BIOLOGICAL OVERVIEW

How do two cells, the progeny from a single cell division, develop different fates? This is the fundamental question of developmental biology. Both Prospero and Numb proteins are asymmetrically distributed to progeny cells. For a more detailed discussion of the mechanics of how this asymmetric distribution of both Prospero and Numb takes place, see the prospero site. The current essay is concerned with the functional result and significance of such an uneven distribution.

Numb protein is asymmetrically distributed to the progeny of the MP2 precursors in the central nervous system, and to the progeny of Sensory organ precursor (SOP) cells in the peripheral nervous system. In the case of MP2 progeny, one of the two develops into an interneuron with an anterior axon projection; the other (the recipient of Numb) develops into an interneuron with a posterior axon projection. In SOP cells, one of the two progeny becomes the precursor for both bristle cells and socket cells; the other (the recipient of Numb) becomes the precursor of both neuron and glial (sheath) cells. Mutation of numb results in a transformation of cell fate: the fate of the cell normally receiving Numb is transformed into that of the Numb deficient cell (Spana, 1995 and Knoblich, 1995).

How does Numb determine cell fate? In addition to the intrinsic Numb signal, extrinsic signals are also required to produce a normal SOP lineage. Loss of either Delta, Notch or Suppressor of Hairless function results in neuron and glial fate, the opposite of the numb loss-of-function phenotype. This suggests that Numb might confer resistence to Notch-mediated signals in the neuron and glial fates (Spana, 1996 and references).

Does Notch signaling similarly alter the fate of MP2 progeny? Odd-skipped protein and a ß-galactosidase enhancer-trap marker were used to identify the two progeny of the MP2 lineage (dMP2 and vMP2, respectively). Mutations of either Delta or Notch transform vMP2 into dMP2. Numb protein is segregated into the dMP2 neuron. Loss of Numb transforms cells from dMP2 to vMP2. This is the opposite of the tranformation found in either Delta or Notch mutants. If the function of Numb were to specify the dMP2 fate, and the function of Delta and Notch were to keep Numb out of vMP2, double mutants (numb and Notch or numb and Delta) ought to show the numb phenotype (two vMP2s). Alternatively, if Delta-Notch signaling induces vMP2 fate, and localization of Numb into dMP2 cells inhibits this signal, then double mutants would show the Delta or Notch phenotype (two dMP2s). In both double mutants the dMP2 phenotype predominates. This indicates that the function of Numb is to antagonize the Delta-Notch signal specifying the vMP2 fate.

Do the physical distributions of Delta and Notch make sense in terms of their presumed function? Delta is not detected in either dMP2 or vMP2, but rather in adjacent mesoderm (in contact with MP2 and its progeny), while Notch is uniformly distributed throughout all cell types, including the dMP2 and vMP2 neurons. There is no sign of asymmetic Notch localization. If Numb functions to block Notch signaling, as is suspected, then the ubiquitous Notch distribution found is consonant with its proposed function. In this case the Notch ligand (Delta) does not have to be present in either dMP2 or vMP2, but can provide its function from adjacent non-neuronal cells (Spana, 1996).

How does Numb oppose Notch signaling? To assess the possibility of a direct physical interaction between Notch and Numb, a yeast two hybrid interaction assay has been carried out. In this experiment genes coding for fragments of each protein are placed in yeast cells, one attached to a coding sequence specifying a DNA binding protein, and the other attached to a coding sequence specifying a transcriptional activator domain. The binding site for the DNA binding domain is placed next to a ß-galactosidase promoter. The fragment attached to the DNA binding domain is thought of as bait; if the bait interacts with the other protein fragment attached to the transcriptional activator, then ß-galactosidase is transcribed. The Notch intracellular domain consists of an N-terminal RAM23 domain, an ankyrin repeat region (serving as a protein interaction domain interacting with Deltex), a C-terminal PEST sequence (serving to promote protein instablity), and a central domain. The N-terminal RAM23 domain does indeed interact with Numb in the yeast two hybrid experiment. The N-terminal phosphotyrosine binding domain of Numb interacts with the N-terminal area of the intracellular region of Notch. The physical interaction of Notch and Numb has been confirmed using an in vitro physical interaction assay (Guo, 1996).

Since Numb can interact with the intracellular domain of Notch, it is presumbed that Numb can interfer with Notch signaling. The next question: what is the target of Notch? A good candidate is tramtrack, which has been shown to be downstream of Numb (Guo, 1995). Is there an alteration of ttk expression due to reduction or overexpression of Notch? Experimental evidence suggests there is. ttk is normally expressed in the sheath cell, one of the products of the sensory organ precursor lineage, but not in the neural cell, the sister of the sheath cell. In Notch mutants, extra neurons have been detected, resulting from a transformation of sheath cells into neurons. ttk is expressed in most cells in the epidermis of the mutant embryo, but not in neurons, including the supernumerary neurons derived from transformation of sheath cells. Thus Notch function is required not only to specify the sheath cell but also to express ttk in this daughter cell of an asymmetric division. In a reciprocal experiment, overexpression of Notch turns on ttk expression in cells that normally do not express ttk. It is concluded that Notch targets ttk presumably downstream of Numb (Guo, 1996).

GENE STRUCTURE

There are two transcripts: maternal and zygotic. The promoters and first exon of the two transcripts differ (Uemura, 1989).

cDNA clone length - maternal - 3.1 kb; zygotic - 3.5kb.

Bases in 5' UTR - zygotic, 791

Exons - two for maternal, two for zygotic

Bases in 3' UTR - 1051 for each, maternal and zygotic

PROTEIN STRUCTURE

The maternal and zygotic proteins differ only in the first 41 amino acids, which are absent from the zygotic protein (Uemura, 1989).

Amino Acids - maternal 515, zygotic 557

Structural Domains

The zinc finger, common to maternal and zygotic forms, has a CHX4-CX12-CX4-C motif, one commonly found in zinc fingers. The amino terminal region (partially deleted in the maternal transcript) has many charged amino acids. Both maternal and zygotic forms have multiple PEST sequences, correlating with rapid protein turnover. An N-terminal domain consists of residues predictive of a phosphotyrosine binding domain (PTB domain) (Uemura, 1989 and Zhong, 1996).

Numb protein has an N-terminal phosphotyrosine binding domain. Asymmetric localization but not membrane localization of both Prospero and Numb in Drosophila embryos is inhibited by latrunculin A, an inhibitor of actin assembly. Deletion of either the first 41 aa or aa 41-118 of Numb eliminates both localization to the cell membrane and asymmetric localization during mitosis, whereas C-terminal deletions or deletions of central portions of Numb do not affect its subcellular localization. The N-terminus of Numb protein contains a consensus site for N-myrstoylation, but mutation of this site suggests that it is not required for association with the cell membrane or for asymmetric localization. Fusion of the first 76 or the first 119 aa of Numb to beta-galactosidase results in a fusion protein that localizes to the cell membrane, but fails to localize asymmetrically during mitosis. In contrast, a fusion protein containing the first 227 aa of Numb and beta-galactosidase localizes asymmetrically during mitosis and segregates into the same daughter cell as the endogenous Numb protein, demonstrating that the first 227 aa of the Numb protein are sufficient for asymmetric localization (Knoblich. 1997).

The Numb protein is involved in cell fate determination during Drosophila neural development. Numb has a protein domain homologous to the phosphotyrosine-binding domain (PTB) in the adaptor protein Shc. In Shc, this domain interacts with specific phosphotyrosine containing motifs on receptor tyrosine kinases and other signaling molecules. Residues N-terminal to the phosphotyrosine are also crucial for phosphopeptide binding to the Shc PTB domain. Several amino acid residues in Shc have been implicated by site-directed mutagenesis as being critical for Shc binding to receptor tyrosine kinases. Homologous mutations have been generated in Numb to test whether, in vivo, these changes affect Numb function during Drosophila sensory organ development. Two independent amino acid changes that interfere with Shc binding to phosphotyrosine residues do not affect Numb activity in vivo. In contrast, a mutation shown to abrogate the ability of the Shc PTB domain to bind residues upstream of the phosphotyrosine virtually eliminates Numb function. Similar results were observed in vitro by examining the binding of the Numb PTB domain to proteins from Schneider S2 cells. These data confirm the importance of the PTB domain for Numb function but strongly suggest that the Numb PTB domain is not involved in phosphotyrosine-dependent interactions. The identity of the PTB domain partner(s) of Numb is not yet known (Yaich, 1998).

date revised: 10 August 98  

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