BIOLOGICAL OVERVIEW

The kuzbanian gene is essential for the partitioning of neural and nonneuronal cells during the development of both the central and peripheral nervous system. In neurogenesis, cells from initially equivalent populations are selected to adopt different fates. The selection of neural cells occurs in a stepwise process controlled by proneural genes that confer neural potential on groups of cells. Subsequently, members of the neurogenic gene class ensure that only a single cell in each group is allowed to achieve its neural potential, whereas the other cells become epidermal. The emerging neural cell inhibits the neural potential of its neighboring cells through a process termed lateral inhibition.

Maternal kuzbanian mutants are characterized by neural hyperplasia (too many cells). Animals with kuz mutant clones exhibit clusters of sensory bristles at positions where single sensory bristles would normally be observed. Stimulation of mutant bristles in a reflex test elicites a leg cleaning response, indicating that mutant clusters contain multiple sensory bristles and not just multiple shafts. This multiple-bristle phenotype has been observed in clones mutant for several neurogenic genes, such as Notch and shaggy/zeste white 3. It is indicative of a failure of lateral inhibition during the development of the peripheral nervous system (Rooke, 1996).

What is the specific role of maternal kuz in lateral inhibition? yellow and crinkle marker mutations were used to mark kuz mutant clones in the adult cuticle. This allowed for the determination of the genotype of individual cells and thus the examination of the autonomy of the kuz mutant phenotype. Such analysis can distinguish between sending and receiving roles for a gene involved in the lateral inhibition process.

A role for kuz in lateral inhibition is suggested by the observation that all sensory bristles in a mutant cluster are mutant; no wild-type bristles are ever present in a cluster. Thus, there is a cell-autonomous requirement for kuz in order for cells to be inhibited from adopting a neural precursor fate. A consistent explanation is that kuz is required in cells to receive an inhibitory signal from the emerging neural cells. Cells in the proneural cluster with wild-type kuz function receive the inhibitory signal and are forced to become epidermal, where kuz mutant cells cannot be inhibited and develop as neural precursor cells (Rooke, 1996).

A second distinct role for kuz was revealed by the same mosaic analysis. All mutant bristle clusters are produced at clone borders, regions mutant cells contact wild-type cells. No bristles are ever produced in clone interiors, either singly or in clusters. Large kuz mutant clones therefore cause bare patches devoid of bristles containing only hair-secreting epidermal cells. This phenotype indicates there is a non-cell-autonomous requirement for kuz in bristle development. One simple explanation is that a kuz-mediated neural-promoting signal is produced by neighbors of the neural precursor. Cells inside the kuz mutant clone develop as epidermal cells, perhaps because they cannot obtain the neural-promoting signal. However, kuz mutant cells on clone borders are supplied by their neighboring wild-type cells with the kuz function necessary to promote neural development (Rooke, 1996).

There is precedence for the idea that a neural-promoting signal is produced by neighbors of a neural precursor. When cells of the procephalic neurogenic region (PNR) are transplanted into the ventral neurogenic region (VNR), a full 84% develop into neural clones. Cells from the VNR give rise to neurogenic clones less frequently (50%) when transplanted back into the VNR. To further verify the idea that the PNR cells are firmly committed to their neural fates, PNR cells were transplanted into the dorsal epidermal anlage (DEA). Surprisingly, the overwhelming majority of the cells transplanted did not differentiate into recognizable neural structures. In contrast, cells from the DEA transplanted into the PNR develop frequently and differentiate, in some instances (almost 25% of the time), into neural types. It was concluded that cells in the VNR could produce a diffusible substance promoting neural development (Stüttem, 1991).

Kuzbanian is also required for axonal extention. Zygotic mutants in kuz have dramatic defects in the development of central nervous system axon pathways, with many axons stalling and failing to extend through the nerve cord. Are the two functions of Kuzbanian truely distinct or do they stem from the same root cause? Three lines of evidence suggest that these are in fact distinct functions of KUZ. (1) In kuz mutants, the expression patterns of various cell surface and nuclear markers are normal (e.g., FasII and Even-skipped). If zygotic loss of kuz were affecting cell fate, one would expect these cell markers to show aberrant expression patterns. (2) The initial axon extensions of various cells are normal, again suggesting that their cell fates have been correctly specified. (3) Phenotypic rescue experiments are consistent with the axon extension phenotype, being unrelated to cell fate defects. In phenotypic rescue, kuz is expressed in neurons and not neural progenitors, and neural expression is sufficient to rescue the axon extention phenotype. It is suggested that maternal kuz expression is sufficient to correctly specify cell fates, while neurons show an additional requirement for zygotic kuz during axon extension (Fambrough, 1996).

What are the biochemical functions of this newest neurogenic gene? Kuz is a metalloprotease-disintegrin with highly conserved mammalian homologs. ADAM (standing for A Disintegrin And Metalloprotease) domains are unique among cell surface proteins in possessing both a potential adhesion domain and a protease domain. The name ADAM honors the dual origins of research that have added to the scientific tree of knowledge concerning these proteins: the fields of fertility and snakes. The first ADAMs described, fertilin alpha and beta, are expressed in spermatogonic cells. One ADAM has been implicated in integrin-mediated sperm-egg binding. Domains in ADAMs are also related to domains found in a family of soluble snake venom proteins, the snake venom metalloproteases. Snake venom metalloproteases and disintegrins promote hemorrhage in snake bite victims. Soluble metalloproteases degrade capillary basement membranes, and soluble disintegrins bind to platelet integrins, thereby inhibiting platelet aggregation (Wolfsberg, 1995).

Disintegrin-like domains are known to serve a cell adhesion function. Disintegrins interact with integrins (see the Drosophila protein Myospheroid) through a disintegrin loop, a thirteen amino acid motif which contains an integrin-binding sequence at its tip (for example, RGD). In the metalloprotease domain three histidines function to bind zinc, a glycine residue to allows for a turn in the polypeptide chain, and a glutamic acid functions as a catalytic residue (Wolfsberg, 1995).

Notch and the disintegrin metalloprotease encoded by the kuzbanian (kuz) gene are both required for a lateral inhibition process during Drosophila neurogenesis. A mutant Kuz protein lacking protease activity acts as a dominant-negative form in Drosophila. Expression of such a dominant-negative Kuz protein can perturb lateral inhibition in Xenopus, leading to the overproduction of primary neurons. This suggests an evolutionarily conserved role for Kuz (Pan, 1997).

Genetic and biochemical evidence shows that Notch is an in vivo substrate for the Kuz protease, and that this cleavage may be part of the normal biosynthesis of functional Notch proteins. However, additional evidence provided below indicates that Delta and not Notch is the target of Kuz (Qi, 1999). Flies were generated that express, under heatshock control, dominant negative Kuzbanian (KUZDN). Heat pulses applied during third instar larval stage result in supernumerary macrochaetes only, while heat pulses applied during early pupal stages result in supernumerary microchaetes only. These time points match the periods when SOPs for each bristle type are selected from pools of equivalent cells, suggesting that KUZDN interferes with lateral inhibition during the selection of SOPs. KUZDN was expressed in the morphogenetic furrow of the developing eye under the control of the rough promoter. Flies carrying the rough/KUZDN transgene have supernumerary photoreceptor cells in each ommatidium. kuz is also required for axonal extension at later stages of neural development. Embryos expressing KUZDN in developing neurons show major defects in axonal pathways, such as disruption of longitudinal axonal tracts (Pan, 1997).

kuzbanian acts genetically upstream of Notch. If kuz should act genetically downstream of N then the combination of an activated form of Notch (Nact) and kuz should display the kuz phenotype of extra microchaetes. Conversely, if kuz acts genetically upstream of N then the combination of Nact and kuz should display the Nact phenotype of missing microchaetes. The combination of Nact and kuz display the Nact phenotype, suggesting the kuz acts genetically upstream of N (Pan, 1997).

In cultured cells expressing Notch, expression of KUZDN abolishes a 100 kDA Notch species while a 300 kDa Notch species is not greatly affected. KUZDN also affects the 100kDa species in third instar imaginal discs. After the induction of KUZDN by heat shock, the 100 kDa species disappears; by 4 hours after induction, the 100 kDa species is almost undetectable, while the 300 kDa species has accumulated to a higher level. Only the 300 kDA species of Notch is detected in kuz null embryos (Pan, 1997).

kuzbanian-mediated cleavage of Drosophila Notch

It has been suggested that this results from a requirement for kuzbanian-mediated cleavage of the Notch ligand Delta (Qi, 1999). Using transgenic Drosophila expressing transmembrane Notch proteins, it has been shown that kuzbanian, independent of any role in Delta processing, is required for the cleavage of Notch. Kuzbanian can physically associate with Notch and removal of kuzbanian activity by RNA-mediated interference in Drosophila tissue culture cells eliminates processing of ligand-independent transmembrane Notch molecules. These data suggest that in Drosophila, kuzbanian can mediate S2 cleavage of Notch (Lieber, 2002).

Pan and Rubin (1997) originally proposed that Kuz cleaves Notch. This proposal is in accord with the cell-autonomous requirement for kuz both in Drosophila and in C. elegans. More recently it has been suggested that the phenotypes resulting from loss of kuz are attributable to its role in the processing of Dl (Qi, 1999), although in that case the requirement for kuz would be non-cell-autonomous. This study shows that Kuz can associate with N, and that removal of kuz activity from S2 cells, which do not express Dl, blocks the processing of ligand-independent gain-of-function N molecules. In vivo, a gain-of-function N molecule that is completely Dl-independent displays an absolute requirement for kuz. These results show that kuz can mediate the cleavage of N, and are therefore in agreement with Pan and Rubin's original proposal. However, whereas Pan and Rubin proposed that kuz mediates S1 cleavage, the data suggest that kuz is responsible for S2 cleavage (Lieber, 2002).

The role of kuz/ADAM10 in the N pathway in vertebrates is uncertain. It has been shown that expression of a dominant-negative form of mouse Kuz causes the overproduction of neurons in Xenopus (Pan, 1997) and inhibits Delta-1-like-induced transactivation of a HES-1 reporter in HeLa cells expressing Notch-1. Yet both S2 and S3 cleavages of a ligand-independent N protein occur in cells derived from kuz mutant mice. Whereas mammals have a single kuz/ADAM10 gene, Drosophila has two: kuz, the focus of this work, and another ADAM10 homolog. Perhaps Drosophila kuz has evolved a function distinct from that of ADAM10 (Lieber, 2002).

Although both LN^LexA and LNR^LexA (mutant LexA tagged N proteins consisting solely of the the soluble cytoplasmic domain of N) function in the absence of the ligand Dl, the activity of LNR^LexA, deleted for a fewer number of amino acids than is LN^LexA, is greater than that of LN^LexA. This difference is not caused by an enhanced affinity of Kuz for LNR^LexA, as both associate equally with Kuz in S2 cells. This suggests that the difference in activity of these gain-of-function N molecules results from an enhanced ability of LNR^LexA to be cleaved by Kuz. In fact, the interaction of LN and LNR with Kuz is no greater than that of wild-type N, and the association of Kuz with any of the N molecules occurs in the absence of Dl. In this regard, the association of Kuz with N is like that of Kuz with ephrin-A2, which forms a stable complex with Kuz prior to Eph receptor binding. The binding of clustered Eph receptors to the Kuz-ephrin-A2 complex activates Kuz and triggers ephrin-A2 cleavage. Likewise, the binding of Dl to the Kuz-N complex could activate Kuz and trigger N cleavage (Lieber, 2002).

A curious result is the difference in Kuz dependence of LNR^LexA and DeltaEGF1-18 LNRLexA (for N^{DeltaEGF1-18 LNRLexA}) in vivo. Two explanations are offered for this observation. The first takes into account the difference in Dl responsiveness between Delta1-18 LNRLexA and LNR^LexA. LNR^LexA, which has reduced activity in the absence of Dl, still retains some function in the absence of Kuz, whereas Delta1-18 LNRLexA, which is completely Dl-independent, cannot function in the absence of kuz. One possible explanation for the discrepancy is that there are two pathways that mediate N cleavage and function in embryos. One pathway requires Dl but is independent of Kuz, and the other pathway requires Kuz but is independent of Dl. LNR^LexA can operate in both pathways, so that upon removal of either Dl or Kuz, LNR^LexA still functions via the alternative remaining pathway. Delta1-18 LNRLexA can only operate in the kuz pathway, so that upon removal of kuz it is nonfunctional. There is, however, no strong evidence pointing to a Dl-independent N pathway involving cleavage in embryos, and given the requirement for Dl in the germ line for the differentiation of follicle cells, the generation of embryos that are maternally Dl null is not straightforward (Lieber, 2002).

Therefore the hypothesis is favored that the difference in kuz-dependence of LNR^LexA and Delta1-18 LNR^LexA in vivo is owing to their differing abilities to be cleaved by TACE. TACE, another member of the ADAM family of metalloproteases, mediates S2 cleavage of mammalian N in vitro. Drosophila S2 cells do not contain any detectable TACE RNA, and exogenous TACE only poorly complements the RNAi-mediated loss of kuz activity. The restoration of some S3 product upon expression of TACE is in accord with the residual activity of LNR^LexA in kuz embryos, and in vitro TACE and N do interact, albeit less well than do Kuz and N. In the absence of a TACE mutant, it is not possible to say for certain whether kuz and TACE have redundant functions, if another member of the ADAM family is responsible for the residual S3 cleavage, or if the residual in vivo activity is caused by the expression of LNR^LexA from a heterologous promoter. Hardly any S3 product was generated from Delta1-18 LNR^LexA by exogenous TACE in S2 cells that had been treated with kuz double-stranded RNA, accounting for the in vivo Kuz-dependence. It is not clear why the ability of exogenous TACE to produce S3-cleaved N differs between LNR^LexA and Delta1-18 LNRLexA; however, in S2 cells, even in the presence of endogenous Kuz, Delta1-18 LNR^LexA is not cleaved as well as is LNR^LexA, suggesting that perhaps differences in the secondary structure of the molecules account for their differing responses to TACE (Lieber, 2002).

The pattern of cleavage products generated by expression of TACE in kuz^- S2 cells also provides an explanation for the in vivo biochemical data, which had seemed to suggest that kuz is responsible for S3 cleavage. It is intriguing that both in kuz embryos and in TACE-complemented kuz^- S2 cells there is an accumulation of a protein the size of S2-cleaved N, which is not efficiently cleaved further to produce S3-cleaved N. It is proposed that although TACE can cleave N at juxtamembrane sites, a large fraction of this cleavage is occurring at a site close to but distinct from the S2 site that allows for efficient S3 cleavage. This suggests that cleavage of N at any juxtamembrane site is not immediately followed by efficient S3 cleavage (Lieber, 2002).

A surprising result is the suppression of the kuz neurogenic phenotype by expression of N^LexA. In fact, the size of the nervous system in N^LexA kuz embryos is smaller than in LNR^LexA kuz embryos. Since kuz embryos are neurogenic, the suppression must result from the overexpression of N. This, along with the fact that the suppression does not involve the association of the cytoplasmic domain of N with Su(H), suggests that in the absence of kuz, overexpressed N is competitively interacting with a protein required for neurogenesis. Wild-type N accumulates to a higher steady-state level on the cell surface than does LN or LNR. It has been shown that expression of the extracellular domain of N can disrupt the establishment of proneural clusters in the developing wing disc (Lieber, 2002).

In summary, it has been shown that in flies S2 cleavage can be mediated by kuz. This contrasts with mammalian data that suggest S2 cleavage occurs via TACE. The discrepancy might be owing to mechanistic differences between flies and mammals as has also been shown for S1 cleavage (Lieber, 2002).

PROTEIN STRUCTURE

Amino Acids - 1239

Structural Domains

Kuzbanian has an N-terminal signal peptide, involved in potentiating secretion, a prodomain, often the site of cleavage in the maturation of proteins, a central metalloprotease domain, a disintegrin-like domain that potentially functions to bind integrins, a cysteine rich domain, a transmembrane domain and a cytoplasmic tail (Rooke, 1996).

date revised: 10 June 2024 

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