Maintenance of neuronal differentiation during development
Lead Research Organisation:
Imperial College London
Department Name: Life Sciences
Abstract
Nervous system development is one of the most complex biological processes in nature. Understanding the molecular mechanism and the genes involved in the differentiation of stem cells into neurons is crucial in deciphering the development of the nervous system. A model widely used to study neuronal stem cell biology is Drosophila melanogaster, which will be adopted in this project, as it resembles aspects of the mammalian system (1). Thanks to the easy culturing techniques and vast genetic toolkit it offers fertile grounds to gain deeper understanding of the genetic regulators and molecular mechanisms behind neuronal development.
The transcription factor Lola is one of several proteins that maintain the differentiated state of neurons. In a study by Southall et al., lola mutants lead to the regression of neurons into a stem-cell like state and the development of tumours (2). These tumours develop due to the tightly regulated programming of the cells being perturbed. Therefore, it is crucial to identify the mechanism maintaining the cell fate and what other proteins are involved in this process. To investigate this, a yeast-two-hybrid screen was carried out to detect protein-protein interactions with Lola. Two of the high confidence hits from this screen were proteins CG7518 and Cap-G which will be the focus of this project (personal communication, Southall). In this study we will exploit the genetic toolkit available in Drosophila to induce overexpression and RNAi knockdown of said proteins and investigate phenotypes in neurons at larval and adult stages. Immunostaining techniques are used on larval and adult brains to image the proteins of interest using confocal microscopy. CRISPR/Cas9 system will be used to create a cell-type specific loss-of-function mutants for both our candidate interactors and observe potential phenotypes. The aim is to characterise CG7518 and its potential interactions with Lola, as its molecular function is still unknown. We hypothesise CG7518 to be a Lola regulator by inhibiting its function, either by retaining it in the cytoplasm, modifying its structure or targeting it for degradation. Preliminary data shows that CG7518 is a cytoplasmic protein ubiquitously expressed in the nervous system throughout all stages of development. This implies that it may have a broader biological role and experiments will focus on whether specific neuronal phenotypes can be observed. Any effects to Lola localisation will be investigated as it is a nuclear protein whilst our candidate is cytoplasmic.
Cap-G is a subunit of the condensin I protein complex and is necessary for condensation, assembly and segregation of chromosomes (3). Its interaction with Lola sparks an interest, as Lola is activated post-mitotically, whilst Cap-G is mainly expressed in dividing cells. This implies that Cap-G may have a temporal-regulated role in the early stages of differentiation, interacting with Lola. To investigate whether Cap-G has a post-mitotic function and a potential role in cell differentiation, several methods are used. Quantitative PCR can be carried out on adult brains to quantify the Cap-G expression in post-mitotic neurons, as no cell division is present in adult brains. Moreover, DamID will reveal the DNA and chromatin binding-sites of Cap-G, offering an insight on its function. The understanding of these Lola interactors will be an addition to the molecular puzzle regulating the maintenance of neuronal differentiation. The implications of this study are relevant not only for our understanding of developmental biology but also to decifer factors involved in tumour initiation and regenerative medicine. Neurological diseases such as Parkinson's and multiple sclerosis would highly benefit from the advancement of knowledge in this field to lead therapeutic progress (4)
The transcription factor Lola is one of several proteins that maintain the differentiated state of neurons. In a study by Southall et al., lola mutants lead to the regression of neurons into a stem-cell like state and the development of tumours (2). These tumours develop due to the tightly regulated programming of the cells being perturbed. Therefore, it is crucial to identify the mechanism maintaining the cell fate and what other proteins are involved in this process. To investigate this, a yeast-two-hybrid screen was carried out to detect protein-protein interactions with Lola. Two of the high confidence hits from this screen were proteins CG7518 and Cap-G which will be the focus of this project (personal communication, Southall). In this study we will exploit the genetic toolkit available in Drosophila to induce overexpression and RNAi knockdown of said proteins and investigate phenotypes in neurons at larval and adult stages. Immunostaining techniques are used on larval and adult brains to image the proteins of interest using confocal microscopy. CRISPR/Cas9 system will be used to create a cell-type specific loss-of-function mutants for both our candidate interactors and observe potential phenotypes. The aim is to characterise CG7518 and its potential interactions with Lola, as its molecular function is still unknown. We hypothesise CG7518 to be a Lola regulator by inhibiting its function, either by retaining it in the cytoplasm, modifying its structure or targeting it for degradation. Preliminary data shows that CG7518 is a cytoplasmic protein ubiquitously expressed in the nervous system throughout all stages of development. This implies that it may have a broader biological role and experiments will focus on whether specific neuronal phenotypes can be observed. Any effects to Lola localisation will be investigated as it is a nuclear protein whilst our candidate is cytoplasmic.
Cap-G is a subunit of the condensin I protein complex and is necessary for condensation, assembly and segregation of chromosomes (3). Its interaction with Lola sparks an interest, as Lola is activated post-mitotically, whilst Cap-G is mainly expressed in dividing cells. This implies that Cap-G may have a temporal-regulated role in the early stages of differentiation, interacting with Lola. To investigate whether Cap-G has a post-mitotic function and a potential role in cell differentiation, several methods are used. Quantitative PCR can be carried out on adult brains to quantify the Cap-G expression in post-mitotic neurons, as no cell division is present in adult brains. Moreover, DamID will reveal the DNA and chromatin binding-sites of Cap-G, offering an insight on its function. The understanding of these Lola interactors will be an addition to the molecular puzzle regulating the maintenance of neuronal differentiation. The implications of this study are relevant not only for our understanding of developmental biology but also to decifer factors involved in tumour initiation and regenerative medicine. Neurological diseases such as Parkinson's and multiple sclerosis would highly benefit from the advancement of knowledge in this field to lead therapeutic progress (4)
Organisations
People |
ORCID iD |
| Tony Southall (Primary Supervisor) | |
| Jie Song (Primary Supervisor) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M011178/1 | 01/10/2015 | 25/02/2025 | |||
| 1656706 | Studentship | BB/M011178/1 | 03/10/2015 | 30/09/2019 |
| Description | Condensin complex has a crucial role in compacting DNA into chromosome during cell division, but its role in post-mitotic cells remains unexplored. In this work we described a novel role for condensin I subunit Cap-G in neurons. We characterised Cap-G expression pattern and localisation in the Drosophila central nervous system in vivo. To understand the function of Cap-G in neurons we generated cell-specific knock-down of Cap-G in neurons. Removal of Cap-G from post-mitotic neurons surprisingly resulted in severe developmental arrest, behavioural defects and an overall disruption in gene expression in the CNS. This is the first known effect of a condensin subunit in a post-mitotic neuron. I investigated whether Cap-G had a neuron-specific role in Drosophila, so I characterised Cap-G DNA binding profiles in neuronal stem cells and neurons, identifying dynamic, cell-specific binding of Cap-G in the CNS. Cap-G binding was depleted at accessible chromatin and known enhancer regions but highly enriched at repressive chromatin sites. Cap-G appears to have a cell-specific gene regulatory role in neurons. Knockdown animals exhibited a downregulation of neuron-specific genes and an upregulation of ectopic, non-CNS- specific genes. Genes bound by Cap-G significantly overlap with the disrupted genes detected in knockdown animals. This works provides novel evidence that condensin I subunit Cap-G in neurons is necessary for healthy nervous system development, transcriptome stability and maintenance of neuronal identity. |
| Exploitation Route | The discovery of a post-mitotic role for Cap-G highlights the importance in studying these chromatin associating proteins in a post-mitotic context to understand their role in development beyond cell division. Cell-binding data and RNA-seq data is freely accessible and can be used by other researchers if required. Fly lines generated are also available upon request. |
| Sectors | Other |
| URL | https://www.biorxiv.org/content/10.1101/2020.01.14.904409v1.article-metrics |