What is migration?

The conversion of stationary epithelial cells into migratory, invasive cells, which is important for normal embryonic development. How do cells achieve this? Cells that are part of a stable epithelium lose their cell-cell and cell-matrix junctions that hold them together and acquire a less rigidly polarised morphology and break away from their neighbours.

Forward genetic analysis
This involves uncovering the genetic basis behind a particular phenotype. Mutant lines are screened for specific defects in the process of interest. Molecular, cellular and chemical characterisation of the affected genes and proteins is then carried out. Reverse genetics: manipulating genes and studying their effect on phenotypes

Border cells are a small group of invasive follicle cells in the Drosophila melanogaster ovary that helped us to gain insight into genes required for migration initiation. Three recently discovered signalling pathways that control different aspects of the migration:

  1. a global steroid hormone that defines timing of migration and coordinates the event with other changes that occur at the same stage of ovarian development
  2. a highly localised cytokine signal that activates the Janus kinase (signal transducer and activator of the transcription (Stat) pathway) and is necessary and sufficient to induce migration
  3. a growth factor related to platelet-derived growth factor (Pdgf) and vascular endothelial growth factor (Vegf) that contributes to guiding the cells to their destination

The border cell model

FIrst up, we are going to talk about the egg chamber development in Drosophila. It starts with a structure called the germarium.

[Anterior tip - follicle cells surround germline stem cells] ->  [Asymmetric div- cystoblast and daughter stem cells] -> [Cystoblast- 16 cells clusters(incomplete cleavage)] -> [Oocyte and 15 nurse cells] -> [Nurse cells are polyploid and provide cytoplasm to oocyte] -> [Follicle cells cover 16-cell cyst] -> [Oocyte moves to posterior and is surrounded by columnar epithelial layer of follicle cells] -> [the rest of follicle cells cover nurse cells (stage 9 rearrangement)] -> [Development of egg chamber] -> [Pair of special cells at each end, called polar cells] -> [polar cells, among 6-10 cells form border cell cluster] -> [These cells round up, detach from the follicle cells and extend processes between the nurse cells and forge their way to the anterior border of the oocyte through the centre of egg chamber] -> [n the final few hours of oogenesis, the nurse cells undergo apoptosis and the follicle cells produce the eggshell]

Identifying border cell migration mutants

slow border cell (slbo) mutants

The stratgy was to screen for female-sterile, transposable P-element insertion mutants. Several allreles of the slbo mutant was found, which showed varying degrees of female sterility, caused by insertion of p-elements into the regulatory regions that control expression of the slbo gene specifically in the ovary. Only defects in mutation - border cell migration defects, resulting in production of numerous unfertilised eggs. Defects - Most severe - Border cells completely fail to initiate migration, and although their nuclei cluster around polar cells, and express border-cell markers, they do not form cluster, or detach. (Null mutations are lethal)

Find out about:

  1. Transposable P-elements
  2. Null mutations

What does the slbo locus encode? The slbo locus encodes a transcription factor called Slbo and this implies that it regulates changes in gene expression for cells to become motile.

Targets of slbo:

  1. DE-cadherin - Homophilic cell-cell adhesion molecule. These molecules are expressed more in border cells at the time of migration in a slbo-dependent manner. WHY? The DE-cadherin molecules provide traction, so the border cells can crawl on the nurse cells. This indicates, cadherins might support not only stable adhesion in epithelial cells but also, dynamic adhesion required for cell migration.

Further: What are the mechanisms that control the dynamics of cell adhesion in migrating cells and are they similar for both cadherin and integrin - mediated adhesion?

  1. Focal adhesion kinase
  2. A zinc finger transcription factor(Jing) and myosin-VI, a pointed end directed motor

Techniques

  1. Clone mosaicism - Since the mutations in these genes are often lethal or pleiotropic, they are difficult to study. This method involves screening for mutations that affect border cell migration defects in mosaic clones. Mosaic clones are groups of homozygous mutant cells in an otherwise heterozygous and viable organism.
  2. Enhancer-promoter(EP) screen - This involves the overexpression or misexpression of random genes in a cell type of interest.

How are border cells specialised from the follicle cells? How does a stationary epithelial follicle cell turn into a migratory and invasive border cell? We pin all our money on the polar cells here. Why? It was observed that polar cells are always at the middle of the border cell clusters, differentiate earlier than the border cells, and in mutations with extra polar cells, more border cell clusters formed.

It is hypothesised that the polar cells produce a signal that might enable neighbouring cells to migrate. However, interestingly, polar cells themselves cannot migrate without the border cells. In experiments, where the outer ring of border cells was obliterated using toxins, the intact polar cells were rendered immotile.

In conclusion, the polar cells seem to recruit neighbouring epithelial cells to cluster around them and to carry them to the oocyte.

Jak-Stat Pathway Start92e is a transcription factor that is activaed by the Jak homologue of Drosophila, known as hopscotch(Hop). How does it work? ![[Pasted image 20231230150331.png]]

The question has been posed as to whether Upd and signalling through the Jak–Stat pathway regulate migra- tion of the cell specifically, or simply specify the fate of the border cells. In support of a function in migration per se, hypomorphic mutations in components of the Jak–Stat pathway result in border-cell clusters with a normal number of cells that express Slbo and other border-cell markers but still fail to migrate. This implies that there are down- stream targets of Jak–Stat signalling that are independent of the Slbo pathway and that are required for migration. Moreover, upd is clearly expressed in the polar cells throughout the migration, which indicates that it continues to function beyond the stage when border-cell fate has been specified. This is reminiscent of the role of the transcription factor Slug in NEURAL CREST cells. Slug is required first for the specification of this migratory cell population and then for them to be able to migrate.

Who regulates when migration starts? Ecdysone is the only steroid hormone found in Drosophila. Ecdysone, functioning through EcR, Usp and Tai, seems to regulate the timing of border-cell migration and coordinates this event with others that occur at the same stage of oogenesis, such as germline development and the general rearrangement of the follicle-cell epithelium. The evidence for this is that, when Ecdysone sig- nalling is globally reduced (for example, using a temperature-sensitive allele of a gene that is required for Ecdysone biosynthesis), oogenesis arrests just before border-cell migration. When Tai function is eliminated specifically from border cells, they fail to migrate 1 . When Tai function is removed from small groups of epithelial follicle cells, the mutant cells fail to rearrange.

The Oocyte releases attractants to guide the Border cells This speculation rises from the fact that border cells always traverse through the inside of the egg chamber to reach the oocyte, even when the oocyte is a little displaced from its normal position. EP screen reveales abnormally high leve;s of expression of pfv1 in the oocyte. It has also been seen that if the nurse ceels exhibit high levels of Pfv1, border cell migration defects are caused. However since the border cell migration and other migration processes like axonal guidance are guided by many classes of chemical attractants, pfv1 mutants are often mid, unless co-occurring with other mutated guidance factors. Indeed, there re other genes that encode attractants in Drosophila, namely the pfv2 and the pfv3 mutants.