Biological Clocks

Mutations in eight genes have strong effects on circadian (~24 hour) locomotor activity rhythms of Drosophila. These rhythms can be compared to human sleep/wake cycles. Indeed human orthologs of three of these Drosophila "clock" genes have been associated with disorders of sleep.

In Drosophila, mutations of the period (per), timeless (tim), double-time (dbt), Clock (Clk), cycle (cyc), shaggy (sgg), vrille (vri) and Par-Domain Protein 1 (Pdp1) loci can lengthen or shorten the period of the locomotor activity rhythms, or can abolish the rhythms altogether. The abundance of per, tim, vri, Pdp 1 and Clk RNA and their encoded proteins changes rhythmically with a circadian period in wild-type flies. Mutations affecting any of the eight genes have corresponding effects on behavioral and molecular rhythms. Thus, molecular rhythms likely drive the behavioral rhythms.

The PER, DBT, TIM and SGG proteins physically interact. Interactions first occur in the cytoplasm between PER and DBT. The latter protein, an ortholog of Casein Kinase 1, phosphorylates PER, triggering rapid PER degradation. After several hours a further interaction with TIM blocks DBT-dependent phosphorylation allowing accumulation of PER. TIM is eventually phosphorylated by SGG, an ortholog of GSK-3, and this permits nuclear localization of DBT/PER/TIM complexes. In the nucleus, PER and TIM eventually dissociate, and TIM-free PER strongly suppresses activity of two transcription factors encoded by Clk and cyc. This regulation is significant because in the absence of nuclear PER these transcription factors activate per and tim expression. As in the cytoplasm, TIM-free PER is phosphorylated by DBT in the nucleus. Consequently transcription of per and tim resumes after an interval of nuclear PER phosphorylation and degradation (view video). Two more cycling transcription factors, Vrille and PDP1, form a second feedback loop by regulating transcription of Clk. Vrille is a repressor of Clk and PDP1 is a Clk activator. Each affects Clk transcription at a different time of day, resulting in oscillating Clk expression.

TIM protein couples this molecular oscillator to the environment because TIM is rapidly degraded following exposure to light. Ordinarily cycles of light and dark restrict accumulation of PER-containing complexes to times after nightfall since TIM is degraded by light and TIM is required to stabilize PER. However, pulses of light occurring in the early evening or just prior to dawn reset the cycle by prematurely eliminating TIM in the nucleus. In this way phase-shifts are induced in the behavioral rhythms. The acute-light sensitivity of TIM requires its phosphorylation by SGG, and function of an unusual photoreceptor, Cryptochrome (CRY).

We have begun to look at programs of gene expression that are regulated by this molecular clock using oligonucleotide microarrays representing all genes (~14,000) of the fly. We find that in the Drosophila head, ~400-500 genes are expressed with a circadian rhythm (see data). This represents 6-7% of the genes that are active in the head. Genes composing this large circadian program influence almost every aspect of the fly's biology, and subsets of these genes are switched on and off with phases representing every hour of the day and night. Mutations of genes composing the clock appear to abolish this program of temporally sequenced gene expression, even when environmental cycles are provided. This indicates a thorough dependence of the temporal program on the identified molecular oscillator.

Neurogenesis

Mutations in the neurogenic genes of Drosophila alter development of the embryonic ectoderm through overproduction of neuroblasts. This occurs at the expense of presumptive hypodermal cells and thus appears to involve rerouting of cell fates. Best characterized of the neurogenic loci is Notch, which codes for a large transmembrane protein. Most of the extracellular domain of the protein is composed of tandem repeats of an epidermal growth factor-like amino acid sequence, and direct interactions with a variety of ligands (including Serrate and Delta) occur through these epidermal growth factor-like elements. Because some ligands are themselves transmembrane proteins, Notch often contributes to ectodermal development through direct cell-cell interactions. The neurogenic genes appear to have parallel roles in development of the ectoderm, mesoderm and endoderm. All of the neurogenic genes are expressed in these germ layers and affect the sorting of their embryonic cell fates.

Several years ago it was observed that antineurogenic phenotypes (underproduction of neuroblasts) can be generated in Drosophila embryos by expressing a truncated form of the Notch protein that includes only its cytoplasmic domain. This domain of Notch was also found to contain nuclear localization signals that function in cultured cells, which led to our proposal that Notch is cleaved in response to ligand to produce a nuclear signal. This has now been verified in cultured cells and in flies. The metalloprotease Kuzbanian (KUZ), cleaves an extracellular Notch sequence close to the transmembrane domain upon ligand stimulation. Following this proteolysis a second, transmembrane Notch cleavage, involving Presenilin and Nicastrin, produces a soluble, intracellular form of Notch that is associated with the transcription factor Suppressor of Hairless (Su(H)). A complex of intracellular Notch and Su(H) then directly promotes specific gene expression to establish cell fate.