Some more pure science from guest blogger LisaJ:
Everyone seems to love a little Sonic Hedgehog around here. Whenever PZ discusses another function that this fascinating gene is capable of, much excitement ensues in the comment posts. So I thought I would take this opportunity to talk a bit about what I study, and how my seemingly unrelated favourite protein pathway is also connected to the Shh gene.
The main protein that I study was originally identified through studies of a pediatric eye cancer, called Retinoblastoma, as loss of function of this protein (termed the Retinoblastoma protein, or pRb for short) was found to be causative in the formation of this tumour type. What we know now is that pRb is required to control normal cell division in all cell types; its functional loss leads to the formation of many types of tumours, and is thought to be involved in the development of at least half of all human cancers. Not only is pRb essential in preventing uncontrolled cell division that can lead to cancer, but it is also essential for embryonic development, as mice deficient for the Rb gene die by about the 15th day of gestation, about 5 days before they would be born.
To explain how pRb functions in the cell, I thought that I could easily pull out a figure from a review paper that diagrams a simplified cell cycle pathway and the protein interactions that take place. But instead, I’m going to show you this beautiful piece of work that a few of my talented previous lab mates created for me one day, as a means of cheering me up after a year’s worth of protein purifications and binding assays did not give me the result I was hoping for. Although a simplistic depiction of cell cycle regulation, I think it really drives home the point well of how pRb functions.
This figure shows that pRb, or ‘RB Boy’ as is the case here, is stopped in his tracks in G1 (Gap 1) phase of the cell cycle. His watch and the stop light ahead of him are telling him to stay put for the moment until the cell’s DNA is fully repaired and ready to be duplicated, at which time he may initiate entry into the DNA synthesis phase (S phase). You’re probably wondering what that is sticking out of his pocket. Well, it’s an E2F transcription factor complex, and pRb binds E2F proteins through a domain that forms a pocket like structure. The manner by which pRb inhibits entry into S phase is by physically binding E2Fs while they are sitting on DNA; this binding interaction inhibits the activation of genes required for DNA synthesis to occur by E2Fs. Once any DNA damage has been repaired and the cell is ready to enter into S phase, a cyclin/cyclin dependent kinase (cdk) complex, shown here by the ‘cyclin/cdk bird’, phosphorylates pRb. This phosphorylation disrupts the physical interaction between pRb and E2F, and thus E2F is free to activate expression of pro S phase genes. These are the basics of how pRb regulates the cell cycle.
As I said previously, we know that pRb, by virtue of its regulation of E2F transcriptional activity, is a very important tumour suppressor. The role of these proteins in regulating tumour development has been, and still is, very heavily studied. We’re also discovering, however, that pRb has other important functions besides regulating the cell cycle and suppressing cancer. For instance, pRb/E2F proteins play an important role in regulating cellular differentiation and cell death, as well as the expansion of stem cells in a number of tissue types, including the haematopoeitic system, intestinal epithelium, and the brain (which I am most partial to). Additional functions that we’ve found for pRb/E2F in the brain include regulation of the differentiation of neurons and neuronal sub types, the migration of neurons through the brain, progenitor cell division, and the patterning of the ventral forebrain. We’re finding some very exciting functions for these proteins in the developing brain, and what’s even cooler is that the mechanisms through which pRb/E2F regulates neural development are emerging to be very similar to those that are involved in cancer development.
So of course, since the Sonic Hedgehog (Shh) gene does so many exciting things, we have also found an important functional link between E2Fs and Shh. When you knock out the E2F4 gene in mice and examine how embryonic development proceeds in the brain, you notice a very similar phenotype to what is seen when you knock out the Shh gene. The brain is quite reduced in size, and you see a large defect in the patterning of the ventral telencephalon (which is a part of the forebrain). Basically, the most prominent ventral structures, which are normally rich in neural stem and progenitor cells, are totally absent. Not surprisingly, these embryos do not survive to birth. You can see this gross defect in the figure below.
What you’re looking at here is an un-dissected embryonic mouse brain, at day 11 of gestation, in the first column, with the brain from a normal mouse on top and a much smaller E2F4 mutant brain on the bottom (TE in this figure stands for ‘telencephalon’). In the second column you are looking at a coronal, or frontal view cross section, of normal and mutant brains at the same embryonic stage; you can see that those bumpy structures (called the ganglionic eminences) present in the bottom half of the normal brain are almost totally absent in the E2F4 mutant.
PZ has discussed here before theimportance of Shh function in patterning the nervous system during embryonic development, by initiating its expression ventrally. What was ultimately determined about this observed E2F-Shh interaction is that the E2F protein is responsible for activating expression of the Shh gene within the ventral telencephalon at the correct time point during embryogenesis. Without this transcriptional activation, Shh is not adequately expressed and forebrain patterning is severely disrupted. This second figure shows really nicely how important the timing of Shh expression by E2F is.
You’re looking first at a side view, and then a frontal view, of a mouse embryo at day 9 of development. The wild type and E2F mutant mice in this experiment have been bred with mice that carry a transgene containing regions of the Shh promoter that are known to be essential for directing expression of Shh specifically to the brain regions that are affected in the E2F mutant. When these promoter ‘enhancer’ regions are bound by activating transcription factors, they direct expression of the beta-galactosidase gene, which is also present on the transgene downstream of the Shh enhancer sequence. Embryos are subsequently stained with a compound called X-gal, which is enzymatically cleaved by beta-galactosidase to produce a bright blue precipitate. Thus, the blue markings that you see in the figure indicate the locations that Shh is expressed in the ventral forebrain, and this expression is almost completely absent in the E2F4 mutant (lower panel). In the third column, you can see that one day later in the developmental time course, Shh expression is starting to appear in the E2F mutant mouse. It’s not an all or nothing effect here, but this seemingly short-lived dysfunction in Shh expression is what leads to the disastrous developmental effects to the animal that are described above.
This is a great example of two molecular pathways that were originally thought to do very different things converging to perform an essential, developmental function together. The number of gene products that function in our cells is huge, and it’s incredible to think of how immensely this complexity is increased when we find that many proteins are performing multiple functions on their own and in cooperation with other proteins or pathways. The wonders of biology never cease to amaze.
E2F/Shh figures from Ruzhynsky et al. Journal of Neuroscience. 2007. 27(22): 5926-35