BIOLOGICAL OVERVIEW

cut expression is detected in a bewildering variety of organs, including external sensory organs, Malpighian tubules, ovary follicule cells and precursors of adult muscle cells. These organs appear to share no common developmental origin, and this only adds to cut's intrigue and the mystery of its function (Blocklinger, 1993).

Cut contains a unique homeodomain, different from all other homeodomains, and three unusual domains. Taken together they define the Cut repeat. In the mammalian Cut counterpart, one Cut repeat is known to have DNA binding activity independent of the homeodomain (Andres, 1994).

The regulation of cut is as complex as any other gene with tissue specific enhancers. Eight tissue specific enhancers have been identified for cut, including wing margin external sensory organs, Malphighian tubules, posterior spiracles and tracheae. These enhancers are distributed over 80 kb upstream from the structural gene (Jack, 1995).cut expression in different organs has appeared downstream of Krüppel, pox neural and perhaps achaete. However, cut expression persists after these other genes become silent (Jack, 1995).

Somatically expressed cut interacts with Notch to regulate egg chamber formation and to maintain germline cyst integrity during Drosophila oogenesis. Communication between the germline and the soma during Drosophila oogenesis is essential for the formation of egg chambers and to establish polarity in the developingoocyte. Cut expression initiates in somatic follicle cells in region 2b of the germarium, at about the same time that follicle cells interleave and surround germline cysts. It persists in all follicle cells, including the polar follicle cells and the interfollicular stalk cells, until about stage 6. Between stages 6 and 9 of oogenesis, Cut expression ceases in all the follicle cells except the polar follicle cells. At about stage 10, Cut expression resumes, first in the anterodorsal follicle cells, then throughout the layer of columnar follicle cells which surround the oocyte, and continues in these cells until stage 14 (Jackson, 1997).

Genetic manipulations of cut activity results in defective packaging of germline-derived cysts into egg chambers and disintegration of the structural organization of oocyte-nurse cell complexes to generate multinucleate germline-derived cells. Although these egg chambers invariably

contain 16 germline-derived nuclei, the total number of ring canals is frequently decreased to 14 or 13. The distribution of ring canals is also abnormal in affected egg chambers. cut null alleles in combination with wimp result in egg chamber defects. wimp encodes the RNA polymerase II 140 kD subunit. Ectopic cut expression produces compound egg chamber. cut interacts genetically with the Notch gene and with the catalytic subunit of Protein kinase A gene during egg chamber morphogenesis. cut null mutations suppress loss of Notch function during oogenesis. Two different cut null mutations reduce the incidence of compound egg chambers found in mutant Notch ovaries. Since cut expression is restricted to the somatic follicle cells and cut mutant germline clones are phenotypically normal, it is proposed that the defects in the assembly of egg chambers and the changes in germline cell morphology observed in cut mutant egg chambers are the result of altered interactions between follicle cells and germline cells. It is suggested that cut participates in intercellular communications by regulating the expression of molecules that directly participate in this process (Jackson, 1997). Interestingly, Notch and cut also interact, albeit synergistically, during the development of the wing margin; the activation of cut expression along the prospective wing margin has been shown to depend on Notch activity (de Celis, 1996, Micchelli, 1997 and Neumann, 1996).

Depending on the tissue observed, cut mutations cause a variety of transformations and defects. External sensory organs are transformed into chordotonal neurons (Blockinger, 1991). Malpighian tubules become bladder-like structures (Liu, 1992). Multiple dendritic organs are transformed from one subtype to another (Brewster, 1995).

If one generalization is to be made, it must be that cut expression is so broad, it may be found in a subset of cell types for any given tissue. Also relevant is the fact that cut is activated at the precursor stage, and expression persists in the differentiated progeny. It would seem that cut is responsible for subtype specificity in a number of cell lineages, possibly for the maintenance of gene expression in differentiated cells. Additionally amazing is the conservation of this gene's structure and function through 600 million years of evolution.

The selector gene cut represses a neural cell fate that is specified independently of the Achaete-Scute-Complex and atonal

The peripheral nervous system of Drosophila offers a powerful system to precisely identify individual cells and dissect their geneticpathways of development. The mode of specification of a subset of larval PNS cells, the multiple dendritic (md) neurons (or type II neurons),is complex and still poorly understood. A morphological categorization of mdneurons reveals three subpopulations: md-da neurons arethe most abundant subclass, which have extensive dendriticarborizations; md-bd neurons have bipolar dendrites; and md-td neurons extend their dendrites along tracheal branches.Within the dorsal thoracic and abdominal segments, two md neurons, dbd and dda1, apparently require the proneural gene amos but not atonal or Achaete-Scute-Complex (ASC) genes. ASC normally acts via the neural selector genecut to specify appropriate sensory organ identities. Dbd- and dda1-type differentiation is suppressed by cut in dorsal ASC-dependent md neurons. Thus, cut is not only required to promote an ASC-dependent mode of differentiation, but also represses an ASC- and ato-independent fate that leads to dbd and dda1 differentiation (Brewster, 2001).

The requirement for proneural gene function is arguablythe most reliable criterion for distinguishing differentsensory cell types. In support of this idea, neurons thatrequire the same proneural gene for their formation havebeen shown to project to the central nervous system in a similar fashion. ASC is required for the formation of all es neurons,for a large number of md neurons, which are lineage-related to es organs, and for most solo md lineages. In contrast, the ch organs and three ventral ch-related md neurons depend on ato. In the absence of both ASC and ato, the only remaining cells of the PNS are two dorsally located md neurons, suggesting that they are dependent on an unknown proneural gene (Brewster, 2001).

Since all the components of the PNS, including these twomd neurons, require the bHLH gene da for their formation, it is likely that the 'missing' proneural gene also codes for a bHLH-containing bindingpartner for Da. Indeed, a likely candidate gene has recentlybeen identified, which apparently is required for the formation of dbd and a dorsal da neuron (Brewster, 2001).

pdm1 and pdm2, two closely linked genes belonging tothe POU family of transcription factors, are co-expressed intwo PNS neurons that are potentially coincident with ASC/ato-independent neurons: dbd and a dorsal md-daneuron. In addition, the ligament cells of lch5also weakly express these genes. To determine if the pdm-expressing cells are ASC-and ato-independent, the pdm1 expressionpattern was examined in ASC and ato single and double mutant embryos.In all three mutant configurations pdm1 expression ispresent in dbd and a dorsal md-da neuron. The latter will be referred to henceforth as dda1 (dorsal da neuron #1). Thus, pdm specifically marks the ASC/ato-independent subclass of PNS neurons (Brewster, 2001).

In order to further characterize ASC/ato-independent mdneurons, other markers expressed in thesecells were sought. en and the lacZ reporter gene from theE7-3-49 enhancer trap line were identifed. In the PNS, en is expressed in one dorsal md neuron as early as stage 11. The E7-3-49 line confers lacZ expression to several PNS cells, including dbd and 2-4 dorsal da-md neurons. Co-incidence of expression of pdm1 with that of en and E7-3-49 was examined in the PNS. Double-labeling for expression of pdm1 and E7-3-49-lacZ reveals that they overlap in dda1 and dbd. Similarly, pdm and en are co-expressed in dda1 but not in dbd (Brewster, 2001).

The expression pattern of the homeobox neural selectorgene cut encompasses all es organs and a large number ofmd neurons (the majority of which are related to es organsby lineage). cut is clearly not expressed in the readily identifiabledbd neuron. Since dbd and dda1 co-express the markersdescribed above and perhaps are specified by the sameproneural gene(s), whether dda1 is indeed negative forcut expression was examined. Embryos double-labeled for cut and E7-3-49-lacZ or pdm show that the pattern of cut expression in thedorsal PNS cluster is complementary to these markers. These results indicate that, unlike the majority of md neurons, dda1 and dbd (along with its siblingglial cell) are specified in a cut-independent fashion (Brewster, 2001).

The observation that dda1 and dbd co-express a numberof cell markers and are both ASC/ato-independent raises thepossibility that these cells derive from a common precursor.In order to address the lineage relation of these cells, random lacZ-expressing clones previously generated using the flp/FRT method were examined. Ofthe four embryos in which the dbd neuron and its accompanying glial cell were clearly lacZ-positive: no dorsal daneuron in the position corresponding to dda1 was co-labeled. These data suggests that dda1 and dbd/sibling glial cells are likely to be generated from two independent precursor cells. The dynamic pattern of amos expression in the dorsal cluster region is consistent with this hypothesis (Brewster, 2001).

The expression of ASC and ato in proneural domains isthought to become restricted to and enhanced in single SOPsduring a process called lateral inhibition, mediated by the'neurogenic' genes. A failure to down-regulate proneural gene expression in most cells of a proneural cluster results in a massive overproduction ofPNS neurons, as observed in neurogenic mutants. Therefore, the number of dbd anddda1 neurons in neurogenic mutants was examined. en- and pdm-expressing PNS neurons are produced in large excess in mutants of Notch, Delta, mastermind and neuralized, and less so inbig brain mutants, similar to the other components of the PNS. These findings suggest that the geneticcontrol mechanism of lateral inhibition is common to allSOPs of the PNS, including the ASC/ato-dependent neurons.The size of ASC proneural clusters that will give rise tothoracic macrochaetes in the adult fly has been estimated at20-30 cells. Due to the close proximity of sensory cells in thePNS and to the limited number of cell-specific markers, thesize of embryonic proneural clusters has been difficult toestimate. Since en is only expressed in dda1, the size of the dda1proneural cluster was assessed by counting the number of en-expressingdorsal PNS cells in neurogenic mutants. In Notch mutants,10 En-positive cells are present on average per cluster (witha range of five to 14 cells), consistent with the estimated number of cells in the amos-expressing proneural clusters (Brewster, 2001).

Since en and pdm are specifically expressed in the ASC/ato-independent PNS, the possibility thatthese two genes may play a role in the formation and/ordifferentiation of dbd and dda1 was examined. (1) pdm1expression was examined in embryos that lacked en function and inembryos in which en was overexpressed via a heat shock-driven transgene. In both en loss- and gain-of-function mutants, a normal number of peripheral pdm-expressing cells was found. Thissuggests that en may not be essential for the formationand correct specification of dda1. It cannot be ruled out,however, that en may have a redundant function in thisprocess. For example, although en⁴ is a strong loss-of-en-function allele, expression of its neighboring sister gene,invected, which can substitute for en function when over-expressed, may not decline in dda1 in the absence of enfunction as it does in most other situations. Alternatively, en may be involved in distinguishing dda1 versus the dbd identity (analogous to the role of cut in specifying es versus ch organ fate). This possibility seemsunlikely, since the overall morphology of dda1, as determined by pdm expression and Nomarski optics, appearsunchanged in en mutants (Brewster, 2001).

(2) en and E7-3-49-driven lacZ expression was examined in embryos that lacked both pdm genes [by using Df(2L)GR4, a genetic deficiency for both genes], and in embryos in which pdm1 or pdm2 were overexpressed using a heat shock vector. In all embryos examined, enand E7-3-49-lacZ expression appeared normal. The characteristic bilateral extensions of dbd were also unchanged in pdm mutants as observed with anti-FasII and 22C10 antibodies. en;Df(2L)GR4 double mutants were generated to determine if dda1 formation is affected when both en and the pdm genes are inactivated. Again, as in the single mutants, double mutant embryos appeared normal at least with respect to dbd formation and morphology. Taken together, en and pdm are unlikely to have a crucial role in the correct specification of the ASC/ato-independent md neurons (Brewster, 2001).

The neural selector gene cut encodes a homeodomain-containing transcription factor and functions as a criticalbimodal switch between different cell fates in the PNS. Incut mutants, es organs are transformed into ch-like organs,as assessed by multiple morphological and immunocytochemical criteria. The ASC-dependent md neurons also acquire an alternative fate in cut mutants. For example, E7-3-49-driven lacZ expression isgreatly expanded in cut mutants and encompasses nearlyall md neurons of the da subtype. In that study, however,it was not clear what type of md fate was in fact assumed bythe new E7-3-49-lacZ-expressing cells (Brewster, 2001).

In order to address this question further, the identity of the transformed md-da neurons in cut mutants was examined with markers for ASC/ato-independent neurons. Similar to E7-3-49, the expression of pdm1 isexpanded to additional neurons in the dorsal and lateralPNS clusters. The cells expressing pdm1 ectopically are also positive for a marker, the E7-2-36 enhancer trap line, which is specific for all md neurons, suggesting that the extra Pdm1-positive neurons are indeed md neurons. Themechanism for restricting ectopic pdm1 but not E7-3-49-lacZ expression to the dorsal and lateral clusters is not known. Overall, these findings are consistent with the interpretation that in cut mutants many ASC-dependent md neurons are transformed towards an ASC/ato-independent rather than an ato-dependent fate (Brewster, 2001).

In contrast to these findings with E7-3-49-lacZ and pdm1expression, when en expression was examined in cutmutants, the pattern of en-expressingPNS cells is unaltered, i.e. there is only one En-positivecell per dorsal cluster. Since en is not expressedin dbd but pdm1 is, the possibility that the invariance ofen expression reflects the acquisition of a dbd rather than adda1 cell fate in cut mutants cannot be ruled out. This possibility seems unlikely, however, since the morphology of supernumeraryPdm1-positive cells is unlike that of dbd, and a marker forthe dbd-associated glial cell (repo) isnot ectopically expressed in cut mutants (Brewster, 2001).

Taken together, it appears that in cut mutants the mdneurons that normally depend on ASC in dorsal and lateralclusters are transformed towards an ASC/ato-independentfate, as determined by pdm and E7-3-49-lacZ expression.However, the postulated cell fate change may be incompletedue to the lack of ectopic en expression. This partial phenotype is not surprising, since cut (null) mutants also exhibitvariability and incomplete phenotypic penetrance withrespect to es organ transformation towards a ch fate. It is thus likely that gene functions other than cut also contribute to the restriction of en and pdm1, similar perhaps to the situation of ato-dependent mdneurons, which do not express cut or pdm/en (Brewster, 2001).

Since ASC-dependent md neurons partially exhibit characteristics of the ASC/ato-independent PNS in cut mutants,it was of interest to see if overexpression of cut results in suppressionof marker gene expression typical of an ASC/ato-independent fate. It has already been shown that cut overexpressionabolishes E7-3-49-driven lacZ expression in the PNS. pdm1expression was examined in cut overexpression embryos, and anearly complete loss of Pdm1 protein was observed in dbd and dda1. Moreover, en expression is also suppressed in virtually all dda1 neurons. Thus, en fails to be ectopically expressed in cut mutants, yet ectopic cut expression is apparently sufficient to repress PNS-specific enexpression in dda1. These findings lend support to thehypothesis that in ASC-dependent md neurons, cut apparently contributes to the repression of an ASC/ato-independent md fate (Brewster, 2001).

GENE STRUCTURE

Bases in 5' UTR - 210

Exons - four

Bases in 3' UTR - 1388

PROTEIN STRUCTURE

Amino Acids - 725

Structural Domains

The Cut protein is a homeodomain homolog. Although it is the most divergent of all Drosophila homeodomain proteins, it shows identity at the nine residues that are invariant in all homeodomain proteins. There are three 60 residue repeats showing 55-68% identity to each other which are not homologous to repeats found in other proteins. These are known as the Cut repeats. There are also four stretches of polyglutamate/aspartate and a number of runs rich in single amino acids (Blochlinger, 1988 and Aufiero, 1994).

The CCAAT displacement protein, the homolog of the Drosophila melanogaster Cutprotein, contains four DNA-binding domains: three CUT repeats (CR1, CR2, andCR3) and the CUT homeodomain (HD). Using a panel of fusion proteins, it was foundthat a CUT repeat cannot bind to DNA as a monomer, but that certain combinationsof domains exhibit high DNA-binding affinity: CR1+2, CR3HD, CR1HD, and CR2HD.One combination (CR1+2) exhibits strikingly different DNA-binding kinetics andspecificities. CR1+2 displays rapid on and off rates and binds preferably totwo C(A/G)AT sites, organized as direct or inverted repeats. Accordingly, onlyCR1+2 is able to bind to the CCAAT sequence, and its affinity is increased bythe presence of a C(A/G)AT site at close proximity. A purified CCAATdisplacement protein/CUT protein exhibits DNA-binding properties similar tothose of CR1+2; and in nuclear extracts, the CCAAT displacement activity alsorequires the simultaneous presence of a C(A/G)AT site. Moreover, CR1+2, but notCR3HD, is able to displace nuclear factor Y. Thus, the CCAAT displacementactivity requires the presence of an additional sequence (CAAT or CGAT) andinvolves CR1 and CR2, but not the CUT homeodomain (Moon, 2001).

These results reveal that cooperation between various CDP/CUT DNA-binding domains can generate at least two DNA-binding activities with distinctly different bindingkinetics and specificities: (1) CR1 and CR2 bind rapidly but transiently to sequences containing two C(A/G)AT sites in either orientation; (2) CR3 and the CUT homeodomain can form a stable complex with the ATCGAT DNA sequence. Moreover, the purified full-length protein binds with higher affinity to oligonucleotides containing two binding sites. Thus, the two bipartite domains CR1+2 and CR3HD can cooperate, albeit weakly, to bind to DNA. Surprisingly, however, the full-length CUT protein binds in vitro with kinetics similar to those of CR1+2, with fast on and off rates. Although a fast on rate was to be expected because of CR1+2, the simultaneous presence of CR3HD should have stabilized the protein on DNA. These results suggest the possibility that CR3HD is not very active in the context of the full-length CDP/CUT protein. This would help explain the lack of extensive cooperativity as noted above. Although such behavior by CDP/CUT in cells could be explained by invoking the phosphorylation of the homeodomainduring the G1 phase of the cell cycle, the same explanation cannot hold in the case of the purified full-length protein that was dephosphorylated in vitro prior to DNAbinding. The possibility is considered that CR1+2, in a manner analogous to high mobility group proteins, may impart a conformational change to DNA thatwould cause the quick release of CR3HD from its adjacent binding site. But when tested as individual proteins, CR1+2 and CR3HD are able to bindsimultaneously to the N19 probe, which contains binding sites for both proteins. Therefore, CR1+2 does not prevent the stable binding of CR3HD to an adjacent site. The results instead suggest that something else, perhaps some conformational constraint, prevents CR3HD from binding to DNA with high affinity in the context of the purified full-length protein; yet the same protein, when present within nuclear extracts in S phase, can stably bind to DNA. The molecular basis for this discrepancy is not known; however, the results clearly point to a difference in the behavior of the CDP/CUT protein in vitro and in nuclear extracts (Moon, 2001).

date revised: 20 Nov 2001  

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