Evidence that Kuzbanian targets Delta

Given the similar phenotypes produced by loss of Notch signaling and loss-of-function mutations for kuz, it has been suggested that Kuz may be involved in the cleavage of N (Pan, 1996). This hypothesis is not corroborated by recent biochemical studies, indicating that the functionally crucial cleavage of N in the trans-Golgi network is catalyzed by a furinlike convertase (Logeat, 1997). These observations raise the possibility that Notch signaling in vivo is modulated by soluble forms of the Notch ligands (Qi, 1999).

A genetic screen to identify modifiers of the phenotpes associated with the constitutive expression of a dominant negative transgene of kuz (kuzDN) in developing imaginal discs has identified Delta as an interacting gene (X. Wu, W. Wang, and T. Xu, unpublished observation reported by Qi, 1999). Flies expressing this dominant negative kuz construct, despite carrying a wild-type complement of kuz, become semi-lethal when heterozygous for a loss-of-function Delta mutation (X. Wu, W. Wang, and T, Xu, unpublished observation reported by Qi, 1999). In contrast, Delta duplications rescue the phenotypes associated with kuzDN. The kuzDN flies display extra vein material (especially deltas at the ends of the longitudinal veins); wing notching (observed with a low penetrance); extra bristles on the notum, and they also have small rough eyes. When kuzDN flies carry three, as opposed to the normal two, copies of wild-type Notch the bristle and eye phenotypes are not affected, nor are the vein deltas altered. However, the kuzDN phenotypes are effectively suppressed by Delta duplications, indicating that a higher copy number of Dl molecules is capable of overriding the effects of the kuzDN construct (Qi, 1999).

Delta has been shown to be cleaved by Kuz in transfected cultured cells, with the release of the extracellular domain of Delta. Sequencing reveals a putative propeptide processing site that is conserved in all Delta homologs. There is a distinct absence of cleaved Delta in kuz minus embryos but no difference in the processing of Notch. The biological activity of Delta extracellular domain can be detected in culture. Ligand-dependent Notch activation has been demonstrated in cortical neurons, which express endogenous Notch receptors, causing morphological changes as well as retractions of neurites. The same effects are observed when neurons are cultured in the presence of the extracellular domain of Delta. The importance of additional cleavages in Dl, the mode of activity of full-length Dl, and whether the second ligand Serate is also processed are critical questions to resolve. It is now apparent that future analysis of Delta in Notch signaling events must consider its potential as a diffusable ligand (Qi, 1999).

The Adam family metalloprotease Kuzbanian regulates the cleavage of the roundabout receptor to control axon repulsion at the midline

Slits and their Roundabout (Robo) receptors mediate repulsive axon guidance at the Drosophila ventral midline and in the vertebrate spinal cord. Slit is cleaved to produce fragments with distinct signaling properties. In a screen for genes involved in Slit-Robo repulsion, the Adam family metalloprotease Kuzbanian (Kuz) was identified. Kuz does not regulate midline repulsion through cleavage of Slit, nor is Slit cleavage essential for repulsion. Instead, Kuz acts in neurons to regulate repulsion and Kuz can cleave the Robo extracellular domain in Drosophila cells. Genetic rescue experiments using an uncleavable form of Robo show that this receptor does not maintain normal repellent activity. Finally, Kuz activity is required for Robo to recruit its downstream signaling partner, Son of sevenless (Sos). These observations support the model that Kuz-directed cleavage is important for Robo receptor activation (Coleman, 2010).

Genetic and biochemical findings support the hypothesis that cleavage of the Robo receptor (rather than its Slit ligand) by the metalloprotease Kuz is important in the context of midline guidance. Loss of Kuz protease activity or of the cleavage site of Robo in vivo results in ectopic crossing of ipsilateral axons because of the loss of Robo-mediated repulsion, whereas an uncleavable form of Slit is able to rescue guidance defects in slit mutants as well as does Slit-FL. Furthermore, biochemical analyses have demonstrated that Robo is a substrate of KuzADAM10 in vitro. Finally, an Sos recruitment assay demonstrates that reduction of endogenous Kuz protease activity attenuates Slit-dependent relocalization of Sos to the plasma membrane, where it acts as a regulator of actin cytoskeletal rearrangement and, presumably, growth cone retraction (Coleman, 2010).

The data suggest a model in which Kuz promotes Robo ectodomain shedding as a mechanism of Robo activation. It is proposed that Kuz cleavage of Robo is initiated by binding of Slit, and that the release of the ectodomain of Robo causes a conformational change in Robo that allows its cytoplasmic domain to associate with Sos via the SH3-SH2 adaptor protein Dreadlocks. Sos is then properly localized in order to exert its effect on cytoskeletal rearrangement (Coleman, 2010).

In light of the strong evidence from vertebrate studies indicating that the different Slit cleavage products have distinct properties, it was surprising to find that an uncleavable form of Slit can rescue slit mutants as effectively as can wild-type Slit. What then is the significance of Slit cleavage? Although the cleavage fragments are clearly present in western blots of total embryonic protein, it is not known where in the embryo this cleavage is occurring. Proteolysis might be important for developmental events other than axon guidance that involve Slit; for instance, muscle migration or attachment, or formation of the heart. Future experiments might shed light on the significance, if any, for Slit proteolysis in these contexts. Even though Slit-FL and Slit-U (incleavable Slite) appear to be largely interchangeable in the experiments presented in this study, it cannot be ruled out that Slit proteolysis plays a role in fine-tuning axon guidance. Slit-FL does not fully rescue slit mutant axon guidance defects, which leaves open the question of whether cleavage is important for those guidance events that are not rescued; for example, the fine-tuning of the lateral positioning of axons (Coleman, 2010).

Although it seems evident that Kuz activity is important for Robo-mediated growth cone retraction, it is unclear how Robo ectodomain shedding is involved in the repulsive process. Both Notch and ephrins are known to be substrates of Kuz, but the role that Kuz plays in their signaling is very different. GPI-linked Ephrin A2 forms a stable complex with ADAM10, although ADAM10 proteolytic activity is only initiated when EphA3, the transmembrane Eph receptor, is present. ADAM10 cleavage of Ephrin A2 can be considered a permissive event, in that it releases the strong Eph-ephrin tether that attaches the two cell surfaces, thereby allowing the EphA3-expressing growth cone to retract. The role of Kuz in Notch signaling is more directly linked to Notch activation. Although the genetic data cannot distinguish whether Kuz acts in a permissive or an activating capacity with respect to Robo signaling, the observation that the expression of dominant-negative ADAM10 blocks Slit-induced recruitment of Sos to the plasma membrane suggests that Kuz/ADAM10 is likely to be important for the association of Robo with its signaling effectors. In other words, it appears that Kuz/ADAM10 contributes to the initiation of Robo signaling events (Coleman, 2010).

If Kuz is indeed playing an activating role in Robo signaling, it should be regulated in a way to prevent continuous repulsive signaling. The most parsimonious explanation for regulation of Kuz activity is that it is Slit dependent. Indeed Notch and Ephrin proteolysis by Kuz is known to be dependent upon ligand binding. Additionally, other studies have demonstrated that ADAM10 substrates, including APP and Notch, are cleaved upon receptor-ligand binding. Tests were performed to see whether Kuz proteolysis of Robo was also dependent upon ligand binding, but unfortunately it was not possible to detect a Slit-induced effect on Kuz-dependent Robo ectodomain shedding in vitro. However, these experiments were performed in Drosophila S2 cells in which both Robo and Kuz were overexpressed, and the normal regulation of cleavage might not be maintained in this context. The possibility also exists that Kuz processing of Robo might be regulated by calcium influx, differential substrate glycosylation events, or substrate oligomerization, as is observed with some ADAM10 substrates. In the future, it will be important to determine if Robo proteolysis is dependent on Slit binding, perhaps by examining, both in mammalian cells and in vivo, the processing of a Robo receptor that cannot bind Slit (Coleman, 2010).

The full-length form of the Drosophila amyloid precursor protein is involved in memory formation

The APP plays a central role in AD, a pathology that first manifests as a memory decline. Understanding the role of APP in normal cognition is fundamental in understanding the progression of AD, and mammalian studies have pointed to a role of secreted APPα in memory. In Drosophila, APPL, the fly APP ortholog, is required for associative memory. This study aimed to characterize which form of APPL is involved in this process. Expression of a secreted-APPL form in the mushroom bodies, the center for olfactory memory, was able to rescue the memory deficit caused by APPL partial loss of function. The study next assessed the impact on memory of the Drosophila α-secretase kuzbanian (KUZ), the enzyme initiating the nonamyloidogenic pathway that produces secreted APPLα. Strikingly, KUZ overexpression not only failed to rescue the memory deficit caused by APPL loss of function, it exacerbated this deficit. Further, in addition to an increase in secreted-APPL forms, KUZ overexpression caused a decrease of membrane-bound full-length species that could explain the memory deficit. Indeed, transient expression of a constitutive membrane-bound mutant APPL form was sufficient to rescue the memory deficit caused by APPL reduction, revealing for the first time a role of full-length APPL in memory formation. This data demonstrates that, in addition to secreted APPL, the noncleaved form is involved in memory, raising the possibility that secreted and full-length APPL act together in memory processes (Bourdet, 2015).

The majority of studies into APP biology have focused on pathogenic mechanisms. However, it remains crucial to understand the normal physiological function of APP, especially as it is possible that APP loss of function elicits early cognitive impairment in AD patients. This study shows that overexpression of secreted APPL rescues the short-term memory deficit caused by a reduction of APPL level. In sharp contrast, overexpression of the α-secretase, KUZ, which produces sAPPL, exacerbates the memory impairment, a phenotype that is likely due to a deficit in full-length APPL protein level. Supporting this hypothesis, it was further demonstrated that expression of a nonprocessed APPL mutant form is able to restore wild-type memory in an APPL partial loss of function background (Bourdet, 2015).

In the past, two main strategies have been considered as therapeutic approaches for AD. First, inhibition of the β- or γ-secretase has been used to achieve an inhibition of Aβ toxic production. However, reduction of Aβ production is not only an ineffective approach for AD, it also can actually promote further pathology, as these enzymes have numerous substrates. A second proposed approach has been to inhibit the amyloidogenic pathway by activating the α-processing of APP. In addition to the potential beneficial inhibition of the amyloidogenic pathway, the advantage of this type of approach is to also increase the production of sAPPα. Indeed, decreased CSF sAPPα levels were found in familial and sporadic AD patients, and correlated with poor memory performance in patients with AD. Thus, in vitro and in vivo studies indicate that sAPPα is downregulated during AD. Numerous analyses have shown that sAPPα ectodomain has neurotrophic and neuroprotective effects in different models of neuronal stress. In addition, sAPPα exhibits memory-enhancing properties. Intracerebroventricular infusion of anti-sAPPα serum was deleterious for memory, while that of sAPPα was beneficial. However, these studies relied on an exogenous excess of sAPPα and mechanisms of action and potential targets remained to be elucidated. With knock-in mice experiments, showed that sAPPα was sufficient to correct the impairments in spatial learning and long-term potentiation that are present in APP KO mice. This study shows in Drosophila that sAPPL is able to fully rescue the STM deficit caused by a reduction in endogenous APPL level, thus establishing that an APPL soluble form plays a role in memory, and giving further support for a role of secreted forms in memory in mammal systems (Bourdet, 2015).

When the fly α-secretase, KUZ, was overexpressed in the adult MB, no STM-enhancing effect was seen and, unexpectedly, KUZ overexpression in the MB of flies with an APPL partial loss of function exacerbated their memory impairment. Thus, KUZ overexpression was actually deleterious for memory, rather than beneficial. These results contrast with a previous study showing that overexpression of the mammalian α-secretase ADAM10 in an AD mice model led to an increase in sAPPα, and was able to overcome APP-related learning deficits. However, these studies showed that α-secretase activation has a positive impact on memory exclusively under conditions where human APP is overexpressed. In wild-type mice, results were not clear because overexpression of either the wild-type or an inactive form of the bovine ADAM10 altered learning and memory. Furthermore, ADAM10 has many substrates, and no evidence was brought to link the memory deficit to APP (Bourdet, 2015).

Interestingly, this study observed that KUZ overexpression decreases membrane nonproteolyzed APPL level, suggesting that its negative impact on memory in APPL LOF flies is linked to a reduction of nonproteolyzed APPL level. Therefore, strategies aimed at increasing APP α-cleavage may not be appropriate as this could provoke a decrease of fl-APP levels that might be deleterious to APP function (Bourdet, 2015).

Transient expression of a constitutive membrane-bound mutant APPL has the capacity to fully rescue the STM deficit caused by APPL partial loss of function. Thus, both sAPPL and fl-APPL appear to be involved in memory processes. This is in apparent contradiction with the observation that mammalian sAPPα was sufficient to correct spatial learning deficit of APP KO mice. However, in this study APP-like proteins APLP1 and ALPL2 were preserved, and as it is known from double KO analyses that the three APP homologs exert functional redundancy, they may have compensated for the loss of essential fl-APP functions. In consequence, one cannot attribute the memory function exclusively to sAPPα (Bourdet, 2015).

If both fl-APPL and sAPPL carry the capacity to restore wild-type STM in APPL partial LOF flies, it is puzzling to observe that KUZ overexpression in this genetic context is deleterious for memory. Indeed, in addition to causing a decrease in fl-APPL, KUZ overexpression leads to a concomitant increase in sAPPL that should be able to complement fl-APPL deficiency. It is suggested that in this context, fl-APPL level is below threshold so that even high levels of sAPPL cannot restore a wild-type memory. This hypothesis is supported by protein quantification experiments showing a 30% decrease in fl-APPL level. Because APPL was extracted from the whole brain, whereas KUZ overexpression was only driven in a subset of neurons, the effective fl-APPL decrease in the MB must be much higher than 30%. In mammalian cells under steady-state levels, ~10% of APP is located at the plasma membrane. APP has long been suggested to act as a cell-surface receptor; however, such a function has not been unequivocally established. Several reports have shown that APP exists as homodimers. Cis-dimerization of APP would represent a potential mechanism for a negative regulation of APP functions and a concomitant impact on Aβ generation via an increase in β-processing. Interestingly, it has been suggested that APP is a receptor for sAPPα as its binding could disrupt APP dimers (Bourdet, 2015).

In Drosophila, it has been reported that the secreted N-terminal ectodomain of APPL acts as a soluble ligand for neuroprotective functions. Furthermore, coimmunoprecipitation experiments from transfected Drosophila MB intrinsic cells revealed a physical interaction between fl-APPL and sAPPL, suggesting that sAPPL could be a ligand for fl-APPL. The current data showing the involvement of both membrane fl-APPL and sAPPL in memory are consistent with the hypothesis that sAPPL could be a ligand for its own fl-APPL precursor (Bourdet, 2015).

In conclusion, these data reveal for the first time a role for membrane fl-APPL in memory, opening new questions about APP nonpathological functions and relations between secreted and full-length forms in memory processes (Bourdet, 2015).

Post-transcriptional regulation

In common with several transcription units of the E(spl)-C, including E(spl)m4, Bearded contains two novel heptanucleotide sequence motifs in its 3' untranslated region (UTR), suggesting that all these genes are subject to a previously un-recognized mode of post-transcriptional regulation. These sequence motifs are called the Brd box (AGCTTTA) and the GY box (GTCTTCC). Like known sequence elements that function in post-transcriptional regulation, both of these motifs are found in a single orientation and specifically in the UTRs of the genes that include them. Many mRNAs are translationally inactive until they undergo additional cytoplasmic polyadenylation, a process controlled by cytoplasmic polyadenylation elements (CPEs). Polyadenylation is implicated in Brd box function. Negative regulation by the Brd box motif affects steady-state levels of both RNA and protein. This result indicates that Brd boxes have an additional role in regulating translation, beyond the effect attributable to transcript level differences. Thus, the Brd 3' UTR confers negative regulatory activity in vivo. This activity is spatially and temporally general, in that most or all cells are able to respond to Brd boxes. This suggests that some genes expressed outside of proneural clusters may be regulated by these motifs as well. Three other genes that encode negative regulators of PNS development also contain these sequences in their 3' UTRs. In particular, kuzbanian (kuz) and extramacrochaetae (emc) each include single Brd boxes, while hairy (h) contains a GY box. emc also includes four copies of a GY box-related sequence (GTTTTCC) in its 3' UTR, which may be relevant for its regulation. kuz has functions in SOP selection and lateral inhibition, so its expression certainly includes proneural clusters. However, emc and h are expressed in spatial patterns that are largely complementary to proneural clusters in the leg and wing imaginal discs, and are thus possible examples of genes regulated by the Brd box (and possibly the GY box) in territories outside the clusters. Interestingly, the Emc and H proteins, as members of the HLH family, are structurally related to the E(spl)-C bHLH proteins. In contrast, kuz encodes a metalloprotease/disintegrin protein of the ADAM family (Lai, 1997 and references).



kuzbanian is broadly expressed in the embryonic CNS, probably in all neurons. KUZ mRNA is also expressed at lower levels in the epidermis (Fambrough, 1996).

Effects of Mutation or Deletion

kuzbanian mutants exhibit defective development of the central and peripheral nervous systems and the adult eye. Adult flies with mutant kuz clones have supernumery bristles in macro- and microchaete positions, as well as missing bristles in these same areas. The regular array of ommatidia is severly disrupted in adult eyes. In mutant eyes the density of photoreceptors is abnormally low, and none are successfully organized into ommatidia. Chimeric ommatidia at the clone border contain a mixture of pigmented, wild-type photoreceptor cells and mutant, unpigmented cells (Rooke, 1996).

There is a requirement for maternal as well as zygotic kuz. In a kuz maternal null embryo with one zygotic copy of kuz a greater proportion of the embryo develops as neural tissue than in wild type. When no maternal or zygotic kuz is present, most cells of the epidermis have developed a neural fate. In kuz maternal null embryos with one zygotic copy of kuz , a small patch of cuticle develops on the dorsal side of the embryo; presumably the remaining cells that fail to produce cuticle adopt a neural fate. In kuz null embryos, only a tiny dot of cuticle develops (Rooke, 1996).

Maternal null embryos with one copy of the zygotic kuz gene shows hyperplasia and disorganization of the CNS on the ventral side of the embryos, which is a phenotype similar to the neurogenic phenotype of Notch null embryos. Hypertrophy of the nervous system is not restricted to the ventral region. Such a severe neuralizing phenotype is similar to that of shaggy/zeste white 3 null embryos (Rooke, 1996).

Zygotic mutant embryos show major defects in the embryonic axon pattern, such as a reduction in the thickness of the longitudinal axon tracts. Staining with antibody to Fasciclin II, expressed on three parallel axon bundles on either side of the CNS midline, shows that this decrease in longitudinal staining and increase in commissural region staining results from a failure of longitudinal bundles to extend through the intercommissural region. Instead the bundles stall before crossing the segment boundary and form masses of axon material in the commissural region. Although the late pattern of axon bundles is highly aberrant in kuz mutants, at least some initial axon projections are normal. Therefore the failure of axon extension observed in the CNS is not due to a failure in the general axon growth machinery. The defects observed in the axon bundles of zygotic mutants also do not appear to be due to failure of proper cell fate determination. Analysis of CNS cell fate markers, such as Even-skipped and Engrailed proteins, reveals no major changes in th patterns of staining cells. It therefore appears that KUZ is required for CNS axon extension (Fambrough, 1996).

The P-element insertion l(2)k01405 was recovered in a mutagenesis experiment seeking to identify second chromosome genes whose mutation leads to larval/pupal lethality. Late lethality is often associated with defects in the growth of imaginal discs, and indeed l(2)k01405 larvae have imaginal discs of reduced size. Two divergent transcription units were identified on either side of the insertion. One corresponds to the kuzbanian gene. kuzbanian genetic mosaics demonstrate that at particular developmental junctures (during neurogenesis, wing margin formation, and vein width specification) kuzbanian is autonomously required in the cell where Notch is activated. kuz phenotypes are similar to those associated with loss-of function mutations of the neurogenic genes. Abnormal regions in the naked cuticles of l(2)k01405 pharate adults correspond to sites where an initial overproduction of sensory mother cells (SMC) is followed by the differentiation of all SMC descendents as neurons. These results indicated that kuz belongs to the neurogenic class of genes. kuz1405 clones in the wing and notum result in the differentiaation of tufts of sensory organs, patches of naked cuticle, thicker veins and deletions of wing material in the proximity of wing margin. These kuz clone phenotypes for vein differentiation, wing margin and sensory organ formation are largely similar to those associated with the loss-of-function alleles of Notch and Suppressor of Hairless. Genetic interactions between kuzbanian and different genes of the Notch pathway indicate that kuzbanian is required upstream of Suppressor of Hairless, as evidenced by the observation that overexpression of Enhancer of split m8 partially corrects the neurogenic phenotype of kuz larvae. The requirement of kuzbanian for signaling by a ligand-dependent Abruptex receptor, but not by a constitutively activated form of Notch, suggests that kuzbanian is involved in the generation of a Notch functional receptor and/or in its activation. However, differences in the phenotypes of loss-of-function Notch and kuzbanian mutations suggest the existence of alternative Kuzbanian-independent mechanisms that generate Notch functional receptors. Thus, whereas N mutant cells have a reduced capacity to proliferate, kuz mutant cells proliferate normally. Moreover, kuz neurogenic phenotype is milder than those associated with the loss of N or Su(H). This suggests that kuz mutant cells have residual active Su(H), which allows part of the sensory mother cell progeny to develop as the epidermal component of sensory organs (Sotillos, 1997).

The receptor protein Notch plays a conserved role in restricting neural-fate specification during lateral inhibition. Lateral inhibition requires the Notch intracellular domain to coactivate Su(H)-mediated transcription of the Enhancer-of-split Complex. During Drosophila eye development, Notch plays an additional role in promoting neural fate independent of Su(H) and E(spl)-C, and this finding suggests an alternative mechanism of Notch signal transduction. Genetic mosaics were used to analyze the proneural enhancement pathway. Proneural enhancement involves upregulation of proneural gene expression in single cells that will become neurons. In Drosophila eye development, Notch (N) is required for proneural enhancement in addition to lateral inhibition. The molecular mechanism of proneural enhancement has not been determined. As in lateral inhibition, the metalloprotease Kuzbanian, the EGF repeat 12 region of the Notch extracellular domain, Presenilin, and the Notch intracellular domain are required. By contrast, proneural enhancement becomes constitutive in the absence of Su(H), and this leads to premature differentiation and upregulation of the Atonal and Senseless proteins. Ectopic Notch signaling by Delta expression ahead of the morphogenetic furrow also causes premature differentiation. It is concluded that proneural enhancement and lateral inhibition use similar ligand binding and receptor processing but differ in the nuclear role of Su(H). Prior to Notch signaling, Su(H) represses neural development directly, not indirectly through E(spl)-C. During proneural enhancement, the Notch intracellular domain overcomes the repression of neural differentiation. Later, lateral inhibition restores the repression of neural development by a different mechanism, requiring E(spl)-C transcription. Thus, Notch restricts neurogenesis temporally to a narrow time interval between two modes of repression (Li, 2001).

In the developing eye, lateral inhibition restricts the proneural gene atonal (ato) to individual R8 photoreceptor cells, which found each ommatidium. Earlier, ato must first have reached levels of activity sufficient to sustain expression by autoregulation, in conjunction with its bHLH heterodimer partner encoded by daughterless (da) and with a zinc-finger protein encoded by senseless (sens). Such 'proneural enhancement' depends on N and Dl but not on Su(H) or E(spl)-C. Clones of cells mutant for the E(spl)-C or for Su(H) lead to neural hyperplasia because they lack lateral inhibition, but clones of cells mutant for N or Dl show reduced neural differentiation because they lack proneural enhancement. These divergent phenotypes show that proneural enhancement occurs by a mechanism distinct from that of lateral inhibition (Li, 2001).

Mosaic analysis with Notch pathway mutations have been used to elucidate the mechanism of proneural enhancement. Requirements similar to those of canonical N signaling for processed forms of Dl, Notch EGF repeats 10-12, and proteolytic processing of the N intracellular domain have been found. Proneural enhancement is independent of any Su(H)-mediated gene activation but is mimicked by the complete absence of Su(H) protein, and this indicates that proneural enhancement depends on the disruption of Su(H)-mediated gene repression (Li, 2001).

The phenotypes of other mutations can be compared to the E(spl) or N phenotypes. A neurogenic mutant phenotype indicates a role in lateral inhibition, not in proneural enhancement. A hyponeural phenotype indicates a requirement in proneural enhancement (Li, 2001).

The neurogenic phenotype of the metalloprotease kuz suggests that processed Dl might be important for lateral inhibition and that unprocessed, transmembrane Dl may not be sufficient. It is unknown what form of Dl is required for proneural signaling. Clones mutant for kuz show neural hyperplasia. The distribution of R8 cells labeled by Boss antibody is intermediate between the distributions of clones null for E(spl) and for N. This indicates either partial loss of lateral inhibition or a weak proneural phenotype that still permits some neurogenesis to occur. Ato expression was examined to distinguish these possibilities. In kuz clones, Ato protein appears at the same time as it does in neighboring wild-type regions, but it remains at a low level. Posterior to the furrow, small clusters of R8 cells express Ato at a higher level, but many fewer cells do so than in E(spl) clones. This shows that proneural enhancement is affected in kuz mutant clones, but to a lesser degree than in N null clones, so that more cells go on to take the R8 cell fate. An intermediate phenotype associated with small clusters of R8 cells results in combination with the kuz lateral-inhibition defect. This is consistent with a role for processed Dl in proneural enhancement as well as in lateral inhibition, although it is important to note that kuz might have roles besides Dl processing. Such roles might include other aspects of N function (Li, 2001).

Axonal growth cones require an evolutionarily conserved repulsive guidance system to ensure proper crossing of the CNS midline. In Drosophila, the Slit protein is a repulsive signal secreted by the midline glial cells. It binds to the Roundabout receptors, which are expressed on CNS axons in the longitudinal tracts but not in the commissural tracts. An analysis of the genes leak (referred to in much of the literature as roundabout 2) and kuzbanian is presented: both genes are involved in the repulsive guidance system operating at the CNS midline. Mutations in leak, were first recovered by Nusslein-Volhard (1984) based on defects in the larval cuticle. Analysis of the head phenotype suggests that slit may act as an attractive guidance cue while directing the movements of the dorsal ectodermal cell sheath. kuzbanian regulates midline crossing of CNS axons. It encodes a metalloprotease of the ADAM family and genetically interacts with slit. Expression of a dominant negative Kuzbanian protein in the CNS midline cells results in an abnormal midline crossing of axons and prevents the clearance of the Roundabout receptor from commissural axons. These analyses support a model in which Kuzbanian mediates the proteolytic activation of the Slit/Roundabout receptor complex (Schimmelpfeng, 2001).

Another complementation group consisting of five alleles that leads to abnormal commissure formation maps to the genomic interval 34C/D. Subsequent complementation analyses indicates that these alleles are EMS-induced alleles of kuzbanian. In kuzbanian mutant embryos, many Fasciclin II positive axons appear to cross the CNS midline. In fact, it appears as if many, if not all, commissural axons abnormally express the Fasciclin II antigen. As in roundabout or leak, the longitudinal axon tracts of kuzbanian mutant embryos are reduced in size but they are generally found in more lateral positions (Schimmelpfeng, 2001).

The kuzbanian phenotype suggests that kuzbanian might be involved in the repulsive signaling system operating at the CNS midline. Other work has also suggested that Kuzbanian participates in the processing of the secreted Slit protein (Brose, 1999). A possible genetic interaction of the two mutations was tested by first analyzing the CNS phenotype of slit kuzbanian/++ embryos. When heterozygous, both mutations do not lead to a detectable embryonic CNS phenotype. In contrast to this, in 12% of the slit/kuzbanian double heterozygote embryos (or in 6% of the neuromeres out of 650 neuromeres counted), a roundabout-like mutant CNS phenotype was found. In embryos lacking zygotic kuzbanian function, in addition to being heterozygous for slit, a kuzbanian-like CNS phenotype emerged. The position of the longitudinal connectives, however, is shifted toward the CNS midline. Removal of one copy of kuzbanian in a slit mutant background does not enhance the slit phenotype (Schimmelpfeng, 2001).

This could be interpreted such that the Kuzbanian protease is required to activate the Slit protein, which serves as a ligand of the Roundabout receptors. Different allelic combinations of roundabout and kuzbanian were analyzed. roundabout;kuzbanian double heterozygote embryos appear wild type. When one copy of kuzbanian is removed in a roundabout mutant background, a roundabout CNS phenotype developes. The phenotype revealed by Fasciclin II staining may be slightly more extreme compared to the roundabout phenotype, since the lateral Fasciclin II positive fascicles are also affected. The overall axon pattern of embryos homozygous for roundabout and kuzbanian appears to be a slightly more severe phenotype compared to the roundabout phenotype, since axons running along the CNS midline were frequently detected. This is also evident following a Fasciclin II staining (Schimmelpfeng, 2001).

slit and kuzbanian also genetically interact. Since slit is required for head formation as well, the cuticle phenotype of kuzbanian mutant larvae was examined. Removal of the zygotic expression did not lead to a head defect as observed for slit or leak mutant larvae. It is presumed that maternal kuzbanian function may mask a head phenotype. Removal of maternal and zygotic kuzbanian function leads to a neurogenic phenotype with no recognizable head structures (Schimmelpfeng, 2001).

In kuzbanian mutants Leak expression is unaffected. To address the question whether Kuzbanian affects the expression of Roundabout an anti-Robo antibody was used. In wild type embryos, the Roundabout protein is always found on the longitudinal connectives. No Roundabout protein is expressed on commissural tracts. In kuzbanian mutant embryos, however, Roundabout can be detected on commissural axons crossing the CNS midline. Thus, Kuzbanian may function to clear Roundabout/Slit receptor-ligand complex from axons crossing the CNS midline (Schimmelpfeng, 2001).

During development, kuzbanian is expressed ubiquitously and most, if not all, CNS neurons appear to express the gene. Expression of a dominant negative Kuzbanian protein in all CNS neurons using the elav promoter leads to a kuzbanian-like CNS axon phenotype. To better analyze which cells in the developing CNS have to express the Kuzbanian protein in order to prevent the inappropriate crossing of the CNS midline by navigating axons, the GAL4 system was used. Using a single-minded GAL4 driver strain, a dominant negative Kuzbanian protein was expressed in all CNS midline cells. In 18% of the embryos no gross CNS defects were observed; in the remaining embryos, inappropriate midline crossing of Fasciclin II positive axons was found. 40% of these embryos displayed an almost roundabout-like CNS axon phenotype. Additionally, the formation of the lateral Fasciclin II positive axon tracts was affected, pointing toward a non-cell-autonomous function of the dominant negative Kuzbanian protein when expressed in the CNS midline. This is also suggested by the observation that in embryos expressing the dominant negative Kuzbanian protein, muscle fibers traverse the CNS dorsally as observed in slit or leak mutant embryos. It was next of interest to find out whether the expression of dominant negative Kuzbanian in the CNS midline cells also affects the clearance of the Roundabout protein from commissural axons. As observed in kuzbanian mutant embryos, Roundabout is found on commissural axon tracts, suggesting that Kuzbanian participates in the down-regulation of Roundabout expression on commissural axons. This also shows that axons can cross the midline despite the expression of Roundabout (Schimmelpfeng, 2001).

This phenotypic analysis has indicated that in kuzbanian mutant embryos, longitudinally projecting axons expressing Fasciclin II inappropriately cross the midline. These commissural axons also ectopically express the Roundabout receptor, which they never do in wild type embryos. Together with the data obtained from analyzing kuzbanian function during Delta-Notch signaling, this may be interpreted either as a requirement of kuzbanian in the CNS midline to generate the fully active Slit protein or it may indicate that Kuzbanian activates one or more Roundabout receptors in the lateral CNS (Schimmelpfeng, 2001).

Therefore, to further elucidate the function of kuzbanian, a dominant negative version was expressed in all CNS midline cells. Interestingly, this results in a mutant CNS phenotype combining phenotypic traits of roundabout and leak: axons and muscle fibers cross the CNS midline. This may indicate a function of Kuzbanian during Slit processing, possibly influencing the formation of the Slit gradient in the developing Drosophila nervous system. Alternatively, it may be interpreted in such a way that the cleavage of the Roundabout/Slit complex is required to facilitate growth cone retraction at the midline. This latter possibility is reminiscent of recent findings regarding the function of Ephrin signaling. The membrane-anchored Ephrin ligands constitute a large family of axon guidance molecules, which upon binding to Eph-receptor tyrosine kinases frequently mediate repulsive signals. In order to form the receptor-ligand complex, which triggers signaling to the cellular cytoskeleton, the two cells involved must adhere and subsequently cannot pull their plasma membranes apart. Only after the membrane anchored Ephrin ligand is cleaved by the Kuzbanian metalloprotease is the retraction process initiated (Schimmelpfeng, 2001).

In wild type embryos, Commissureless is expressed in the CNS midline cells and down-regulates the expression of Roundabout on commissural axons. It has been suggested that this down-regulation is required in order to allow crossing of contralateral projecting axons. In roundabout mutant embryos, axons frequently cross or even recross the CNS midline since the midline repellent Slit cannot be perceived. Conversely, high levels of roundabout expression result in a commissureless mutant phenotype. In kuzbanian mutant embryos, axons are able to cross the midline despite the expression of Roundabout on commissural axons. The local expression of a membrane-bound dominant negative Kuzbanian protein in the CNS midline mimics this phenotype. Thus, kuzbanian functions at the CNS midline in the clearance of the Roundabout receptor from commissural axons. This process may involve calmodulin and Sos. In the slit;kuzbanian double mutant, too, axons cross the midline and Roundabout protein is found on the surface of commissural axons. Furthermore, in kuzbanian mutants, the function of roundabout appears to be reduced since axons cross the midline, indicating that only the activated Slit/Roundabout complex can induce its clearance from commissural axons (Schimmelpfeng, 2001).

The ADAM metalloprotease Kuzbanian is crucial for proper heart formation in Drosophila

A collection of EMS mutagenized fly lines was screened in order to identify genes involved in cardiogenesis. The present work studied a group of alleles exhibiting a hypertrophic heart. The analysis revealed that the ADAM protein (A Disintegrin And Metalloprotease) Kuzbanian, which is the functional homologue of the vertebrate ADAM10, is crucial for proper heart formation. ADAMs are a family of transmembrane proteins that play a critical role during the proteolytic conversion (shedding) of membrane bound proteins to soluble forms. Enzymes harboring a sheddase function recently became candidates for causing several congenital diseases, like distinct forms of the Alzheimer disease. ADAMs also play a pivotal role during heart formation and vascularisation in vertebrates, therefore mutations in ADAM genes potentially could cause congenital heart defects in humans. In Drosophila, the zygotic loss of an active form of the Kuzbanian protein results in a dramatic excess of cardiomyocytes, accompanied by a loss of pericardial cells. The data presented suggest that Kuzbanian acts during lateral inhibition within the cardiac primordium. Furthermore a second function of Kuzbanian in heart cell morphogenesis is discussed (Albrecht, 2006).

Studies on the function of ADAMs in vertebrates indicate that some of the members of the gene family are involved in cardiac development. Mice, lacking functional ADAM19, exhibit severe defects in cardiac morphogenesis, e.g. ventral septal defects, abnormal formation of the aortic and pulmonic valves and abnormalities of the cardiac vasculature. Recently, it was shown that TACE (ADAM17) is essential for cardiac valvulogenesis in mice, likely by its sheddase function on EGFR. Interestingly, mice lacking a functional TACE die at birth with an enlarged fetal heart with increased myocardial trabeculation and reduced cell compaction, mimicking the pathological changes of noncompaction of ventricular myocardium. In addition, larger cardiomyocyte cell size and increased cell proliferation were reported in ventricles of the TACE knockout mouse hearts. Cardiac restricted overexpression of a non-cleavable, transmembrane TNF/tumor necrosis factor, a major substrate of TACE, in mice provokes a hypertrophic cardiac phenotype (Albrecht, 2006).

Although it is clear that some mammalian ADAMs are crucial for heart development, a role for ADAM metalloproteases during Drosophila heart formation has not reported so far. This study provides multiple lines of evidence that the metalloprotease Kuzbanian/ADAM10 is crucial for cardiogenesis in flies. Screening a collection of mutagenized Drosophila lines for displaying heart phenotypes and the subsequent analysis of selected alleles revealed that mutations in the kuzbanian genes cause a hyperplasic heart (Albrecht, 2006).

Loss of Kuzbanian function and subsequently abrogation of Notch signalling explains the heart phenotype of kuzbanian mutant embryos. Notch activity was found between 6 and 10 h of development in the dorsal mesoderm, from which the cardioblasts, the pericardial cells, and the lymph glands arise. During this time, Notch-dependant lateral inhibition is responsible for selecting cardiac precursors within the dorsal mesoderm. Elimination of Notch during the first half of this period, using a temperature-sensitive Notch mutant, results in substantially more cardioblasts, pericardial cells and lymph gland progenitors. Reducing Notch function between 8 and 10 h causes an excess of cardioblasts and a concomitant loss of pericardial cells, reflecting a crucial function of Notch for cell fate specification. In embryos carrying a deficiency for Notch [Df(1)N81K1], the number of heart cells is strongly increased, pericardial cells are missing, and cardioblasts fail to assemble into a regular tube, reflecting a combined effect on cell number and cell fate. The role of Notch for a particular subset of pericardial cells, the EPCs, has been investigated. Mutants lacking the maternal and zygotic Notch revealed initially enlarged clusters of EPC progenitors. This finding is consistent with the current observation that panmesodermal driven KuzDN causes a loss of Even-skipped expressing pericardial cells at later stages, a phenotype, which was also found in embryos mutant for mastermind, a downstream effector of Notch signalling. Hypertrophy of cardioblasts was also reported using Nts-1/Df(1)N81K1 embryos, but in this case the number of pericardial cells (Zfh1-expressing cells) was reported as being normal. These studies have shown that Notch is required in the dorsal mesoderm (stage 11 to 12) to regulate the initial commitment of cells to the cardioblasts and pericardial cell fate, but also to regulate the choice between the cardioblast and pericardial fate. In addition, a biphasic recruitment of Notch signalling in heart development has been proposed. First, Notch is needed during lateral inhibition for selecting heart precursors in a field of equally competent cells within the dorsal mesoderm, which essentially covers the function of Notch described above. Second, Notch is involved in asymmetric cell division giving rise to specific heart progenitors. The second requirement of Notch is restricted to those heart cells that arise from asymmetric cell division, in conjunction with additional components of the asymmetric cell division machinery, including Sanpodo, Numb and Inscuteable (Albrecht, 2006).

Previous reports have shown that the Notch receptor is a main substrate of Kuzbanian. Proteolytic cleavage of the membrane anchored Notch receptor is interrupted in the absence of Kuzbanian, which finally results in inactivation of Notch signalling. Thus, the observed heart phenotype of the identified EMS-induced kuzbanian alleles is caused by interruption of Notch signalling in the cardiac primordium, respectively. Therefore, a similar phenotype of kuzbanian, mastermind and Notch mutant embryos is expected. Indeed, the five kuzbanian and the three mastermind EMS alleles identified in the current screen for heart mutants as well as the previously described kuzbanian alleles kuz3 and kuz29-4 exhibit cardiac malformations resembling particular aspects of cardiac defects described for Notch mutants. The phenotypes are not completely identical, which can be explained by the maternal product that masks most of the very early recruitments of Kuzbanian. Indeed, embryos lacking any maternally derived Kuzbanian product and contain no zygotic copies of kuzbanian have a early neurogenic phenotype, which is even more severe than strong Notch phenotypes. Therefore, it is assumed that the zygotic kuzbanian mutants reflect a later requirement of Kuzbanian, manifested in the cardiac mesoderm when the maternal product is diminished in this tissue. The cardiac mesoderm develops as cardioblasts to the expense of pericardial cells (and lymph gland cells) in kuzbanian mutant embryos. It is postulated that Kuzbanian plays a dual role during cardiogenesis, first for the selection of cardiac progenitors during Notch dependent lateral inhibition and second for cell fate specification during Notch dependent asymmetric cell division. This explains the strong excess of all subtypes of heart cells and the significant loss of pericardial cells. The excess of cardioblasts firstly arises, because in the absence of Notch dependent lateral inhibition too many progenitors are selected within the dorsal mesoderm. This phenotype is further enhanced in later development when remaining pericardial cells (those that arise from the asymmetric lineage) are transformed into cardioblasts as a consequence of an abrogated Notch dependent asymmetric cell division. This explains why the hyperplasia of the heart is more prominent in segments A2–A8 than in T3 to A1, because the anterior cardioblasts arise from symmetric cell divisions only. If conversion of the pericardial cell fate into the cardioblast cell fate takes place, it occurs in the posterior heart lineage, resulting in a stronger hyperplasia in this heart region as seen in kuzbanian mutant embryos (Albrecht, 2006).

A model predicts that Kuzbanian is essential for heart development by effecting Notch signalling, which is further supported by the fact, that another key component of Notch signalling was retrieved from the screen, based on a nearly identical phenotype. Three alleles of the mastermind gene, which encodes a downstream effector of the Notch signalling pathway, were found. Mastermind has been isolated in various screens as a modifier of Notch signalling and acts on the molecular level via a direct interaction to the ankyrin repeats of the intracellular form of Notch. As in kuzbanian, mastermind mutant alleles give rise to strong excess of cardioblasts and a reduced number of pericardial cells (Albrecht, 2006).

Interestingly, mutations in another component of Notch signalling pathway reveals a slightly different phenotype. liquid facets, which encodes a Drosophila Epsin orthologue, is reported to be responsible for endocytosis and trafficking of the Notch ligand Delta in the signalling cell. Liquid facets homozygous embryos reveal a hyperplasic heart, but a normal number of pericardial cells. The excess of cardioblasts in liquid facets arises due to a preferential expansion of the tinman expressing subtype of cardioblasts (which arise from symmetric cell division), whereas the number of seven-up expressing cardioblasts (which arise from asymmetric cell division) is normal. Moreover, the additional cardioblasts presumably develop at the expense of fusion competent myoblasts from the dorsal mesoderm. In contrast, kuzbanian mutant embryos show an increased number of seven-up expressing cardioblast, although the overall excess of cardioblasts is, similar to the findings in liquid facets mutants, preferentially due to the expansion of the tinman expressing cardioblasts. One explanation is that Kuzbanian is required for the selection of both cardioblast cell types, whereas Liquid facets is not. Additionally, the maternal contribution of Kuzbanian present in kuzbanian mutant embryos might be sufficient for the selection of the correct number of the Seven-up (Svp) progenitors, but not for the symmetrically derived Tinman cardioblast progenitors. Shortly later, absence of Kuzbanian then affects the Notch dependent asymmetric cell division in the Seven-up lineage, resulting in a moderately increased number of Seven-up cardioblasts. This hypothesis is confirmed by observation of a maximum of four Svp cells per hemisegment in the kuzbanian mutant background, fully explainable by an effect on asymmetric cell division in this cell lineage. It should also be mentioned that Liquid facets is reported to be critical for endocytosis and trafficking of Delta in the signalling cells, whereas Kuzbanian is crucial not only for proteolytic processing of Notch, but presumably also for processing Delta as well. As for kuzbanian, maternal contribution of liquid facets likely masks some requirements of this molecule and hampers a direct comparison of the zygotic phenotypes (Albrecht, 2006).

To date, morphogenesis of cardiac cells is not very well understood. After specification, cardioblasts align bilaterally along the a–p axis and migrate together with the overlaying ectoderm towards the dorsal midline. During this process, all cardioblasts become flattened, polarised cells. The heart lumen is formed when the trailing edges of the cardioblasts of either side bend around and contact each other at the dorsalmost position. Recently, a growing number of genes controlling morphogenesis of cardioblasts has been identified. Among these, cell adhesion molecules play a pivotal role during the alignment of cardioblasts (faint sausage), adhesion of opposing cardioblasts and lumen formation (E-cadherin), and maintenance of the normal heart morphology at late embryonic stages (laminin A). For the alignment and migration of cardioblasts towards the dorsal midline, the Toll receptor acts as a critical cell adhesion component. The Drosophila type IV collagen-like protein Pericardin is a crucial factor for the alignment of cardioblasts and the formation of an organised heart epithelium . The polarity of cardioblasts is a prerequisite to form the organised heart tube. Recently, it was shown that the T-box transcription factor neuromancer (Nmr1/H15 and Nmr2/Midline) is required for establishing the polarity of heart cells, most likely by regulating genes that are responsible for the transition of an unpolarised cardioblast progenitor into a flattened polarised cardioblast. A number of studies have shown that Kuzbanian/ADAM10 plays a role in cell–cell communication and cell adhesion. For example, ADAM10 binds Ephrin2A, a protein with a role in neuronal repulsion, is necessary for shedding EGFR ligands and is involved in cleavage of N-cadherin and regulation of cell–cell adhesion. In Drosophila, Kuzbanian mediates the transactivation of EGF receptor as shown by in vivo studies. Kuzbanian is also important for border cell migration in the Drosophila ovary (Albrecht, 2006).

In kuzbanian mutant embryos initial heart cell differentiation takes place. This includes the specification of different subtypes of heart cell, as shown by the use of specific molecular markers, the alignment of cardioblasts on both sides of the heart primordium and the initiation of heart beating. In late embryos (stage 16/17), an abnormal morphology of cardioblasts is observed: an uncoordinated arrangement of ostia-forming cardioblasts and, instead of a single heart lumen, additional lumen-like structures. These findings point to the possibility that Kuzbanian might have a function for final heart cell morphogenesis, e.g. by processing substrates other then Notch. Evidence has been provided that Kuzbanian is involved in the repulsive guidance system in the CNS and interacts genetically with Slit. Interestingly, the Slit/Robo signalling pathway plays a pivotal role in polarity and morphogenesis control of cardioblasts as well, pointing to the possibility that one of these molecules might be a substrate for Kuzbanian. This would explain the heart cell morphogenesis phenotypes seen in kuzbanian mutant embryos. In embryos that express a dominant negative Kuzbanian form driven late in the heart cardioblast, morphogenesis is not significantly affected. This indicates that the drivers used so far are inappropriate to separate different functions of Kuzbanian. Furthermore, it cannot be excluded that the induction of morphogenetic processes requires the Kuzbanian-dependant Notch signalling pathway quite early. This would hamper significantly the separation of Kuzbanian functions using various driver lines and the dominant negative Kuzbanian form. Nevertheless, it remains to be clarified if the described effects on heart cell morphology are primarily due to the absence of Kuzbanian function or occur as a secondary consequence of hyperplasia (Albrecht, 2006).

This paper has shown that Kuzbanian (ADAM10) plays an essential role in heart development. Members of the ADAM gene family in vertebrates are also known to be critical for cardiogenesis. Interestingly, mice lacking ADAM17 exhibit a cardioblast proliferation phenotype as well, besides other defects, indicating a conserved function of ADAM proteins in heart development. It is assumed that Kuzbanian might have additional functions, e.g., as a sheddase on unknown substrates in the cardiac mesoderm, which has to be proven in future studies (Albrecht, 2006).

Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression

During neural lineage progression, differences in daughter cell proliferation can generate different lineage topologies. This is apparent in the Drosophila neuroblast 5-6 lineage (NB5-6T), which undergoes a daughter cell proliferation switch from generating daughter cells that divide once to generating neurons directly. Simultaneously, neural lineages, e.g. NB5-6T, undergo temporal changes in competence, as evidenced by the generation of different neural subtypes at distinct time points. When daughter proliferation is altered against a backdrop of temporal competence changes, it may create an integrative mechanism for simultaneously controlling cell fate and number. This study identified two independent pathways, Prospero and Notch, which act in concert to control the different daughter cell proliferation modes in NB5-6T. Altering daughter cell proliferation and temporal progression, individually and simultaneously, results in predictable changes in cell fate and number. This demonstrates that different daughter cell proliferation modes can be integrated with temporal competence changes, and suggests a novel mechanism for coordinately controlling neuronal subtype numbers (Ulvklo, 2012).

The NB5-6T lineage utilizes two distinct mechanisms to control daughter cell proliferation. In the early part of the lineage, pros limits daughter cell (GMC) proliferation, whereas in the late part canonical Notch signaling in the neuroblast further restricts daughter cell proliferation, resulting in a switch to the generation of neurons directly. The switch in daughter cell proliferation is integrated with temporal lineage progression and enables the specification of different Ap neuron subtypes and the control of their numbers (Ulvklo, 2012).

The data on Notch activation in the NB5-6T lineage, using both antibodies and reporters, indicate progressive activation in the neuroblast: weak at St10-11 and more robust from St12 onward. Thus, Notch activity coincides with the proliferation mode switch. How is this gradual activation of Notch in the neuroblast controlled? NB5-6T undergoes the typical progression of the temporal gene cascade, with Cas expression preceding strong Notch activation. Thus, one possible scenario is that the late temporal gene cas activates the Notch pathway. However, analysis of the E(spl)m8- EGFP reporter shows that this Notch target is still activated at the proper stage in cas mutants. Although this does not rule out the possibility that other, unknown, temporal factors might regulate Notch signaling, it rules out one obvious player, cas. Alternatively, as Notch signaling is off when neuroblasts are formed (a prerequisite for neuroblast selection), Notch activation in the neuroblast at later stages might simply reflect a gradual reactivation of the pathway. Although such a reactivation might at a first glance appear too imprecise, it is possible that the specificity of this particular Notch output -- proliferation control -- might be combinatorially achieved by the intersection of Notch signaling with other, more tightly controlled, temporal changes (Ulvklo, 2012).

Pros and Notch control daughter proliferation in different parts of NB5-6T, and no evidence of cross-regulation between these pathways was found. The limited overproliferation of the lineage when each pathway is separately mutated results not from redundant functions, but rather stems from the biphasic nature of this lineage. Specifically, in pros mutants, Notch signaling is likely to be on in all 'A' type sibling daughter cells, as Numb continues to be asymmetrically distributed between daughter cells. Thus, Notch signaling in 'A' cells may preclude each 'A' cell from dividing even once. This notion is in line with recent studies showing that postmitotic Notch activated cells ('A' cells) within the Drosophila bristle lineages are particularly resilient to overexpression of cell cycle genes. Similarly, in Notch pathway mutants, as Ap cells now divide (in essence becoming GMC-type cells), Pros will still play its normal role in these 'GMCs' and limit their proliferation to a single extra cell division. However, in kuz;pros double mutants, Ap cells are relieved of both types of daughter cell proliferation control and can thus divide for many additional rounds. This notion also applies to early parts of the NB5-6T lineage and probably to the majority of other VNC lineages, as indicated by the extensive overproliferation of the entire NB5-6T lineage, and to the general overproliferation of the VNC. However, based on the findings that neither the Notch pathway nor pros controls neuroblast identity or its progression, it is postulated that these large clones contain a single, normally behaving NB5-6T neuroblast. In fact, the neuroblast is likely to exit the cell cycle and undergo apoptosis on schedule, as neither of these decisions depends upon pros or the Notch pathway. Of interest with respect to cancer biology is that the findings point to a novel mechanism whereby mutation in two tumor suppressors (e.g., Pros and Notch) cooperate to generate extensive overproliferation: not by acting in the same progenitor cell at the same time, but by playing complementary roles controlling daughter cell proliferation (Ulvklo, 2012).

As an effect of alternate daughter cell proliferation patterns, both vertebrates and invertebrates display variability in neural lineage topology. Similarly, progenitors in these systems undergo temporal changes in competence, as evident by changes in the types of neurons and glia generated at different time points. Hence, the temporal-topology interplay described in this study is likely to be extensively used and to be conserved in mammals. As a proof of principle of this novel developmental intersection, single and double mutants were examined for kuz and nab, thereby independently versus combinatorially affecting temporal progression and daughter cell proliferation. Strikingly, these mutants show the predicted combined effect, with the appearance of additional Ap1/Nplp1 neurons beyond those found in each individual mutant (Ulvklo, 2012).

If programmed proliferation switches are conserved, how might such a topology-temporal interplay become utilized in mammals? There are several examples in which different clusters/pools/nuclei of neurons of distinct cell fate are generated from the same progenitor domain in the developing mammalian nervous system. Such pools often contain different numbers of cells, but the underlying mechanisms controlling the precise numbers of each subtype are poorly understood. Based on previous studies in a number of models, at least three different mechanisms can be envisioned. Based on the current study, a novel fourth mechanism is proposed, whereby alteration of daughter cell proliferation is integrated with temporal progression to control subtype cell numbers. These four mechanisms are not mutually exclusive, and given the complexity of the mammalian nervous system it is tempting to speculate that all four mechanisms are utilized during development (Ulvklo, 2012).


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kuzbanian: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Developmental Biology | Effects of Mutation

date revised: 20 April 2015

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