Genetic and Cellular Regulation of Mammalian Brain Development
Summary: Alexandra Joyner studies genetic and cellular events that orchestrate development and patterning of the central nervous system in mammals.
A number of different strategies are used to lay down the basic three-dimensional organization, or pattern, of different regions of the central nervous system (CNS). In one, an "organizing center" within the tissue directs the development of surrounding cells, often by forming a gradient of secreted molecules. In other cases, separation of two cell populations into compartments allows adjacent regions to develop independently. Our long-standing interest has been development of the midbrain, which receives visual and auditory input, and the cerebellum of the anterior hindbrain, which regulates motor control. We have focused on determining how an organizing center located at the midbrain/hindbrain junction regulates anterior/posterior patterning of the midbrain and anterior hindbrain. A second organizer we study expresses the secreted factor Sonic Hedgehog (SHH) and regulates dorsal/ventral patterning of the embryonic CNS and postnatal growth of the cerebellum. Mutations in genes in the SHH pathway in humans cause neurological diseases, as well as cerebellum tumors. We have determined how three GLI transcription factors respond to a gradient of SHH protein and induce cell types in a concentration-dependent manner.
Anterior/Posterior Patterning of the Embryonic Mid/Hindbrain
Experiments performed in chick embryos more than a decade ago showed that the isthmus that separates the midbrain and hindbrain can induce midbrain or cerebellar structures in ectopic locations in the brain. This suggested that anterior/posterior patterning of the midbrain and anterior hindbrain is controlled by a centrally located isthmus organizer that instructs the cells on one side to become the midbrain cell types and on the other side to become the cerebellum. We have used genetic approaches in mice to determine what positions the organizer at the mid/hindbrain junction and how the secreted organizer molecule fibroblast growth factor 8 (FGF8) functions to pattern the two tissues.
A set of transcription factors, including the Engrailed (EN) proteins, are expressed across most of the midbrain and cerebellum, and the secreted factors FGF8 and WNT1 are expressed in the organizer (Figure 1). We and others have shown that FGF8 has organizer activity because it can mimic the effect of transplanting organizer tissue and induce midbrain or cerebellum development. The transcription factors GBX2 and OTX2 are the earliest proteins whose expression divides the neural plate into two domains with a common border where the organizer will form, and they are required, respectively, for midbrain and cerebellum development.
Our genetic studies in mouse have shown that Otx2 and Gbx2 are not required to induce the organizer gene Fgf8 but are required to position its expression domain, and thus the organizer. We used a new mouse genetic technique to remove Gbx2 after the organizer forms and found that Gbx2 is no longer required for cerebellum development but is still necessary to position Fgf8 and Wnt1 expression properly. The first role of Gbx2 is to prevent Otx2 expression in the hindbrain and thus allow cerebellum development. Once the organizer is established, Gbx2 is no longer required for cerebellum development but does have a role in regulating organizer gene expression. We recently developed a novel technique for marking embryonic cells and following them into the adult (fate mapping); this showed that the Otx2/Gbx2 border is a cell-lineage restriction that inhibits midbrain and cerebellum cells from mixing. Thus, the organizer forms at a compartment border and then influences cells on either side to form different tissues.
We have also explored the question of how FGF8 patterns the midbrain and anterior hindbrain. At least two different isoforms of FGF8 protein, and two related FGF proteins, FGF17 and FGF18, are expressed in the organizer. We found that two different isoforms of FGF8 have different activities: FGF8a induces midbrain development, whereas FGF8b induces a cerebellum. Furthermore, FGF8b induces FGF17/18 in the midbrain, and these proteins have midbrain-inducing activity. Using a novel assay in which brain tissues are cultured with FGF-soaked beads, we demonstrated that different genes respond to each FGF protein at particular concentrations. Based on our studies, we propose that patterning of the midbrain and cerebellum involves a series of events, the first being establishment of the Otx2 and Gbx2 domains. En, Fgf8, and Wnt1 are then induced by an unknown factor in the presumptive "midbrain/cerebellum" domain, and OTX2 and GBX2 restrict Fgf8 and Wnt1 to their appropriate domains. The organizer molecule FGF8b then induces Fgf17/18, and the expression patterns of these and other genes is refined through reciprocal feedback loops in which FGF8 is a key factor. Depending on the concentration and isoform of FGF that a cell receives, different regions of the midbrain form, or different regions of a cerebellum.
Regulation of the Size and Pattern of Cerebellum Folds
The cerebellum develops primarily after birth and forms a distinct set of folds that are conserved across species, with a simple cellular architecture. We are interested in determining what genes and cellular events direct foliation, since each fold regulates a particular set of motor activities. SHH expressed in Purkinje cells regulates proliferation of the major cell type (granule cells) that causes foliation after birth (Figure 2). We have studied mice that lack genes in the SHH pathway and found that SHH signaling regulates the size of the folds. We have shown that En1 and En2 play at least two roles in cerebellum development: regulation of the size of the embryonic pool of cells that will form the cerebellum, and direction of a normal pattern of foliation. We are carrying out studies that remove En function at different times to determine whether these two roles are linked and how the concentration of EN protein regulates cerebellum size and foliation.
Ventral Patterning of the CNS
SHH is both required for development of ventral CNS structures and sufficient to induce cell types in a concentration-dependent manner. We have used the mouse spinal cord as a model system to study the mechanism by which SHH patterns the ventral CNS and to determine how each of the three mammalian GLI transcription factors (GLI1, GLI2, GLI3) function to regulate SHH target genes. The GLI-like protein Ci in flies is regulated by Hh: when Hh is present, Ci activates target genes; when Hh is absent, Ci represses genes. We have studied whether each GLI protein also has two functions regulated by SHH. Our previous analysis of mice lacking each Gli gene suggested that GLI2 primarily functions to activate genes in response to SHH, whereas GLI3 is primarily a repressor that must be inhibited by SHH to allow ventral cells to form. Gli1, on the other hand, is not necessary for spinal cord development, but enhances the activator role of GLI2.
Recently we have used a genetic tool in mice to compare directly the normal function of each GLI protein during development. We have tested whether one Gli gene can compensate for the other by replacing Gli2 with Gli1 or Gli3. In this way the inserted gene is expressed precisely as the other gene, whereas normally these genes have different expression patterns. Our studies showed that low-level expression of Gli1 in place of Gli2 is sufficient to replace all the functions of Gli2, but at higher levels Gli1 inappropriately activates genes. This study and others demonstrated that GLI2 is normally only required to activate genes and does not function as a repressor. Although it is a potent repressor, GLI3 also contributes some activator function, but it cannot activate the spectrum of genes activated by GLI2. GLI2 and GLI3 also activate transcription of Gli1; thus GLI1 expression is a readout of SHH activity. We combined this with our novel fate-mapping technique to mark and determine the ultimate fate of cells responding to SHH at specific times during development. A study in the limb demonstrated that the response of cells to SHH protein is regulated by cellular factors, and thus a simple gradient of response is not seen beside sources of SHH (Figure 3). Our studies demonstrate that during evolution the various functions of an ancestral GLI protein have been distributed among three mammalian GLI proteins, and each has unique activator and repressor functions.
Some of our work was supported by a grant from the National Institute of Child Health and Human Development.