Understanding Cerebellar Pattern Formation

I am Kathy Millan. I'm an Assistant professor at the University of Chicago in the Department of Human Genetics and also in the Department of Neurology. And I work on brain development, and specifically I work on Cere Butler development, the cerebellum, the part of the brain back here at the back of the skull.

And it's most famous for its role in motor development, but it turns out it's involved in a lot of things. And it's a really interesting part of the brain Pattern Formation is the development of pattern in a biological system. And it's one of these words that developmental biologists use kind of in a squishy way to mean almost anything.

And it's not really clear what it means. I think every developmental biologist would give you a different version of what it means to me. What it means is how does the brain, or in particular the cerebellum, know from top and bottom, how do, how does you define left and right?

How do you decide which part of the structure is going to be, which part? And once you've made those critical, fundamental early embryonic decisions, how do you bring all of the cellular structure together to form the exquisitely patterned part of the brain that we call the Cerebellum? So we're really Interested in cerebellar development and pattern information in the developing cerebellum.

Because the cerebellum is an exquisitely patterned tissue. It's got not that many cell layers and not that many cell types. So relative to other parts of the brain, it's especially, well, it's a relatively simple structure.

And also because the cerebellum has a very specific function, or one of many functions is motor coordination. When mice or humans have cerebellar abnormalities, one of the resulting phenotypes is motor uncoordination. And so over the a hundred years or so of mouse genetics, there have been multiple spontaneous mouse mutants that had been discovered and characterized to some extent, but also just isolated and sitting on the shelf at Jackson Labs where they're mice with motor abnormalities.

And a large number of those mice, I think there's over about 60 spontaneous strains have cerebellar developmental abnormalities. And that resource is a really valuable rich resource to look at what are the developmental mechanisms that drive cerebellar pattern formation. So one thing my lab is focused on is to take all the tools that we have from the Human Genome Project and the Mouse Genome Project, and use those molecular reagents to go and find the genes that are mutate mutant in all of those spontaneous mutants.

And by finding those mutants, we take a phenotype centric approach to finding genes. So we're not randomly taking genes and knocking them out. We're finding phenotypes and finding the genes because by using that approach, we're specifically finding genes that are particularly relevant to the system of interest.

So our lab and many other labs have cloned a number of these spontaneous mouse mutants. And those spontaneous mouse mutants have led to really interesting new biology. Because we're taking this phenotype driven approach.

We're not having any preconceptions about what kind of genes we're going to find. We're not specifically looking for dal ho analogs or channel mutations. We are getting to the genes that the phenotypes dictate.

And because of that, we're learning a lot of new biology. So for example, one of the genes or one of the phenotypes that my lab has worked on and together when I was a postdoc with another colleague, Jim Linig, we worked on a mouse mutant called dre, which is German for spinner or spinning like a top. And these mouse mutants first arose in 1929 in Germany and have been accumulating at Jackson Labs.

There are multiple alleles of this particular locus, and it was known that the cerebellum was incorrectly formed. So all the cerebellar cell types were there, but the pattern of the cerebellum was abnormal. The middle part was missing the middle part's called the verus.

And we discovered that there was an early embryonic phenotype in these mice that led to that adult phenotype. And the phenotype was a result of mutation in a gene called LMX one A, which stands for limb homeo domain containing box one. So L LMX one A gene is actually turned into a really interesting and important gene in developmental biology because is one of the most critical genes in defining what is up in the central nervous system or what is dorsal.

And without having this rare mutation, nobody actually knew what this gene did. In fact, it had been found for other reasons in insulin producing cells in the pancreas. And other people had been studying it for potential role in diabetes.

But in fact, it turns out that the LMX one aging really by itself has no role in pancreatic development or eyelet cell development. And in fact, its main role in development is driving dorsal patterning in the central Nervous system. When we first cloned the DR Gene, the LMX one A gene, we realized that it's only expressed in the dorsal part of the central nervous system.

And in fact, it's not only expressed in the dorsal part of the central nervous system or the the roof plate that it's called just around the cerebellum. It's actually expressed in the dorsal part of the central nervous along the whole axial length of the mouse embryo. And in fact, it's expressed along the whole axial length of every vertebrate that we've looked at so far.

And because it's expressed dorsally everywhere, we reasoned that it must have a role dorsally in other parts of the, in the central nervous system. And so we actually spent a lot of time characterizing the phenotype of the DR mice in the spinal cord because the spinal cord is actually much better model system for understanding CNS development than the cerebellum because it's much less three dimensionally complicated. It's got a lot less three dimensional structure.

Switching to the chick, we were actually able to take advantage of a large number of reagents to figure out what the role of LMX one A was in the spinal cord. And from the spinal cord, we switched back to the cerebellum. And frankly, I don't think without the technology of chick ation, we really would've had a great understanding of what the LMX one a gene does, a postdoc in my laboratory, Victor Chiko, was the person who really took these experiments and ran with them.

And he was the person who actually figured out what the LMX one a gene does, and its primary role in central nervous system by using the Chick electroporation system. And I honestly don't think that we would've been able to make as much advance as we did without chick electroporation. So Chick electroporation is a really valuable technology because you can over express genes rapidly in the developing chick nervous system, get a result after two or three days of incubation and get analyzable phenotypes.

If you were to actually try and do gene manipulations in mice for transgenic animals that would take several months and for knockouts, et cetera, that would take at least a year and a half. And so the chick ation system is a really valuable system because you can manipulate gene expression and you can find out phenotypes rapidly. So the LMX one aging when it's over expressed in the developing chick nervous system, in particular, the developing spinal cord, caused the whole dorsal part of the central nervous system or the spinal cord to turn into a dorsal part of the central nervous system or the roof plate.

And so what that told us in combination with a bunch of other experiments that Victor did was that LMX one A by itself is responsible for driving the whole differentiation program of the roof plate. And that's important because the roof plate's a critical signaling center in the developing nervous system. If you don't have a roof plate, you don't secrete morphogen to tell adjacent neural tube cells to turn into dorsal sensory neurons.

And without those signals, those cells go into a default state of an intermediate cell type. And you lose most of the dorsal neurons in the, in the, in the spinal cord, but also actually in the developing cerebellum. And I don't think we would've easily been able to come to that conclusion using any other system except the chicken Neural tube.

We're really interested In cerebellar development, not only because of the basic biology in in in mouse systems and also in the chick, but also humans have cerebellar malformations. And those human cerebellar malformations are really poorly understood. And so in collaboration with a colleague, Dr.William Dobbins, one of the world's experts in human brain malformations, together, we've initiated a project to find human cerebellar malformation genes.

And we're taking several approaches. One of the biggest things that we've done is we've built a DNA database and a clinical database where we've recruited over 500 patients with defined human cerebellar malformations and had collected images of MRIs and also correlated those with DNA samples so that we can use that as a resource for gene hunting because it turns out that human cerebellar malformations are not well categorized at all. And I would honestly say that out of every 10 scans we receive for through our studies, at least one of those scans is some malformation that's never been described and has not really been recognized before.

The most common human cerebellar malformation is called Dandy Walker malformation, and it's the one that's most recognized by clinicians. But it turns out it's sort of a garbage diagnosis of the human cerebellar world. Any physician or many physicians, when they see an abnormal cerebellum classified as dandy walker.

And so we recruit patients into our Dandy Walker malformation study, and we find all kinds of different interesting cerebellar malformations. So we're using this resource to find new cerebellar developmental genes, because as a developmental biologist, what I think is that humans are one great big mutagenesis experiment. They're one great source for finding new developmental genes.

So we're taking several approaches to find those genes. One of those approaches is to take genes that we've figured out in mouse that have roles in cerebellar development and sequencing those genes in humans. And in fact, we have just recently identified the very first human LMX one A mutation.

So so far there's Been really limited applications of our basic science research into clinical research. But that's really rapidly changing. As we're finding new and new genes, we can provide new genetic counseling information to families of affected children.

And frankly, that's where I see the biggest impact Of our research. So my Vision for the field of five year in five years is that a lot more gene discovery will have happened both by characterization of mouse models and also by work in human cerebellar malformations. We're gonna know more about the set of genes that are involved in cerebellar development.

And then the goal is gonna have to be how do those genes interact with each other, and how do those genes drive the actual biology? How do they drive all the different cellular events that take place to make a cerebellum? And I think the cerebellum's really an ideal model for the whole central nervous system because frankly, there are only five to seven principle neuronal cell types.

They're all arranged in a various stereotypical manner. We're learning more and more about the molecules that drive the specification and the early differentiation of those progenitors. We're understanding more and more about how those cells proliferate.

So I think more than five years from now, but ultimately I see the field being able to go tube development all the way through cerebellar development to achieve the mature form of the cerebellum. And I think because the cerebellum's a relatively simple system, we have a hope of actually figuring out the molecules and the developmental events that drive the formation of the entire central of the entire cerebellum. And I think that's really important, not only for the field of genetic diagnosis in humans, and it's important for the field of cerebellar development, but I think we're also gonna have applications of those mechanisms to other parts of the brain.

The cerebellum's a relatively simple structure, but many of the developmental events aren't going to be exclusive to the cerebellum. We can take what we learned from the cerebellum to other more complicated regions of the central nervous system like the cerebral cortex, and take the lessons and the pathways that we learned and figure out how those apply to the rest Of the brain.