New Insights into the Regulatory Network Controlling Neuroblast Migration in C. elegans

Neuron migration is a critical process during the development of the nervous system. Proper migration of neurons leads to proper function while incorrect migrations can lead to neurodevelopmental disorders such as epilepsy and mental retardation. Therefore it is worthwhile to study the genetic mechanisms that control migrating neuroblasts.The model organism Caenorhabditis elegans provides a strong model to study migrating neuroblasts. C. elegans have a complete nervous system containing 302 neurons and an extensive genetic toolkit to work out genetic mechanisms involved. The Q neuroblasts, consisting of QR and QL, are bilaterally symmetrical neuroblasts that are born near the posterior of the animal. QR is born on the right side of the animal and will migrate towards the anterior. QL is born on the left side of the animal and migrates towards the posterior. The migrations are highly stereotypical. Given the directional differences of QR and QL and highly stereotypical migrations the Q neuroblasts provide an excellent assay to study neuroblast migrations.The migration of the Q neuroblasts is divided into two control pathways, consisting of short-range initial migrations, followed by long-range migrations. The initial migrations are controlled via a Wnt-independent pathway. Both QR and QL are born between the hypodermal seam cells V4 and V5. Between 3 hours to 4.5 hours post hatching QR begins migrating anteriorly on top of V4. At the same time QL migrates posteriorly on top of V5. This process is regulated by UNC-40/DCC, MIG-21 and PTP-3/LAR to promote the correct directional migrations of QR and QL. There are other factors involved, including the Fat-like cadherins CDH-4 and CDH-3.After the initial migrations the long-range migrations of QR, QL and their descendants are controlled via canonical and non-canonical Wnt signaling. Following the initial migrations EGL-20/Wnt is secreted and activates the canonical Wnt pathway in QL leading to the expression of MAB-5/Hox, which is necessary to drive QL migration towards the posterior. QR does not activate the canonical Wnt pathway, resulting in no MAB-5/Hox expression driving QR migration towards the anterior. The remaining Wnt ligands cwn-1, cwn-2, lin-44, and mom-2 are expressed along the anterior-posterior axis on the animal and act through the non-canonical Wnt pathway in a redundant fashion to ensure proper migrations of the Q neuroblast descendants. Though this system has been extensively studied there is still much left to explore about the genetic mechanisms controlling migration of the Q neuroblasts.After a brief introduction to Q neuroblast migration in chapter I, chapter II dives into a newly described role of the putative RNA-binding protein ETR-1/CELF1 in Q neuroblast migration. The gene etr-1 encodes for an RNA-binding protein most similar to CELF1 in mammalian systems. CELF1 is part of the CELF-family of proteins, a large family of RNA-binding proteins that process messenger RNA. We show that ETR-1 is required for proper Q neuroblast migration, and acts in the muscle cells in a non-autonomous fashion. ETR-1 does not act in the initial migration Wnt-independent pathway but appears to be acting with Wnt signaling to control long-range migrations. We also describe a new method to detect deletions in the genome caused by CRISPR-Cas9 mediated genome editing.In chapter III we present possible targets of ETR-1/CELF1. The CELF family of proteins have roles in mRNA processing, including regulating translation, transcript stability and alternative splicing. Therefore we were interested in identifying targets of ETR-1 in C. elegans. We isolated muscle cells using fluorescence-activated-cell-sorting (FACS) and performed RNA-sequencing on isolated RNA. Using statistical software we were able to identify differential exon usage and differential expression of transcripts when comparing wild-type to etr-1 mutant muscle cells. We screened for AQR and PQR migration defects using RNAi to identify genes required for proper migration. Interestingly we identified unc-52/Perlecan to be required for AQR and PQR migration, which is a new phenotype. These data will help gain insight on how ETR-1 acts in Q neuroblast migration, as well as new factors.In chapter IV we detail a new role of the Caenorhabditis elegans tropomyosin homolog lev-11. Tropomyosin is a known target of CELF proteins and was identified lev-11 in our RNA sequencing study described above. We observed that RNAi knockdown of lev-11 resulted in AQR and PQR migration defects. Taken together these data lev-11 was a good candidate for further study. We show that lev-11 mutation results in an AQR under-migration phenotype. Preliminary data show that etr-1 suppresses the lev-11 under-migration phenotype. We also show that lev-11 may not be acting with either mig-14 or egl-20 in regulating AQR and PQR migration. These data can help describe a new role of lev-11 in C. elegans as well as a new role of tropomyosin.In chapter V we present a role of the Fat-like cadherin CDH-3 in Q neuroblast migration. cdh-3 was identified in an RNA sequencing study, identifying genes that were enriched in sorted Q neuroblasts. cdh-3 was considered a candidate because it was enriched in Q neuroblasts, and the other Fat-like cadherin in the genome, CDH-4, is involved in controlling Q neuroblast migration. We show that mutations in cdh-3 result Q neuroblast migration defects as well as AQR and PQR migration defects. Our data suggests that cdh-3 appears to be acting genetically with unc-40 and ptp-3 controlling the initial migrations of QR and QL.In summary the results presented in this dissertation provide new data about the regulatory network at play controlling the migrations of the Q neuroblasts and their descendants. We have described new roles of genes which furthers our understanding of the genetic mechanisms at play. These new data give insights into the complex genetic networks at play during development.

Grantor dc:publisher

University of Kansas

Year dc:date.issued

2020

Author dc:creator

• Ochs, Matthew Eric

Advisor dc:contributor.advisor

• Lundquist, Erik

• Molecular biology
• C. elegans
• Genetics
• Migration
• Neuroblast

Statement dc:rights

• Copyright held by the author.

Language dc:language.iso

en

Dc Identifier Other

http://dissertations.umi.com/ku:17343

OAI identifier oai:identifier

oai:kuscholarworks.ku.edu:1808/39238