B^V358A displays normal GEF activity............................................................... 102

4.3.2 CRISPR engineered Arl13b^V358A/V358A mice express cilia-excluded ARL13B protein................................................... 103

4.3.3 ARL13B^V358A protein is undetectable in cilia in mouse embryonic fibroblasts............................................................... 106

4.3.4 Arl13b^V358A/V358A mice are viable and fertile............................................................... 111

4.3.5 ARL13B^V358A permits normal embryonic development and Shh signaling............................................................... 113

4.3.6 ARL13B regulates ciliary enrichment of Shh components from outside cilia............................................................... 114

4.3.7 ARL13B regulates ciliary enrichment of ARL3 and INPP5E from within cilia............................................................... 116

4.3.8 Ciliary Arl13b is required for normal cell ciliation and ciliary length............................................................... 116

4.4 Discussion............................................................... 119

 

CHAPTER 5 AN ANALYSIS OF OBESITY AND CYSTIC KIDNEYS IN Arl13b^V358A/V358A AND Arl13b^V358A/hnn MICE............................................................... 124

5.1 Summary............................................................... 125

5.2 Introduction............................................................... 125

5.3.1 Cilia dysfunction is linked to the formation of kidney cysts and kidney disease............................................................... 126

5.2.2 Mutations to cilia genes can cause obesity............................................................... 128

5.3 Results............................................................... 129

5.3.1 Early characterization of cystic kidney phenotypes in Arl13b^V358A mutant mice................................................................ 129

5.3.2 Female and Male Arl13b^V358A mutant mice weigh significantly more than control mice................................................... 132

5.3.3 Arl13b^V358A carrying mice have impaired glucose tolerance............................................................... 136

5.3.4 Arl13b^V358A carrying mice have impaired insulin response............................................................... 138

5.3.5 Analysis of ARL13B expression in mouse embryos............................................................... 141

5.3.6 Using cFos expression to investigate hypothalamic regions’ differential response to insulin ................................................... 142

5.4 Discussion and Future Directions............................................................... 143

5.4.1 ARL13B has a cilia-specific function in kidney to prevent cyst formation............................................................... 144

5.4.2 Could cilia dysfunction in the periphery be contributing to obesity and diabetes in Arl13b^V358A mutant mice?............................................................... 145

5.4.3 Examining central control of satiety and feeding in Arl13b^V358A mutant mice............................................................... 146

 

CHAPTER 6 THE RELATIONSHIP OF ARL13B AND Smoothened............................................................... 149

6.1 Summary............................................................... 150

6.2 Introduction............................................................... 150

6.3 Results............................................................... 154

6.3.1 Arl13b^hnn Smo^cbb embryos are mor severely affected than either single mutant............................................................... 154

6.3.2 Cell fates requiring high levels of Shh are diminished in Arl13b^hnn Smo^cbb mutants............................................................... 157

6.3.3 SMO is absent from cilia in Arl13b^hnn Smo^cbb mutants and cells............................................................... 160

6.3.4 SMO^N223K receptor activation by SMO agonist (SAG) is impaired............................................................... 161

6.4 Discussion............................................................... 163

 

CHAPTER 7 PERSPECTIVES AND DISCUSSION............................................................... 167

7.1 Dissecting the ciliary processes that regulate SMO trafficking and SMO activation............................................................... 168

7.1.1 SMO ciliary exit is regulated by a common GPCR ubiquitination pathway............................................................... 168

7.1.2 SMO activation conformations are modulated by dynamic association with oxysterols............................................................... 171

7.1.3 ARL13B function in its essential regulation of SMO and Shh pathway............................................................... 174

7.1.4 Final thoughts on Smo^cbb............................................................... 176

7.2 Cilia-excluded ARL13B^V358A reveals cilia specific and tissue specific functions of ARL13B............................................................... 177

7.3 Final Conclusions............................................................... 178

8.0 References............................................................... 180

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Evolution and Development Biology, Neuroscience
Keyword	Cilia Arl13b Sonic Hedgehog Smoothened
Committee Chair / Thesis Advisor	Tamara Caspary, Emory University
Committee Members	Randy Hall, Emory University Richard Kahn, Emory University Peter Wenner, Emory University Victor Faundez, Emory University

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