BIOLOGICAL OVERVIEW

Disabled was identified on the basis of its dosage sensitive interactions with Drosophila's Abl oncogene (Henkemeyer, 1987; Gertler, 1989), a non receptor tyrosine kinase that has SH2 and SH3 protein interactions domains. Flies that are Abl^- die as fully formed adults either in the pupal case (pharate adults) or shortly after eclosion; they have normal external structures other than roughened eyes (Henkemeyer, 1987). Given the mild phenotypes of abl mutant animals, it was possible to design genetic screens to identify mutations in genes that enhance or suppress the Abl mutant phenotypes. It was hypothesized that in a genetic background sensitized by Abl mutations, a 50% reduction in the level of a protein that is regulated by Abl might be sufficiently detrimental to shift the lethal phase from the pharate adult stage to an embryonic or early larval stage. This effect is termed haploinsufficiency dependent on an Abl mutant background (HDA). The genes identified are not haploinsufficient themselves but manifest their effects when the fly is also mutant for Abl. Disabled and prospero are two of the genes identified by this strategy. Although Abl mutants exhibit no visible defects in the embryonic central nervous system (CNS), animals that are doubly mutant for Abl and Dab die as embryos and fail to form proper axonal connections in the CNS. The Dab expression pattern is similar to that of Abl, including increased levels of Dab protein in CNS axons and body wall muscles. Analysis of the Dab sequence predicts that Dab contains sites for tyrosine phosphorylation similar to the major autophosphorylation site in Abl. Thus Dab is a putative substrate for the Abl tyrosine kinase (Gertler, 1993).

Given the genetic interaction between Abl and Disabled, and the potential for a physical interaction between Abl and Dbl, the physical interaction of Disabled with the intracellular domain of Notch (Giniger, 1998) might provide a basis for understanding the developmental role of Abl and Dbl. Described below is evidence that Notch and Abl mutations interact synergistically to produce synthetic lethality and defects in axon extension. These axonal aberrations cannot be accounted for on the basis of changes in cell identity because the Notch/Abl interaction has not been shown to cause neurogenic or anti-myogenic phenotypes. Notch is present in the growth cones of extending axons, and the Abl accessory protein Disabled binds to a signaling domain of Notch in vitro. It is therefore speculated that Disabled and Abl may play a role in Notch signaling in Drosophila axons, perhaps by binding to the Notch intracellular domain (Giniger, 1998).

The gross morphology of the nervous system is typically normal in N/Abl embryos, but specific axon tracts fail to develop. Axonal defects are observed in all of the nerve tracts that are known to require Notch, i.e., the CNS longitudinal tracts between neuromeres and the lateral portion of the ISN. In contrast, longitudinal tracts within each neuromere and commissural tracts appear normal, as do the dorsal and ventral portions of the ISN. The penetrance (fraction of embryos affected) and expressivity (number of affected hemisegments per affected animal) of the N/Abl axonal phenotype depend on the particular combination of alleles used (Giniger, 1998).

In principle, the axonal defects observe in mature N/Abl embryos could reflect a failure either to form axon tracts or to maintain them. Moreover, if the defect is in the initial development of the axon, it could be due to the absence of required substratum cells, the absence or improper identity of the neurons themselves, or else the failure of the actual guidance machinery of the growth cone. To discriminate among these possibilities, the development of pioneer neurons and substratum cells were examined directly for affected axon tracts. The initial extension of pioneer axons was examined in N/Abl embryos. Consistent with the terminal phenotype, the combined MP fascicle, the first to form between successive neuromeres, is obviously aberrant from a very early stage (st 13). In contrast, both the anterior and posterior commissures appear to develop normally, as do the longitudinal tracts within the neuromeres. The substratum cells for affected axon tracts were examined. The MP fascicle projects between neuromeres on a specific Fasciclin II-expressing glial cell, LG5, and this is present in affected hemisegments. In the PNS, the direct cellular substratum for ISN extension in the lateral part of the embryo is a cluster of lateral peritracheal cells that lie along the trachea. Examination of stalled motor axons in an N/Abl embryo shows that the nerve frays and stalls precisely as it attempts to grow along the trachea. Since substratum cells for affected axon tracts are present in N/Abl embryos, the pioneer neurons themselves were examined. The positions and cell body morphologies of the sensory neurons in the PNS provide sensitive assays for the identities of these cells, and these typically appear to be wild type. The neuron aCC that pioneers the ISN and innervates the most dorsal muscle (muscle 1) is readily apparent in N/Abl embryos. The neurons that pioneer the MP fascicle within the CNS are MP1, pCC, dMP2, and vMP2, and cells whose positions and axonal morphologies are appropriate for these cells can be seen in affected hemisegments of N/Abl embryos (Giniger, 1998).

The observation of morphologically normal pioneer neurons and substratum cells in N/Abl embryos is surprising, since perturbation of cell identity seems a priori to be the simplest explanation for the axonal defects in these embryos. Molecular markers for the development of affected pioneer neurons were therefore examined to determine whether their identities were disturbed in some more subtle way. This analysis suggests that most of the axonal defects in Notch/abl embryos cannot be accounted for on the basis of observed transformations of pioneer neuron identity. The converse question was therefore asked: whether Notch-dependent transformations of pioneer neuron identity are sufficient to produce axonal defects like those observed in N/Abl embryos. Indeed they are not. The data show directly that the Notch-dependent perturbations of cell identity induced in temperature-shifted N^ts embryos are not sufficient to produce the axonal defects observed in these embryos. These results therefore provide strong evidence that the requirement for Notch in axon patterning reflects a function of the protein at the time of axon outgrowth, genetically separable from the role of Notch in the establishment of cell identity (Giniger, 1998).

Abl is localized to developing axons: it is thought that Abl works in the axon directly to control cytoskeletal organization and function. Might Notch also act in the axon to control axon extension directly? Notch is known to be present in mature nerves, but its presence in developing nerves, and specifically in growth cones, has not been investigated. Since Notch expression in substratum cells interferes with visualizing growth cones in situ, the localization of Notch protein was examined in primary Drosophila neurons cultured in vitro. Primary fly embryo neurons were differentiated in culture and analyzed either by indirect immunofluorescence with anti-HRP, to characterize neuronal morphologies, or with anti-Notch. All samples were also labeled with anti-Elav, to verify that the cells being examined were neurons. Notch protein is clearly detected on the entire cell surface, including extending axons, and on a variety of bulbous, spiked, and flattened structures at the tips of axons, which have the appearance of growth cones. To test further whether the Notch-containing structures at the ends of axons are bona fide growth cones, cell preparations were double labeled for Notch and for a known growth cone marker, kinesin-ß-galactosidase. Notch protein is present on the growth cones of axons extending in culture (Giniger, 1998).

What might be the physical basis of the N/Abl genetic interaction? It is unlikely that the absence of Abl is affecting Notch protein levels, since Western analysis of extracts from homozygous abl^- females detects wild-type amounts of Notch protein. Such a mechanism would be expected to alter Notch-dependent cell identities as well as cell morphologies, and this does not generally occur. Might Abl bind Notch directly? This seems unlikely. While Abl contains a variety of protein interaction domains, Notch does not resemble its known ligands. It has recently been shown that the Drosophila Numb protein includes a PTB domain that binds two sites in the intracellular domain of Notch, even when Notch is not phosphorylated. Recalling that the Abl-interacting gene disabled includes a PTB domain closely related to the Numb PTB, and which like Numb can bind to nonphosphorylated targets, a test was performed to discover whether Dab can bind the intracellular domain of Notch in vitro (Giniger, 1998).

Three experiments demonstrate that the PTB domain of Drosophila Disabled binds directly to the intracellular domain of Notch in vitro. (1) Initially, beads bearing a glutathione S-transferase (GST) fusion of the Dab PTB domain were incubated in an extract of total embryo protein. Western analysis of the protein bound by Dab shows that GST-Dab selects Notch protein out of an embryo lysate, whereas GST alone binds only a small amount of Notch nonspecifically. (2) It was next asked what portion of Notch is recognized by Dab. Four protein fragments, each of which represents a distinct functional domain from the intracellular tail of Notch, were expressed. These are the RAM23 region (amino acids 1766-1896); the ankyrin repeats (amino acids 1896-2109); the PEST/OPA region (amino acids 2262-2606), and the notchoid region (amino acids 2612-2703). The four proteins were translated in vitro in reticulocyte lysates and assayed for binding to GST-Dab as above. Of the four Notch domains, only the RAM23 peptide binds to GST-Dab, while none of the four bind to GST alone. This pattern is similar but not identical to the pattern of Notch binding to the Numb PTB: like Dab, the Numb PTB binds to the Notch RAM23 domain but not to the ankyrin repeats or PEST/OPA region. Unlike Dab, Numb does bind to the notchoid domain. (3) Finally, to determine whether the Dab-Notch interaction is direct, a stable and soluble N-terminal fragment of the Notch intracellular domain (amino acids 1767-2235) was purifed from bacteria and its binding to the purified Dab PTB domain was assayed. The Notch intracellular domain is precipitated by GST-Dab beads but not by GST alone, demonstrating that the purified Dab PTB domain can bind directly to purified Notch intracellular domain in vitro (Giniger, 1998).

The data above demonstrate that Notch interacts genetically with Abl and biochemically with Disabled. These results beg the question whether Notch interacts genetically with disabled. Since isolated dab alleles were not available, their genetic interactions with Notch could not be tested directly. It was asked, however, whether flies that are triply heterozygous for all three mutations, Notch, abl, and dab, display any synthetic phenotypes. Flies were constructed that were both heterozygous for a strong Notch allele (N⁸ or N^55e11) and for one of two unrelated chromosomes that bear strong mutations of both Abl and dab. All pairwise combinations cause defects in eye development, giving rise to flies with rough eyes reminiscent of the defective eyes observed in Abl homozygotes (Giniger, 1998).

Given the genetic and biochemical evidence for Abl-Dab interaction, it is attractive to speculate that Dab may act as an adaptor protein that links Notch to Abl in response to a signal from Delta. Recruitment of Abl by Notch would in turn engage the actin cytoskeleton via mechanisms similar to those that have been studied in vertebrate systems. The notion that Notch may use distinct signaling pathways to control different downstream events is consistent with analysis of other signaling receptors. For example, receptor tyrosine kinases typically bind and activate a complex array of intracellular signaling proteins upon ligand induction, and different downstream signaling pathways are often responsible for different aspects of the induced phenotype. Finally, there is extensive precedent for receptors that control cell fate in some developmental contexts and cell motility or axon extension in others (Giniger, 1998 and references).

The interaction of Disabled and Notch might not be the last word in the complex signaling functions involved in axon guidance. Disabled has also been shown to interact with Drk, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2. Drk is required during signaling by the Sevenless receptor tyrosine kinase (Sev). One role for Drk is to provide a link between activated Sev and the Ras1 activator Sos. The ability of activated Ras1 to bypass the requirement for Sev function during R7 development has suggested that the primary function of Sev is to activate Ras. However, the model suggesting that the sole function of activated Sev is to bind Drk-Sos has been questioned by genetic studies that suggest the existence of multiple intracellular signaling pathways downstream of Sev. For example, although the association of Drk and Sos does not depend on the carboxy (C)-terminal SH3 domain of Drk, mutations that affect this domain partially compromise Sev signaling. Furthermore, a C-terminal SH3 domain-truncated Drk cannot rescue the lethality associated with homozygous drk mutations. These data suggest that Drk-binding proteins other than Sos may play important roles in signaling by Sev and other RTKs. The possibility that Drk performs functions other than binding to Sos has been investigated by identification of additional Drk-binding proteins. The phosphotyrosine-binding (PTB) domain-containing protein Disabled binds to the Drk SH3 domains. Taken together, these results indicate that Dab is a novel component of the Sev signaling pathway (Le, 1998).

Disabled has been implicated in other RTK signaling pathways. A murine DAB-related protein, mDAB1, has been identified as a tyrosine-phosphorylated protein that binds to the non-receptor protein tyrosine kinase Src. Recently, several reports have shown that mice lacking mDAB1 function have neuronal defects similar to those seen in reeler mice, including abnormal cortical lamination resulting from disruptions of neuronal migration processes. These results suggest that mDAB1 might participate in a signaling pathway triggered by REELIN, a secreted protein released near the targets of migrating neurons. The neuronal defects associated with Drosophila and mouse dab mutations and the identification of DAB as a putative adaptor protein acting downstream of the receptor tyrosine kinase Sev suggest that Dab may function downstream of many RTKs, including ones required for proper development of the Drosophila central nervous system (Le, 1998).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 2411

Structural Domains

Amino acids 462 - 673 are removed by alternative splicing. There are 10 potential sites for tyrosine phosphorylation. The 1521-amino acid Abl protein contains one match to the tyrosine phosphorylation consensus (residue 543), which corresponds to the in vivo autophosphorylation site for vAbl. Of the 10 potential tyrosine phosphorylation sites in Dab, 8 are clustered within a 170-amino acid interval that also contains the three 11-amino-acid stretches of alternating acidic and arginine resides (Gertler, 1993). Like the known vertebrate Disabled homologs, Dab is a novel protein (Gertler, 1999).

date revised: 15 October 99

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