The Ciliary Protein Arl13b Regulates Axon Guidance in the Developing Mouse Hindbrain 公开
Suciu, Sarah (Fall 2020)
Published
Abstract
This dissertation covers questions of how localization of a protein known to be associated within the privileged environment of the primary cilium affects signaling and signal transduction. The focus of my work is a regulatory GTPase called ARL13B, a protein that is well known to enrich within the primary cilium. ARL13B has well-established roles in Sonic Hedgehog (Shh) signaling, a pathway that is critical for development which requires the primary cilium to regulate cell fate and proliferation. In mouse, Arl13b-deletion results in Shh-dependent defects in cell fate and proliferation. Recessive point mutations in ARL13B result in the ciliopathy Joubert Syndrome (JS), which presents with developmental delays, intellectual disability, and hypotonia and is diagnosed by the hindbrain malformation the molar tooth sign. This hindbrain defect is caused by a small, underdeveloped cerebellum and midline crossing failure of the superior cerebellar peduncles (SCPs), a projection neuron-based white matter tract that projects from the cerebellum across the midline of the brain into the opposite hemisphere thalamus. Transcription-dependent Shh signaling regulates cerebellar proliferation, so the small cerebellum is explainable by ARL13B mutations resulting in reduced Shh signaling. However, the mechanism by which cilia-associated proteins such as ARL13B impact the regulation of axon guidance remains unclear.
A potential mechanism resides in the additional, distinct role of Shh signaling that still requires the obligate transducer SMO, however, is transcription-independent. Transcription-independent Shh signaling regulates both commissural axon guidance in the neural tube and fibroblast migration towards Shh. Since ARL13B mutations result in axon guidance defects in JS, I investigated whether dysregulated Shh signaling in linked to the failure of SCPs to cross the midline of the brain. Consistent with this, my data shows SCPs lacking SMO or ARL13B display axon guidance defects in the mouse brain. I examined SCP projections in two additional Arl13b alleles: a cilia-excluded variant, Arl13bV358A and a JS-causing mutation, Arl13bR79Q. Both ARL13B variants were sufficient for SCP guidance, indicating that ARL13B functions outside the cilium to regulate projection neuron guidance. To better understand the relationship pf Arl13b and transcription-independent Shh signaling I examined fibroblasts. Fibroblasts lacking SMO or ARL13B display decreased migration toward Shh. Interestingly, fibroblasts lacking cilia, or expressing a non-ciliary SMO mutant, show increased migration toward Shh. These results suggest that there may be a previously unknown, non-ciliary role for these cilia-associated proteins in the regulation of transcription-independent Shh signaling. Taken together, my data indicate that the mechanism of ARL13B action differs depending on whether signaling takes place within or outside of the primary cilium.
Table of Contents
Chapter 1 Arl13b, Cilia, and Shh Signaling__________________________________________1
1.1 Introduction_______________________________________________________________2
1.2 Overview of cilia___________________________________________________________5
1.2.1 Cilia structure and trafficking__________________________________________5
1.2.2 Cilia are required for neurodevelopment_________________________________10
1.2.3 Ciliary signaling takes place within the privileged environment of the primary cilium____________________________________________________________11
1.3 Overview of Shh__________________________________________________________12
1.3.1 The critical developmental pathway Shh signaling functions within as well as outside the cilium___________________________________________________12
1.3.2 The cilia-associated GTPase Arl13b regulates transcription-dependent and -independent Shh signaling___________________________________________19
1.4 Roles of cilia, Shh signaling, and Arl13b during development_____________________22
1.4.1 Neural cell fate specification requires signal transduction via the primary cilium____________________________________________________________22
1.4.2 Neural cell proliferation requires Shh signal transduction via the primary cilium____________________________________________________________26
1.4.3 Interneuron and neuron migration require the primary cilium________________29
1.4.4 Neuronal axon guidance requires cilia-associated proteins and signaling pathways_________________________________________________________31
1.5 Joubert Syndrome overview________________________________________________36
1.5.1 Joubert Syndrome characteristics and diagnosis___________________________36
1.5.2 Genetic complexity of Joubert Syndrome________________________________38
1.6 The evolving connection of cilia to neuronal mechanisms of human disease_________39
1.7 Dissertation roadmap______________________________________________________41
Chapter 2 Materials & Methods__________________________________________________43
2.1 Mice____________________________________________________________________44
2.2 Western blots_____________________________________________________________45
2.3 Fibroblast migration assays_________________________________________________46
2.4 Dissection and culture of embryonic spinal cord________________________________47
2.5 Diffusion tensor imaging (DTI)______________________________________________49
2.6 Tract tracing injections and analysis_________________________________________53
2.7 CLARITY and tissue clearing_______________________________________________56
2.8 Analysis of cerebellar hemisphere and vermis size______________________________57
Chapter 3 Measuring cell response to Shh as an attractive cue__________________________59
3.1 Abstract_________________________________________________________________60
3.2 Introduction______________________________________________________________60
3.3 Results__________________________________________________________________65
3.3.1 Lentiviral infection in MEFs drives expression of Arl13b constructs___________65
3.3.2 Fibroblast migration assays are inconsistent______________________________68
3.3.3 Commissural axon dissection and culture________________________________73
3.4 Discussion_______________________________________________________________74
Chapter 4 Visualizations of SCP guidance__________________________________________76
4.1 Abstract_________________________________________________________________77
4.2 Introduction______________________________________________________________77
4.3 Results__________________________________________________________________83
4.3.1 Deletion of Smo results in a larger Corpus callosum tract___________________83
4.3.2 Deletion of Arl13b or Smo results in lessened SCP FA at decussation_________85
4.3.3 Deletion of Smo results in a larger SCP tract_____________________________89
4.3.4 Anterograde injections indicate SCPs lacking Smo or Arl13b failed to project dorsally_____________________________________________________91
4.3.5 Decussation in SCPs lacking Smo or Arl13b is disorganized_________________92
4.3.6 Clarity imaging is a promising method to image SCPs in whole brain with appropriate injected tracer____________________________________________95
4.3.7 SCP retrograde decussation characterization______________________________96
4.3.8 SMO is required for normal SCP projection to the dorsal thalamus___________101
4.3.9 ARL13B is required for normal SCP projection to the dorsal thalamus________106
4.3.10 ARL13B does not function from within cilia to mediate SCP guidance_______106
4.3.11 SCP projection in mice expressing a Joubert-causing allele,
Arl13bR79Q_____________________________________________________110
4.4 Discussion______________________________________________________________112
Chapter 5 Perspective_________________________________________________________117
5.1 Introduction_____________________________________________________________118
5.2 Preliminary results indicate Arl13b plays a critical role in cerebellar development_____________________________________________________________119
5.2.1 Arl13b may function as a GTPase to regulate cerebellar vermis size__________119
5.2.2 Global cerebellar hypoplasia in mice lacking Ar13b in all neurons___________122
5.2.3 Arl13b functions from within cilia to regulate cerebellar vermis size__________124
5.2.4 Cilia are required to regulate cerebellar development______________________126
5.2.5 Summary________________________________________________________126
5.3 Perspective______________________________________________________________127
5.4 Connecting cilia to axons via shared microtubule-based structures and modifications___________________________________________130
5.5 Future Directions________________________________________________________135
References__________________________________________________________________137
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