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 developing oocyte. 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 genetic pathways 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 md neurons reveals three subpopulations: md-da neurons are the most abundant subclass, which have extensive dendritic arborizations; 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 gene cut 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 arguably the most reliable criterion for distinguishing different sensory cell types. In support of this idea, neurons that require the same proneural gene for their formation have been 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 two md neurons, require the bHLH gene da for their formation, it is likely that the 'missing' proneural gene also codes for a bHLH-containing binding partner for Da. Indeed, a likely candidate gene has recently been 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 to the POU family of transcription factors, are co-expressed in two PNS neurons that are potentially coincident with ASC/ato-independent neurons: dbd and a dorsal md-da neuron. In addition, the ligament cells of lch5 also weakly express these genes. To determine if the pdm-expressing cells are ASC-and ato-independent, the pdm1 expression pattern was examined in ASC and ato single and double mutant embryos. In all three mutant configurations pdm1 expression is present 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 md neurons, other markers expressed in these cells were sought. en and the lacZ reporter gene from the E7-3-49 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 selector gene cut encompasses all es organs and a large number of md neurons (the majority of which are related to es organs by lineage). cut is clearly not expressed in the readily identifiable dbd neuron. Since dbd and dda1 co-express the markers described above and perhaps are specified by the same proneural gene(s), whether dda1 is indeed negative for cut expression was examined. Embryos double-labeled for cut and E7-3-49-lacZ or pdm show that the pattern of cut expression in the dorsal PNS cluster is complementary to these markers. These results indicate that, unlike the majority of md neurons, dda1 and dbd (along with its sibling glial cell) are specified in a cut-independent fashion (Brewster, 2001).

The observation that dda1 and dbd co-express a number of cell markers and are both ASC/ato-independent raises the possibility 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. Of the four embryos in which the dbd neuron and its accompanying glial cell were clearly lacZ-positive: no dorsal da neuron 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 is thought to become restricted to and enhanced in single SOPs during 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 of PNS neurons, as observed in neurogenic mutants. Therefore, the number of dbd and dda1 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 in big brain mutants, similar to the other components of the PNS. These findings suggest that the genetic control mechanism of lateral inhibition is common to all SOPs of the PNS, including the ASC/ato-dependent neurons. The size of ASC proneural clusters that will give rise to thoracic macrochaetes in the adult fly has been estimated at 20-30 cells. Due to the close proximity of sensory cells in the PNS and to the limited number of cell-specific markers, the size of embryonic proneural clusters has been difficult to estimate. Since en is only expressed in dda1, the size of the dda1 proneural cluster was assessed by counting the number of en-expressing dorsal PNS cells in neurogenic mutants. In Notch mutants, 10 En-positive cells are present on average per cluster (with a 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 that these two genes may play a role in the formation and/or differentiation of dbd and dda1 was examined. (1) pdm1 expression was examined in embryos that lacked en function and in embryos 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. This suggests that en may not be essential for the formation and correct specification of dda1. It cannot be ruled out, however, that en may have a redundant function in this process. 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 en function 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 seems unlikely, since the overall morphology of dda1, as determined by pdm expression and Nomarski optics, appears unchanged 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, en and 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 critical bimodal switch between different cell fates in the PNS. In cut 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 is greatly expanded in cut mutants and encompasses nearly all md neurons of the da subtype. In that study, however, it was not clear what type of md fate was in fact assumed by the 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 is expanded to additional neurons in the dorsal and lateral PNS 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. The mechanism 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 pdm1 expression, when en expression was examined in cut mutants, the pattern of en-expressing PNS cells is unaltered, i.e. there is only one En-positive cell per dorsal cluster. Since en is not expressed in dbd but pdm1 is, the possibility that the invariance of en expression reflects the acquisition of a dbd rather than a dda1 cell fate in cut mutants cannot be ruled out. This possibility seems unlikely, however, since the morphology of supernumerary Pdm1-positive cells is unlike that of dbd, and a marker for the dbd-associated glial cell (repo) is not ectopically expressed in cut mutants (Brewster, 2001).

Taken together, it appears that in cut mutants the md neurons that normally depend on ASC in dorsal and lateral clusters are transformed towards an ASC/ato-independent fate, as determined by pdm and E7-3-49-lacZ expression. However, the postulated cell fate change may be incomplete due to the lack of ectopic en expression. This partial phenotype is not surprising, since cut (null) mutants also exhibit variability and incomplete phenotypic penetrance with respect 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 md neurons, 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 suppression of marker gene expression typical of an ASC/ato-independent fate. It has already been shown that cut overexpression abolishes E7-3-49-driven lacZ expression in the PNS. pdm1 expression was examined in cut overexpression embryos, and a nearly 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 en expression in dda1. These findings lend support to the hypothesis that in ASC-dependent md neurons, cut apparently contributes to the repression of an ASC/ato-independent md fate (Brewster, 2001).

Turtle functions downstream of Cut in differentially regulating class specific dendrite morphogenesis in Drosophila

Dendritic morphology largely determines patterns of synaptic connectivity and electrochemical properties of a neuron. Neurons display a myriad diversity of dendritic geometries which serve as a basis for functional classification. Several types of molecules have recently been identified which regulate dendrite morphology by acting at the levels of transcriptional regulation, direct interactions with the cytoskeleton and organelles, and cell surface interactions. Although there has been substantial progress in understanding the molecular mechanisms of dendrite morphogenesis, the specification of class-specific dendritic arbors remains largely unexplained. Furthermore, the presence of numerous regulators suggests that they must work in concert. However, presently, few genetic pathways regulating dendrite development have been defined. The Drosophila gene turtle belongs to an evolutionarily conserved class of immunoglobulin superfamily members found in the nervous systems of diverse organisms. Turtle is differentially expressed in Drosophila da neurons. Moreover, MARCM analyses reveal Turtle acts cell autonomously to exert class specific effects on dendritic growth and/or branching in da neuron subclasses. Using transgenic overexpression of different Turtle isoforms, context-dependent, isoform-specific effects were found on dendritic branching in class II, III and IV da neurons. Chromatin immunoprecipitation, qPCR, and immunohistochemistry analyses demonstrated that Turtle expression is positively regulated by the Cut homeodomain transcription factor, and genetic interaction studies demonastrated that Turtle is downstream effector of Cut-mediated regulation of da neuron dendrite morphology. These findings reveal that Turtle proteins differentially regulate the acquisition of class-specific dendrite morphologies. In addition, a transcriptional regulatory interaction between Cut and Turtle was established, representing a novel pathway for mediating class specific dendrite development (Sulkowski, 2011).

Analyses of tutl-GAL4 and Tutl protein expression reveal differential expression levels in da neuron subclasses. The highest levels of tutl expression were observed in the more complex class III and IV da neurons, whereas moderate levels of Tutl expression were observed in class II da neurons, followed by weaker levels in class I da neurons. This differential expression pattern correlates with certain aspects of morphological complexity by which these neurons have been subdivided into classes. Specifically, the highest levels of Tutl expression correlate with the appearance of numerous, short processes extending from longer primary branches characteristic of class III and IV da neurons. Interestingly, the transcription factor Cut, a known regulator of dendritic growth, shows a strikingly similar pattern of expression in da neurons In addition to differential Tutl expression in da neuron cell bodies, Tutl localization to dendrites and axons was observed (Sulkowski, 2011).

Consistent with differential Tutl expression levels, novel, cell autonomous and class specific defects were observed in da neuron dendrite morphogenesis in tutl mutants. In class III da neurons, tutl mutation had objectively the most severe phenotype. A significant reduction in both branching and length was observed. Overall, class III da neurons mutant for tutl appeared smaller than their wild-type counterparts, with reduced branching and a reduction in number and length of their characteristic 'spine-like' processes. In fact, by directly analyzing the length of terminal branches, it was possible to show that tutl is required for full extension of these processes. MARCM analyses further revealed that class IV and class II da neurons each require Tutl for normal dendrite development. However, Tutl appears to regulate different aspects of morphogenesis in each class. For the complex, space-filling dendrites of class IV neurons, Tutl is required for dendrites to reach the appropriate length and establish full dendritic field coverage. In contrast, class II neurons require Tutl primarily to promote an appropriate number of branches (Sulkowski, 2011).

Many of the findings reported herein sharply contrast with those of a previous study (Long, 2009) in which a very similar set of experimental analyses were performed to investigate tutl function in da neurons. Among the differences in findings between studies is the question of tutl function in class I da neurons. A previous study implicated Tutl in restricting class I neuron dendritic branching complexity (Long, 2009), however, in general, this study found that Tutl is largely dispensable for class I branching. In light of this striking difference, the previous studies were repeated using the same tutl²³ mutant allele, and the analyses were extended to incorporate additional novel allelic combinations and to include all three class I da neuron subtypes, however the results of the previous study in any class I neuron could not be reproduced. These results demonstrate that, contrary to previous findings, Tutl function is largely dispensable for class I dendrite development and moreover, indicate that allelic variation is not a contributing factor to the observed differences between studies at least for class I neurons (Sulkowski, 2011).

In addition, this study found that tutl exerts differential effects on class II and III dendritic branching and growth, whereas a previous study (Long, 2009), using the tutl²³ mutant allele, failed to reveal any significant effects on class II and III dendrite development. The basis for the differences between the two studies are not entirely clear, however, distinctions between studies include the use of independent tutl alleles, suggesting the potential of allelic variation with respect to phenotypic effects, as well as the fact that the previous study focused exclusively on analyses of the class II ddaB and class III ddaA neurons, whereas the present study examined effects on class II or III neurons as a whole incorporating data from all subtypes within an individual class. Finally, while previous reports, using the tutl²³ allele, suggested that Tutl was required to mediate dendritic self-avoidance in class IV da neurons, this study could not detect any significant effect on self-avoidance. Ultimately, the basis for the observed phenotypic differences with respect to self-avoidance remain unclear, however may again potentially be attributable to allelic variation as independent alleles were used in each study (Sulkowski, 2011).

Isoform-specific Tutl overexpression was found to differentially regulate dendrite branching in class II, III, and IV da neurons, however produced no significant defects in class I da neuron dendrite development. Consistent with these findings, the Long (2009) study reported an inhibition of class IV dendrite branching upon overexpression of the membrane bound Tutl RE40452 protein isoform and observed no defects in class I dendrite development upon Tutl RE40452 overexpression. Interestingly, the current results demonstrate that overexpression of one of the previously characterized non-membrane bound Tutl isoforms, AT02763, inhibited terminal dendritic branching in class IV ddaC neurons, whereas overexpression of two membrane-bound Tutl isoforms, HL01565 and LD28224, promoted terminal dendritic branching in ddaC neurons. Given that both membrane bound and soluble isoform overexpression inhibit class IV dendrite branching, whereas two other membrane bound isoforms promote branching there is no simple relationship in terms of soluble vs. trans-membrane Tutl isoforms in regulating class IV dendritic branching. Moreover, analyses of isoform-specific overexpression in class II and III da neurons further illustrate the complexity of Tutl function in mediating class specific dendritic branching. Overexpression of the soluble AT02763 and GH15753 isoforms in class II neurons promotes dendritic branching, whereas the membrane bound isoforms had no effect. In contrast, in class III neurons, overexpression of AT02763 and GH15753 isoforms in class III neurons had opposing effects on dendritic branching and again overexpression of the membrane bound forms had no effect. Consistent with these isoform-specific dendritic effects, a recent study demonstrated Tutl is required for normal CNS axon development and found that specific Tutl protein isoforms function as attractive axon guidance cues and promote axon branching and invasiveness (Al-Anzi, 2009). The Al-Anzi study demonstrated that overexpression of the membrane bound Tutl isoforms, HL01565 and LD28224, produced increased branching of ISNd motor neurons and sprouting of extra axonal processes in retinal neurons similar to the increased dendritic branching observed with overexpression of these Tutl isoforms in class IV da neurons. Moreover, this study demonstrated that soluble Tutl isoforms (e.g., AT02763) can act as attractive axon guidance cues suggesting Tutl may also exhibit non-cell autonomous functions. However, overexpression of AT02763 in class IV da neurons led to inhibition of dendrite branching, suggesting a potential repulsive function for dendrite branching. Multiple functioning of a gene in both dendrite and axon growth is not without precedent. Another conserved IgSF member, the receptor Roundabout (Robo), first discovered for its regulation of axonal midline crossing has recently been shown as necessary for proper dendrite growth in the CNS and PNS. In da neurons, Robo serves to restrict dendrite outgrowth, consistent with its role as a receptor of repulsive signals. Collectively, these isoform-specific overexpression studies suggest that distinct Tutl isoforms may function in a complex context-dependent, cell-type specific manner to regulate dendritic branching morphology. It is possible that these various Tutl isoforms function in concert to 'fine tune' class specific dendritic branching complexity (Sulkowski, 2011).

The Cut homeodomain transcription factor was among the first genes shown to regulate class specific da neuron dendrite morphogenesis; however, to date the pathway through which it functions has been unknown. Although Cut was shown to influence the level of the Knot transcription factor expression, a direct transcriptional relationship was not established. The current studies reveal that Tutl expression is positively regulated by the Cut. Cut specifically binds to the tutl promoter sequence and acts as a transcriptional activator of Tutl expression in da neurons; this constitutes a novel transcriptional regulatory mechanism by which Tutl may mediate class-specific dendrite morphogenesis. Moreover, genetic interaction studies reveal that Tutl functions as a downstream effector of Cut-mediated regulation of a da neuron dendrite morphogenesis (Sulkowski, 2011).

Expression in class I da neurons, albeit at low levels, established that Cut is not the sole transcriptional regulator of Tutl since Cut is not normally detectable in class I neurons. Consistent with this conclusion, this study and that of Long (2009) were able to detect Tutl protein expression in cut mutant MARCM clones. Collectively, these results indicate that Cut is not absolutely or solely required for Tutl expression in da neurons, however this does not preclude the potential that Cut contributes to transcriptional regulation of Tutl in da neurons as the data indicate. Interestingly, ectopic expression of Cut in class I da neurons leads to a significant increase in dendritic branching complexity, whereas overexpression of Tutl in class I da neurons has no demonstrable effect on dendritic complexity, although the current genetic interaction data reveal that Tutl is required for the observed Cut overexpression effects on class I dendritic complexity. These data suggest that Cut regulation of Tutl expression in class I neurons alone is insufficient to modulate dendritic morphology. This raises the possibility that Cut may positively regulate transcription of both Tutl and a putative partner molecule and thus the gain of function dendrite phenotype observed with ectopic Cut expression is the result of co-upregulation of both Tutl and an as yet unidentified Tutl-interacting protein. In support of this possibility are recent findings in which the deletion of the Tutl carboxy-terminal domain (CTD) of the longest known membrane bound Tutl isoform, RE40452, failed to produce specific loss of function defects in dendrite morphogenesis (Long, 2009), whereas tutl mutations in general produce defects suggesting that a potential partner, possibly a co-receptor, may be required to mediate downstream signaling essential for normal class specific dendrite development. Other members of the IgSF require cis or trans interactions to perform cellular functions. For example, Robo-1 interacts with DCC via conserved cytoplasmic regions to neutralize the attractive effect of Netrin-1. Furthermore, axonal repulsion following Slit binding appears to rely on interactions between Robo-1 and the intracellular phosphoprotein Enabled, which modulates cytoskeleton dynamics. As Cut has been shown to modulate dendritic complexity through the actin cytoskeleton, it would be interesting to determine if potential cytoskeletal changes accompany tutl mutation. The differential changes based on neuronal class that tutl mutants display may manifest via different changes in the underlying dendritic cytoskeletal architecture (Sulkowski, 2011).

The Drosophila tutl gene shares evolutionary homology with both humans (KIAA1355) and mice (Dasm1). The data indicate that Tutl is required for the differential regulation of class specific dendrite morphogenesis including dendritic extension and branching in class II-IV da neurons. These results are consistent with initial studies of Dasm1 which was found to be required in promoting dendrite outgrowth in cultured rat hippocampal neurons (Shi, 2004). However, later studies using a Dasm1 genetic knockout did not observe the reported morphological defects, nor any overt behavioral phenotype. A possible explanation for this discrepancy is the presence of the co-expressed and closely related ortholog IgSF9b which may contribute to functional redundancy. As such, the contribution of mammalian homologs to mediating dendrite morphogenesis remains somewhat unclear. Future studies examining double knockout animals will likely be needed to fully elucidate the contribution of mammalian Tutl homologs to the regulation dendrite development. Due to its apparent lack of functional redundancy, further studies of the Tutl/Dasm1/KIAA1355 family of proteins using Drosophila may be a more direct route to further study this evolutionarily conserved gene family (Sulkowski, 2011).

Similar to Tutl, evolutionarily conserved Cut-related proteins in vertebrates have likewise been implicated in regulating class-specific dendrite morphogenesis. Previous studies have demonstrated that Cut-related protein, CDP, is functionally conserved in regulating dendrite morphogenesis as expression of CDP can rescue cut mutant dendritic defects in Drosophila da neurons and ectopic expression of CDP can phenocopy dendritic defects observed with ectopic Cut expression. Moreover, a recent study has implicated the Cut-related, Cux1 and Cux2 genes, in regulating dendritic branching, spine morphology, and synaptogenesis in upper layer neurons of the cortex. Moreover, these analyses implicate Cux1 and Cux2 in regulating subclass-specific mechanisms of synapse regulation providing further evidence for the evolutionarily conserved role of Cut-related proteins in the specification of class-specific dendritic morphologies. The fact that both Tutl- and Cut-related proteins display evolutionarily conserved roles in directing dendrite morphogenesis, coupled with the findings of a transcriptional regulatory interaction between Tutl and Cut, together with genetic interaction data indicating Tutl functions as a downstream of effector of Cut in da neuron dendrite morphogenesis suggests the potential that murine Cut homologs may also function in transcriptionally regulating the murine Tutl homolog, DASM1 and/or the highly orthologous IgSF9b. Ultimately, elucidating the molecular mechanisms by which Tutl and Cut mediate differential effects on dendrite development will require additional studies aimed at the identification of Tutl-interacting proteins which may be distinct in individual neuron subclasses and identification of other downstream effectors of Cut transcriptional regulation in da neurons (Sulkowski, 2011).

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 Cut protein, contains four DNA-binding domains: three CUT repeats (CR1, CR2, and CR3) and the CUT homeodomain (HD). Using a panel of fusion proteins, it was found that a CUT repeat cannot bind to DNA as a monomer, but that certain combinations of domains exhibit high DNA-binding affinity: CR1+2, CR3HD, CR1HD, and CR2HD. One combination (CR1+2) exhibits strikingly different DNA-binding kinetics and specificities. CR1+2 displays rapid on and off rates and binds preferably to two C(A/G)AT sites, organized as direct or inverted repeats. Accordingly, only CR1+2 is able to bind to the CCAAT sequence, and its affinity is increased by the presence of a C(A/G)AT site at close proximity. A purified CCAAT displacement protein/CUT protein exhibits DNA-binding properties similar to those of CR1+2; and in nuclear extracts, the CCAAT displacement activity also requires the simultaneous presence of a C(A/G)AT site. Moreover, CR1+2, but not CR3HD, is able to displace nuclear factor Y. Thus, the CCAAT displacement activity requires the presence of an additional sequence (CAAT or CGAT) and involves 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 binding kinetics 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 homeodomain during 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 DNA binding. The possibility is considered that CR1+2, in a manner analogous to high mobility group proteins, may impart a conformational change to DNA that would cause the quick release of CR3HD from its adjacent binding site. But when tested as individual proteins, CR1+2 and CR3HD are able to bind simultaneously 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: 12 August 2023  

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