The Role of Arl13b and Non-Canonical Sonic Hedgehog Signaling in Joubert Syndrome Open Access

Mariani, Laura Elizabeth (2016)

Permanent URL:


The human ciliopathy Joubert Syndrome (JS) can be caused by mutations in ARL13B, a gene that codes for a cilia-associated protein regulating canonical, transcriptional Sonic hedgehog (Shh) signaling. While many symptoms and signs of JS can be easily explained by defects in cilia and/or canonical Shh signaling, the key diagnostic criterion of JS is of unknown molecular etiology. In order to receive a diagnosis of JS, patients must exhibit an anatomical malformation of the hindbrain white matter known as the molar tooth sign (MTS), which involves abnormal axon guidance in the projection neurons that give rise to the superior cerebellar peduncle (SCP). Indeed, JS patients show aberrant axon guidance in many brain regions. The mechanism through which cilia-associated proteins can affect axon guidance is unknown. However, in addition to its canonical functions, Shh signaling can act through a non-canonical pathway to regulate axon guidance in some neurons. This dissertation tests the hypothesis that mutations in ARL13B give rise to JS-associated anatomical abnormalities by misregulating Shh-dependent axon guidance. I first examine how Arl13b mutations affect Shh signaling in mouse embryonic fibroblasts. Previous work has shown that total loss of Arl13b significantly disrupts transcriptional Shh signaling. In this study, I show differential effects of JS-causing mutations and other mutations in Arl13b on cilia and Shh signaling. Using an assay of Shh-dependent fibroblast migration, I give the first evidence that shows Arl13b is involved in non-canonical Shh signaling. Surprisingly, I find that Arl13b need not be present in cilia in order to execute some of its functions, and that mutations disrupting cilia localization of Arl13b have counterintuitive effects on canonical (cilia-associated) and non-canonical (cilia-independent) Shh signaling. I next ask whether Arl13b is specifically involved in the development of axons in neurons known to use Shh as a guidance cue, and whether perturbing Arl13b or Shh signaling in hindbrain projection neurons can recapitulate the phenotypes seen in JS. Using mouse genetic models and cultured neurons, I show that a) loss of Arl13b in the mouse brain produces phenotypes consistent with JS, b) loss of either Arl13b or the Shh pathway protein Smoothened (Smo) in the neurons of the SCP produces defects in midline crossing, and c) loss of Arl13b affects axonal development in spinal commissural neurons, which use Shh as a guidance cue. In summation, this work indicates that Arl13b plays multiple roles in regulating both canonical and non-canonical Shh signaling and suggests a mechanism through which JS-causing mutations in ARL13B can affect the development of white matter tracts leading to the MTS.

Table of Contents

1 Introduction 1

1.1 Joubert Syndrome: A ciliopathy caused by ARL13B mutations 3

1.1.1 Genetics of JS 5

1.1.2 Shh-dependent features of JS 6

1.1.3 Unexplained features of JS 8

1.2 Cilia proteins and Sonic hedgehog signaling 9

1.2.1 Canonical Shh signaling pathway components 10

1.2.2 Molecular mechanisms of Shh signaling in cilia 13

1.2.3 Arl13b and Shh signaling 18

1.3 Non-canonical Shh signaling 19

1.3.1 Shh-dependent axon guidance 20

1.3.2 Shh-dependent fibroblast migration 21

1.4 Unanswered questions in JS etiology and Shh signaling 22

1.4.1 Hindbrain development 23

1.4.2 Non-ciliary role of ciliopathy proteins 25

1.5 Summary and objectives 27

2 Materials and Methods 28

2.1 Mouse embryonic fibroblast protocols and assays 28

2.1.1 Mouse lines and genotyping 28

2.1.2 Generating primary MEFs 28

2.1.3 Immortalizing MEFs 29

2.1.4 Site-directed mutagenesis 30

2.1.5 Generating lentivirus 31

2.1.6 Expression validation of Arl13 mutations 32

2.1.7 FACS purification of lentiviral MEF lines 33

2.1.8 Stereological analysis of fluorescent micrographs 34

2.1.9 Western blots 36

2.1.10 Quantitative real-time PCR 37

2.1.11 Migration assays 38

2.1.12 Statistical analysis 39

2.2 Axon guidance protocols and assays 40

2.2.1 Mouse lines and genotyping 40

2.2.2 In vivo tamoxifen-induced recombination 40

2.2.3 Mouse embryo histology 41

2.2.4 Mouse brain histology 41

2.2.5 Mouse ex vivo DTI 42

2.2.6 Arl13b knockdown in vitro 43

3 Role of Arl13b in Shh signaling in mouse embryonic fibroblasts 44

3.1 Introduction 44

3.2 Results 47

3.2.1 Mutations in Arl13b affects its cilia localization and have 47

dominant effects on cilia length

3.2.2 Arl13b mutations have dominant effects on transcriptional Shh 51

signaling Tamoxifen-induced recombination 52 AAV-Cre recombination 54

3.2.3 Arl13b mutations affect cilia in an Arl13b null background 54

3.2.4 Arl13b mutations affect Shh signaling in an Arl13bhnn background 57

3.2.5 Variability in the transcriptional response to Shh 58

3.2.6 Arl13b mutations affect Shh-dependent Smo enrichment in cilia 60

3.2.7 Arl13b mutations affect non-transcriptional Shh signaling in MEF 62 migration

3.3 Discussion 64

3.3.1 Dominant effects of Arl13b mutations 65

3.3.2 Variability in transcriptional Shh response may indicate differences 66

in negative feedback

3.3.3 Cilia localization of Arl13b is required for only a subset of its 69


3.3.4 Summary and Future Directions 71

4 Arl13b and Shh-dependent axon guidance: a model for Joubert Syndrome 72

4.1 Introduction 72

4.2 Results 73

4.2.1 Arl13b regulates axon guidance in neurons associated with JS 73

phenotypes Loss of Arl13b causes abnormal hindbrain development 73 Loss of Arl13b or Smo causes anatomical changes in the SCP as 76

measured by DTI

4.2.2 Arl13b regulates axon development in neurons that use Shh as a 80

guidance cue Arl13b regulates spinal commissural neuron axon 80

guidance in vivo Loss or knockdown of Arl13b potentiates spinal 80

commissural neuron axon outgrowth

4.3 Discussion 82

4.3.1 Abnormal white matter tracts in Arl13b and Smo mutants 82

4.3.2 Loss of Arl13b alters guidance and outgrowth in spinal 85

commissural neuron axons

4.3.3 Summary and future directions 86

5 Perspectives 88

5.1 Ciliary and non-ciliary Arl13b regulates Shh signaling through distinct 88


5.2 Arl13b and receptor traffic 90

5.3 Common factors regulating ciliogenesis, migration, and axon guidance 92

5.4 Defects in Shh-dependent neural development as a model for Joubert 95


References 98

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
Subfield / Discipline
  • English
Research Field
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files