single-minded

DEVELOPMENTAL BIOLOGY

Embryonic

Single-minded transcript is first found at the cellular blastoderm in mesectodermal precursors of the midline of the CNS. These precursors form two single longitudinal rows of cells, one on either side of the embryo. Through the process of gastrulation [Image], these rows of cells are brought together in the ventral midline. An annulus of single-minded expressing cells is also found around the presumptive anterior midgut where it passes through the brain, between the supra- and subesophageal ganglia (Crews, 1988). Transcripts are also found in the posterior midgut and proctodeum.

By eleven hours of development, protein is found more in midline glial cells than in neuronal elements and in a subset of cells of the foregut (Nambu, 1990).

Expression of single-minded is found in a subset of ventral muscle precursor cells. Null mutations of single-minded result in an alteration of the ventral oblique muscles; muscle fibers form inside the embryo above the central nervous system. This defect is due to a mislocalization of a subset of mesodermal precursor cells. The muscle defect is a consequence of the influence of the central nervous system on ventral muscle development (Lewis, 1994).

Molecular and functional analysis of Drosophila single-minded larval central brain expression.

Developmental regulatory proteins are commonly utilized in multiple cell types throughout development. The Drosophila single-minded (sim) gene acts as master regulator of embryonic CNS midline cell development and transcription. However, it is also expressed in the brain during larval development. This paper demonstrates that sim is expressed in three clusters of anterior central brain neurons: DAMv1/2, BAmas1/2, and TRdm and in three clusters of posterior central brain neurons: a subset of DPM neurons, and two previously unidentified clusters, which were termed PLSC and PSC. In addition, sim is expressed in the lamina and medulla of the optic lobes. MARCM studies confirm that sim is expressed at high levels in neurons but is low or absent in neuroblasts (NBs) and ganglion mother cell (GMC) precursors. In the anterior brain, sim⁺ neurons are detected in 1st and 2nd instar larvae but rapidly increase in number during the 3rd instar stage. To understand the regulation of sim brain transcription, 12 fragments encompassing 5′-flanking, intronic, and 3′-flanking regions were tested for the presence of enhancers that drive brain expression of a reporter gene. Three of these fragments drove expression in sim⁺ brain cells, including all sim⁺ neuronal clusters in the central brain and optic lobes. One fragment upstream of sim is autoregulatory and is expressed in all sim⁺ brain cells. One intronic fragment drives expression in only the PSC and laminar neurons. Another downstream intronic fragment drives expression in all sim⁺ brain neurons, except the PSC and lamina. Thus, together these two enhancers drive expression in all sim⁺ brain neurons. Sequence analysis of existing sim mutant alleles identified three likely null alleles to utilize in MARCM experiments to examine sim brain function. Mutant clones of DAMv1/2 neurons revealed a consistent axonal fasciculation defect. Thus, unlike the embryonic roles of sim that control CNS midline neuron and glial formation and differentiation, postembryonic sim, instead, controls aspects of axon guidance in the brain. This resembles the roles of vertebrate sim that have an early role in neuronal migration and a later role in axonogenesis (Freer, 2011).

Previous studies have demonstrated that sim is expressed in three anterior clusters of neurons in the 3rd instar larval brain. This paper shows that these three clusters are the DAMv1/2, BAmas1/2, and TRdm neurons. Neurons that are Sim⁺ are present in 1st, 2nd, and 3rd instar larvae at three discrete positions in the anterior brain. It is likely that the cells in 1st and 2nd instar larvae correspond to the identified 3rd instar clusters, but this has not been directly shown. All five NBs that give rise to these neurons are Class I NBs, in which the NB generates GMCs, each of which divides once to give rise to two neurons. Axons of the DMAv1/2 and BAmas1/2 neurons ultimately cross the midline, whereas the TRdm axons project ipsilaterally and do not cross. The specific larval and adult functions of these neurons are unknown (Freer, 2011).

This paper describes three additional clusters of Sim⁺ neurons that reside on the dorsal/posterior side of the brain. The dorsal-most cluster can be identified as a subset of DPM neurons, which are derived from a Type II NB that generates transit-amplifying progenitors. Another cluster of neurons could not be unambiguously identified, and was tentatively named PLSC. These cells are also likely to be derived from Type II NBs, since they are relatively dispersed. The third cluster is a small group of neurons, which also were not previously identified, and are tentatively named PSC. The Sim⁺ DPM, PLSC, and PSC neurons all send axonal projections across the midline (Freer, 2011).

Previous analysis of the sim^J1–47 allele showed defects in adult walking behavior. These defects were interpreted as an inability to coordinate movement and are consistent with a lack of interhemispheric communication. Consistent with this notion is the occurrence of five clusters of sim⁺ neurons in the central brain that cross the midline and could be communicating information that coordinates movement. Analysis of the sim^J1–47 adult central complex neuropil revealed a disorganization of the axons that cross the midline, providing a cellular rationale for the behavioral defects. However, it is unclear whether this phenotype reflects an absence of sim function or a hypomorphic condition. In addition, it is unknown whether the underlying phenotype affects neurogenesis or axonogenesis, since either could result in the observed adult phenotype. The sim^J1–47 phenotype may also be due to an indirect non-autonomous effect of sim on other neurons or axons. This study used MARCM to examine the sim null mutant phenotype in DAMv1/2 neurons. After sequencing exonic DNA from 13 sim mutants, three were selected that were derived from different genetic backgrounds and are likely to be null mutants. All three mutants possess in–frame stop codons that should produce truncated Sim proteins. sim⁸ is predicted to produce a protein only 12 amino acids long, whereas sim² and sim^BB68 should produce proteins that terminate in the PAS-2 domain and lack Sim activation domains. Previous work has shown that the absence of the sim activation domains results in a protein that cannot activate transcription in vivo. Consistent with these molecular defects, all three mutants showed similar defects. In all mutant clones, the neurons and initial axonal projections appeared normal, indicating no obvious effects of sim on neurogenesis and neurite outgrowth. However, in 7/12 mutant clones, there were clear axon fasciculation defects. In wild-type clones, the axons from all DAMv1/2 neurons extended across the midline as a single, tightly fasciculated bundle, whereas in sim mutant larvae there were more than one bundle and the axons appeared frayed (Freer, 2011).

While provocative, the sim axonal and behavioral phenotypes raise a number of issues. The first is that while axon guidance defects are observed for the DAMv1/2 neurons, it is unknown whether the behavioral defects are the result of this defect, since the sim^J1–47 mutation may also affect sim function in the other anterior and posterior Sim⁺ brain neurons, the optic lobes, and the midline cells of the ventral nerve cord. It is also is possible that reductions in sim could control additional aspects of terminal differentiation and neurotransmission, which could also contribute to the behavioral phenotype. Another potential developmental role is that the sim⁺ cells themselves do not physiologically contribute to locomotion, but their axons may pioneer the axons of other neurons that control movement. Finally, since only about half of the cells in each cluster were mutant, the presence of genetically wild-type axons mixed with sim mutant axons could mask the severity of the phenotype. These issues can ultimately be resolved using targeted expression of various transgenes affecting sim function, differentiation, and neurotransmission. Axon guidance defects can be assayed in the other sim⁺ neurons using MARCM, but behavioral phenotypes will need to be addressed by targeting disruptions of sim function specifically to each neuronal cluster. The ability to do this was one of the goals of this study, although the Gal4 lines that were generated still generally lack sufficient specificity to fully address this issue. Nevertheless, what is clear is that sim controls proper axonal patterning, but not neurogenesis, of the sim⁺ DAMv1/2 neurons (Freer, 2011).

Mammalian Sim1 and Sim2 and Drosophila sim play multiple roles in development, both in the CNS and in other cell types. Within the CNS, each plays a role in neurogenesis or neural migration and later in axonogenesis. Drosophila sim controls neurogenesis of embryonic CNS midline cells and differentiation of the optic lobe laminar neurons. In mammals, Sim1 plays a prominent role in neuronal migration in the hypothalamus. Additionally, the murine Sim1 and Sim2 genes are expressed in the mammillary body and control axonogenesis. In wild-type mice, the Sim1⁺Sim2⁺ mammillary body cells extend axons along the principal mammillary tract (PMT) that project to the thalamus and tegmentum via the mammillotegmental (MTEG) and mammillothalamic (MTT) tracts. Genetic experiments indicated that the MTEG and MTT are greatly reduced in Sim1Sim2 double mutant embryos and, to a lesser degree, in Sim1 single mutant embryos. Normally, the PMT extends along the ipsilateral side of the developing brain, but in Sim1Sim2 mutant embryos, the axons abnormally cross the midline. This suggests that the mammillary body axons no longer respond to a midline-directed repellent in Sim mutant embryos. Consistent with this interpretation, Sim was shown to normally repress expression of Rig-1/Robo3, a gene that antagonizes Slit-mediated repulsion. Consequently, upregulation of Rig-1/Robo3 in Sim mutant embryos results in the loss of PMT repulsion by the midline (Freer, 2011).

The Drosophila sim DAMv1/2 axonal defect differs from the mammalian Sim mutant defect in that the DAMv1/2 axons show fasciculation defects. Significantly, targeting appears roughly correct, since mutant axons branch and migrate toward the midline. Presumably, sim regulates the expression of one or more genes involved in controlling axonal fasciculation, although the identities of those genes are unknown. There are a number of Drosophila cell adhesion proteins that have been implicated in axon fasciculation, including Fasciclin II, Roughest, and Cadherin-N. One possible explanation for the sim phenotype is that Sim positively regulates the levels of cell adhesion/fasciculation proteins, and when their levels drop below a certain point, defasciculation can occur. Alternatively, there exists a class of genes that are anti-adhesive, such as beaten path and protein tyrosine phosphatases, which may normally be repressed or silenced by Sim, and, thus, in sim mutants become active and promote defasciculation. Further insights into the mechanisms that govern axon guidance of Sim⁺ cells will require identifying the relevant transcriptional targets of Sim (Freer, 2011).

Comprehensive transgenic analysis of the sim regulatory region identified enhancers for all postembryonic sim⁺ brain neurons. The P261 fragment drives expression in the anterior central brain, DPM, PLSC, and medulla neurons. The B2.4 fragment drives expression in the PSC and lamina. Thus, these two fragments, which do not overlap in expression, account for all of the central brain and optic lobe expression. In addition to the P261 enhancer, an adjacent fragment, Q255, drives expression in TRdm, DPM, and the medulla, and another proximate fragment, M582, drives medulla expression. Thus, in some cases, there are multiple enhancers that contribute to expression in specific brain neurons. However, the sim enhancer fragments have not yet been sufficiently subdivided into subfragments capable of driving the expression of each individual brain cell type. In some cases, this may not be possible if expression in different cell types share transcription factor binding sites. In addition, another enhancer in the A1.0 fragment, which resides in the 5′-flanking sequences, is autoregulatory and drives expression in all larval brains cells (Freer, 2011).

The genomic arrangement of the sim brain enhancers provides insights into the mechanisms that control sim expression. RT-PCR data strongly suggest that the brain enhancers function through P_L. While the upstream A1.0 autoregulatory enhancer interacts with P_L in a straightforward manner, the B2.4 and F1.4 enhancers are downstream of P_L and P_E and must skip over P_E to interact with P_L. No relevant brain enhancers were found 3′ to the sim transcription unit, although enhancers in fragments J2.5 and K2.6 with brain expression patterns that do not overlap with sim⁺ neurons are present in the 3′-flanking region. They may control expression of genes 3′ to sim. ChIP-chip embryonic protein analysis of Drosophila insulators by modENCODE has revealed strong binding of the CTCF, Mdg4, and Su(Hw) proteins to a site just 3′ of the sim transcription unit. These proteins may act as insulators to block enhancers 3′ to sim from interacting with upstream sim promoters. Similarly, an insulator site just 3′ to the pic gene may act to insulate pic enhancers from acting on sim promoters, and vice-versa (Freer, 2011).

Effects of Mutation or Deletion

single-minded mutants are embryonic lethals. Denticle bands on all segments are narrowed, and there is a fusion of the ventral arms of the head skeleton as well as the anal plates. Transverse commissures, missing in the ventral nervous system of sim mutants, show aberrant projection of longitudinal pathways, collapsing at the midline (Thomas, 1988). All the sim-positive cells delaminate. All neural and glia cells of the midline are missing in sim mutants (Crews, 1988).

The relative contribution of individual mesectodermal cell (MEC) lineages to CNS midline morphogenesis has been examined in mutations that disrupt commissure formation in Drosophila. The absence of commissures, leading to longitudinal tract collapse, is seen in embryos mutant for the genes single-minded and slit. MEC lineages did not survive in single-minded mutant embryos, in contrast to the survival of all MEC lineages in slit mutant embryos. The midline glial cells are displaced and appeared ultrastructurally normal in slit mutant embryos, yet the presence of the MG is not sufficient to generate commissures. Commissure formation requires correct MEC cytoarchitecture, dependent upon slit activity (Sonnenfeld, 1994).

Mutations in the genes spitz (spi), Star (S), single-minded, pointed (pnt), rhomboid (rho) (all zygotic), and sichel (sic) (maternal), collectively called the spitz group, cause similar pattern alterations in ventral ectodermal derivatives of the Drosophila embryo. The cuticle structures lacking in mutant embryos normally derive from longitudinal strips of the ventro-lateral blastoderm. Defects are found in the median part of the central nervous system. In addition, the nerve cells expressing the even-skipped protein appear abnormally arranged. These results suggest that groups of cells from the same region, including both epidermal and neural precursor cells, require spitz-group gene activity for normal development. The members of the spitz group differ from one another: sim affects a more median strip of the ventral ectoderm than the other zygotic genes and pnt causes separation rather than deletion of pattern elements. As shown by pole cell transplantations, spi and S are also required for normal development of the female germ line, while sim, rho, and pnt appear to be exclusively zygotically expressed. Of all the spitz-group genes, sim appears to have the most specific effect on the embryonic pattern (Mayer, 1988).

The CNS midline of Drosophila should not be considered as an isolated autonomous entity but as an organizing center for the rest of the CNS. Cells located at the midline of the developing central nervous system perform a number of conserved functions during the establishment of the lateral CNS (the rest of the CNS as distinguished from the midline). The midline cells of the Drosophila CNS are required for correct pattern formation in the ventral ectoderm (which gives rise to the rest of the CNS) and for induction of specific mesodermal cells. The midline cells are also required for the correct development of lateral CNS cells. Embryos that lack midline cells through genetic ablation show a 15% reduction in the number of cortical CNS cells. A similar thinning of the ventral nerve cord can be observed following mechanical ablation of the midline cells. A number of specific neuronal and glial cell markers have been identifed that are reduced in CNS midline-less embryos, as for example in single-minded embryos, in early heat-shocked Notch(ts1) embryos or in embryos where the midline cells have been mechanically ablated. Genetic data suggest that both neuronal and glial midline cell lineages are required for differentiation of lateral CNS cells. One marker, the rR226 enhancer trap insertion, reveals a reduction in the number of marker positive cells in midline ablated embryos. Loss of orthodenticle, a gene expressed specifically in midline neurons, results in the degeneration of many midline neurons. Compared to wild type, the number of rR226-positive cells is reduced in otd mutant embryos. Likewise, in embryos lacking pointed transcript P2, the number of rR226-positive cells decreases. The lateral CNS phenotype of single-minded mutant embryos can be rescued by transplantation of midline cells as well as by homotopic expression of single-minded. Midline cells transplanted laterally in the neuroectoderm show a tendency to migrate back to the midline and do not remain at the point of implantation. sim expression in the entire neuroectoderm results in a routing of cells towards different midline cell fates (glial or neuronal) strictly according to their position in the segment. Thus, upon sim expression in the neuroectoderm, midline glial cells form in the anterior part of the segment, whereas neuronal cell types form in the posterior part of the segment. Whereas the lateral midline neurons remain where they are induced, the ectopic midline glial cells migrate toward axonal tracts, which might be comparable to the normal migration of the midline glial cells toward commissural axons. Ectopic midline cells are able to induce enhanced expression of some lateral CNS cell markers such as rR226. This effect is not due to secreted Spitz, as ectopic expression of Spitz does not enhance rR226 expression. It is thus concluded that the CNS midline plays an important role in the differentiation or maintenance of the lateral CNS cortex (Menne, 1997).

Isolation and analysis of tango mutants reveal CNS midline and tracheal defects, and gene dosage studies demonstrate in vivo interactions between single-minded::tango and trachealess::tango. Defects in CNS midline neurons and glia were examined using enhancer trap reporters. In wild-type embryos, the AA142 enhancer trap is expressed in an average of 3.5 midline glia per segment by stage 14 of embryogenesis. In tango mutant embryos, there is a reduction in the number of stained midline glia to approximately one cell per segment. The X55 enhancer trap gene stains the ventral unpaired median neurons (VUMs) and the median neuroblast (MNB) and its progeny in the ventral region of the CNS. In tango mutant embryos, the number of VUM neurons and MNB progeny are reduced in number (60% of wild-type) and do not migrate into the ventral regions of the ventral cord. The role of tango in tracheal development was examined by staining tango mutant embryos with monoclonal antibody 2A12, which stains the lumen of the tracheal tubes. tango mutant embryos are shown to have a variety of tracheal defects. Experiments with heterozygotes show that tango interacts genetically with both trachealess and single minded (Sonnenfeld, 1997).

Hairless plays an important role as the major antagonist in the Notch signaling pathway in Drosophila. It appears to be a direct inhibitor of the signal transducer Su(H). Hairless encodes a pioneer protein that has been dissected in a structure-function analysis; a series of deletion constructs was tested for wild type and gain of function activity in the fly as well as for Su(H) binding. In this way, the Hairless protein was subdivided into the absolutely essential Su(H)-binding domain, important N- and C-terminal domains and a central antimorphic domain. A construct C2 that deletes the Su(H) binding domain has some activity during wing development, suggesting that Hairless has additional functions apart from Su(H) binding. For example, overexpression of the C2 deleted protein causes a novel, net-like wing phenomenon that cannot be explained by Su(H) inhibition. The central acidic domain may mark a repression domain of the Hairless protein required for silencing Hairless function, e.g. for releasing Su(H) from a H/Su(H) complex. It is speculated that the C-terminal region comprises an interactive surface for additional components involved in H function. Therefore, Hairless protein might have additional functions apart from Su(H) binding and may antagonize Notch mediated cell-cell communication in a more complex way than currently anticipated (Maier, 1997).

CNS midline to mesoderm signaling in Drosophila

The dorsal median cells are unique mesodermal cells that reside on the surface of the ventral nerve cord in the Drosophila embryo. The Buttonless homeodomain protein is specifically expressed in these cells and is required for their differentiation. Properbuttonless gene expression and dorsal median cell differentiation require signals from underlying CNS midline cells. Thus, dorsal median cells fail to form in single-minded mutants and do not persist in slit mutants. Through analysis of rhomboid mutants and targeted rhomboid expression, it has been shown that the EGF signaling pathway regulates the number of dorsal median cells. wingless-patched double mutants exhibit defects in the restriction of dorsal median cells to segment boundaries and alterations in CNS midline cell fates (Zhou, 1997).

In addition to prominent CNS midline expression, the single minded gene is also expressed in clusters of mesodermal cells that arise adjacent to the midline, migrate laterally during germ band retraction and contribute to ventral muscle fibers. The EGF signaling pathway regulates this set of mesodermal cells that expresses rhomboid. Mesodermal expression of rho is transient and eliminated well before muscle fiber formation, suggesting that at least some of the muscle defects in rho mutants may result from earlier patterning defects in specific mesodermal precursor cells. While the function of sim in developing is unclear, it is of interest that the mesodermal sim expression is conserved in vertebrates. Thus, the mouse Sim-1 gene is prominently expressed in paraxial mesodermal cells of the developing lateral somitic compartment. Taken together, these data define a novel neuroectoderm to mesoderm signaling pathway and suggest that unique mesodermal cell types are specified by a combination of midline and segmental cues (Zhou, 1997).

Drosophila Single-minded represses gene transcription by activating the expression of repressive factors

The Drosophila single-minded gene controls CNS midline cell development by both activating midline gene expression and repressing lateral CNS gene expression in the midline cells. The mechanism by which Single-minded represses transcription was examined using the ventral nervous system defective gene as a target gene. Transgenic-lacZ analysis of constructs containing fragments of the ventral nervous system defective regulatory region have identified sequences required for lateral CNS transcription and midline repression. Elimination of Single-minded:Tango binding sites within the ventral nervous system defective gene does not affect midline repression. Mutants of Single-minded that remove the DNA binding and transcriptional activation regions abolish ventral nervous system defective repression, as well as transcriptional activation of other genes. The replacement of the Single-minded transcriptional activation region with a heterologous VP16 transcriptional activation region restores the ability of Single-minded to both activate and repress transcription. These results indicate that Single-minded indirectly represses transcription by activating the expression of repressive factors. Single-minded provides a model system for how regulatory proteins that act only as transcriptional activators can control lineage-specific transcription in both positive and negative modes (Estes, 2001).

The relationship between Sim and Vnd in the CNS midline cells was examined by immunostaining embryos with both anti-Sim and anti-Vnd. Vnd protein is first seen at embryonic stage 5 in the presumptive mesectoderm and ventral neuroectoderm, preceding the appearance of Sim protein. Sim protein appears during gastrulation (stage 6) in the mesectoderm and overlaps with Vnd protein. During stages 6-9, both Sim and Vnd are colocalized in the CNS midline cells, while Vnd protein continues to be present in cells of the ventral neuroectoderm. By the end of stage 9 and during stage 10, Vnd protein is absent in the CNS midline cells, while Sim protein remains. The absence of Vnd protein is preceded by the reduction of VND RNA at stage 8. These results show that Sim and Vnd proteins overlap within the mesectoderm during several stages and that a considerable lag exists between the appearance of Sim protein and the loss of Vnd protein (~2 h). There is also a substantial lag between the appearance of Sim protein and the loss of midline VND RNA (~1 h). The delay in vnd repression after initial Sim appearance is consistent with an indirect mechanism of repression (Estes, 2001).

Three general models of Sim-mediated repression were tested: (1) Sim directly represses target genes by binding their DNA and repressing transcription in association with a corepressor(s); (2) Sim does not bind DNA of target genes but interacts with positively acting factors preventing their action, and (3) Sim represses indirectly by activating transcription of genes encoding repressive factors. Several complementary experiments demonstrate that midline repression requires activation of repressive gene expression by Sim (Model 3). Ectopic expression experiments utilizing mutant forms of Sim demonstrate that the basic region, PAS domain, and C-terminal regions are all required for both transcriptional activation and repression. Removal of the PAS domain also abolished the ability of Sim to form dimers with Tgo, suggesting that Tgo is necessary for repression. More informative is Db-Sim. This mutant protein was able to dimerize with Tgo and the protein complex accumulates in the nucleus. However, neither midline transcription nor repression occurs, presumably due to the inability of the Sim:Tgo dimer to bind DNA. This argues against a model in which Sim interacts with an activator protein in a non-DNA-binding mode (Model 2) and instead suggests that DNA binding is required for Sim repression (Model 1 or Model 3). However, analysis of the vnd gene using lacZ transgenes indicates that Sim:Tgo binding sites are not required for midline repression (Model 1); mutation of the single CNS midline element (CME; ACGTG) in fragment 2.5RB or mutation of three CMEs in 5.3RS does not affect lacZ expression. Transient transfection experiments have shown that CMEs are relevant targets of Sim:Tgo binding, and in vivo analyses of five different genes have shown that the CME functions in vivo as a Sim:Tgo binding site. However, it remains possible that Sim:Tgo could bind a variant sequence within the vnd gene. Arguing against this are the results indicating that Sim represses indirectly by activating transcription (Estes, 2001).

The C-terminal region of Sim that follows the PAS domain contains multiple transcriptional activation domains. Removal of the C-terminal 211 aa eliminates those activation domains and additional residues. The DeltaC-Sim protein is unable to activate midline transcription or repress vnd expression, even though it dimerized with Tgo and the complex accumulates in nuclei. This is consistent with Sim repressing vnd expression by activating the transcription of repressive factors. However, it is also possible that there is a domain within the C-terminal region that could directly mediate repression. Fusing the VP16 activation domain onto DeltaC-Sim and functionally assaying the fusion protein in vivo tested this. The results show that addition of the VP16 activation domain restores the ability of DeltaC-Sim to activate transcription and repress vnd. These experiments demonstrate that vnd repression correlates with the ability of Sim to activate transcription (Model 3). Another construct removed the Sim AAQ repeat region (a repeating stretch of 10 Ala-Ala-Gln repeats followed by several imperfect repeats). Its deletion does not affect the ability of Sim to dimerize with Tgo, accumulate in nuclei, activate transcription, or repress vnd. Although striking in sequence, its function remains a mystery. The combination of the vnd-lacZ and ectopic Sim-mutant experiments demonstrate that Sim does not directly repress or inhibit vnd gene expression but, instead, activates transcription of genes that encode repressive factors consistent with the third model of repression. This model is also consistent with the delayed timing of vnd repression seen in early embryonic development (Estes, 2001).

A 0.5-kb region necessary for repression maps between -3.6 and -3.1 in the vnd regulatory region. This repression of vnd occurs variably throughout the midline but is seen consistently between embryos. The lack of uniform repression suggests that other elements residing in the vnd gene may help control the maintenance of repression (Estes, 2001).

Midline repression by Sim functions by activating transcription of one or more genes that, in turn, repress transcription of genes normally expressed in the lateral CNS. The nature of these repressive factor genes and how they function are unknown, although plausible candidate genes exist. Since E(spl) proteins repress lateral CNS expression, members of this family are candidates for midline repressors, and several are expressed in the CNS midline cells early in development (m5, m7, and m8). In this scheme, Sim:Tgo would activate factors that would modify or interact with E(spl) proteins to repress proneural gene activity in the midline. The vnd upstream regulatory region contains numerous E(spl) consensus binding sites, although none of the sites lie within the 0.5-kb fragment shown to be important for repression. Since the E(spl) proteins reside in midline cells well before repression occurs, it is unlikely that Sim is required for initial E(spl) transcription, although maintenance of their expression is a possibility. Other potential repressors have not yet been identified (Estes, 2001).

It is unclear what role the CMEs have within the vnd regulatory region. They are not involved in vnd repression nor do they seem to play a role in vnd embryonic CNS expression. The sequences immediately flanking the four CMEs within the vnd regulatory region were compared to CMEs found within genes known to be important for midline activation by Sim. The consensus for sites within genes positively activated by Sim is (A/T)ACGTG, while the consensus for the CMEs within the vnd regulatory region is GACGTG (three of four sites had a G at the first residue). Otherwise, the sequences varies widely and no consensus was found among the sequences flanking the CMEs of Toll, sim, slit, rhomboid, and breathless nor among the sequences flanking the CMEs found within the vnd regulatory region. It may be the larger context of the vnd regulatory region that prevents Sim from interacting with these sites to affect transcription. It is also possible that the CMEs are bound by bHLH-PAS proteins and utilized for postembryonic expression of vnd (Estes, 2001).

Three discrete regions ( -5.3 to -4.2; -4.2 to -3.1, and -3.1 to -2.8) within the 2.5RB domain of the vnd upstream regulatory sequences are necessary for vnd-like expression. 2.5RB was examined for sequences related to the consensus binding sites of known transcription regulators of vnd. Genetic analysis has shown that Dorsal and Twi are required for vnd activation and Sna for mesodermal repression, and vnd is positively autoregulated. Four putative Dorsal binding sites are located between -5.3 and -4.2 and none are observed between -4.2 and -2.8. Seven putative Sna sites are observed: two between -5.3 and -4.2; four between -4.2 and -3.1, and one between -3.1 and -2.8. Two of the Sna sites possess embedded E boxes (CANNTG sequences) that can enhance gene expression. Twi E-box sites show a weak and short consensus sequence and are difficult to identify by sequence alone. Nonetheless, seven putative Twi E-box sites lie between -5.3 and -4.2. These sites have been shown to bind Twi protein when present in other genes (Estes, 2001).

The sites lie close to the Dorsal binding sites, suggesting cooperative binding of Dorsal and Twi. Sequences required for vnd autoregulation are localized within 8.1 kb upstream of the vnd transcription unit. Although not rigorously tested for autoregulation, the 2.5RB transgene shows a similar pattern of expression compared to 8.1HV, suggesting that autoregulatory sequences may be present. At least 15 potential Vnd binding sites are scattered throughout the entire 2.5RB region, consistent with a direct autoregulatory role for Vnd. In summary, sequence analysis of the vnd regulatory region as defined by deletional analysis suggests that Dorsal, Twi, and Sna directly initiate vnd expression and that Vnd directly autoregulates. However, biochemical experiments to test transcription factor binding, coupled with transgenic analysis of DNA containing mutated binding sites, are required to test the functional significance of these sites (Estes, 2001).

It is concluded that midline cell formation occurs by the concerted activation of genes required for midline cell development and repression of genes normally expressed in the lateral CNS. While there are examples of transcription factors that can directly activate and repress (e.g., Dorsal, Kruppel, and the glucocorticoid receptor), the sim mode of activating directly and repressing indirectly may be a common mechanism of lineage-specific gene control (Estes, 2001).

he CNS midline cells control the spitz class and Egfr signaling genes to establish the proper cell fate of the Drosophila ventral neuroectoderm

The spitz class genes, pointed (pnt), rhomboid (rho), single-minded (sim), spitz (spi) and Star (S), as well as the Drosophila Epidermal growth factor receptor (Egfr) signaling genes, argos (aos), Egfr, orthodenticle (otd) and vein (vn), are required for the proper establishment of ventral neuroectodermal cell fate. The roles of the CNS midline cells, spitz class and Egfr signaling genes in cell fate determination of the ventral neuroectoderm were determined by analyzing the spatial and temporal expression patterns of each individual gene in spitz class and Egfr signaling mutants. This analysis shows that the expression of all the spitz class and Egfr signaling genes is affected by the sim gene, which indicates that sim acts upstream of all the spitz class and Egfr signaling genes. Overexpression of sim in midline cells fails to induce the ectodermal fate in the spi and Egfr mutants. In contrast, overexpression of spi and Draf causes ectopic expression of the neuroectodermal markers in the sim mutant. Ectopic expression of sim in the en-positive cells induces the expression of downstream genes such as otd, pnt, rho, and vn, which clearly demonstrates that the sim gene activates the Egfr signaling pathway and that CNS midline cells, specified by sim, provide sufficient positional information for the establishment of ventral neuroectodermal fate. These results reveal that the CNS midline cells are one of the key regulators for the proper patterning of the ventral neuroectoderm by controlling Egfr activity through the regulation of the expression of spitz class genes and Egfr signaling genes (Chang, 2001).

Novel behavioral and developmental defects associated with Drosophila single-minded

In Drosophila, the development of the midline cells of the embryonic ventral nerve cord depends on the function of the bHLH-PAS transcription factor Single-minded (Sim). However, the expression domain of sim is also found anterior and posterior to the developing ventral cord throughout the germ band. Indeed, mutations in sim were identified based on their characteristic cuticle phenotype. Eight abdominal segments (A1-A8) can be easily seen in the larval cuticle, while three more can be identified during embryogenesis. Cells located in A8-A10 give rise to the formation of the genital imaginal discs, and a highly modified A11 segment gives rise to the anal pads that flank the anus. sim is expressed in all these segments and is required for the formation of both the anal pads and the genital imaginal discs. A new temperature-sensitive sim allele allowed an assessment of possible postembryonic function(s) of sim. Reduction of sim function below a 50% threshold leads to sterile flies with marked behavioral deficits. Most mutant sim flies are only able to walk in circles. Further analyses have indicated that this phenotype is likely due to defects in the brain central complex. This brain region, which was previously implicated in the control of walking behavior, expresses high levels of nuclear Sim protein in three clusters of neurons in each central brain hemisphere. Additional Sim localization in the medullary and laminar neurons of the optic lobes may correlate with the presence of ectopic axon bundles observed in the optic lobes of sim mutant flies (Pielage, 2002).

sim expression is first evident during the cellular blastoderm stage in a strip of cells flanking the mesodermal anlage. The majority of these cells will later divide to generate the neurons and glial cells found at the midline of the ventral nerve cord. However, it is important to note that sim expression exceeds the neurogenic region from which the nerve cord will form. At the posterior, sim expression extends into abdominal segment 10, where it can be detected until the end of stage 11. The fate of these cells is presently unknown. Possibly, the ectodermal midline cells provide inductive signals influencing the developing neighboring tissues, which appears to be a more general feature of the midline cells. Within the CNS, the midline cells act as an organizing center controlling the patterning of axons by providing attractive and repulsive cues. Furthermore, the midline cells regulate the number and differentiation of cortical neurons and mesodermal cells (Pielage, 2002).

The anal pad anlage can be labeled by Eve expression and forms immediately posterior to Sim-expressing cells. In sim mutants, the Eve expression domain shifts toward the midline and meets at the midline. Possibly, the gap in Eve expression in wild type embryos allows the formation of the anal slit. In sim mutants, the posterior midgut invaginates normally and the proctodeum initially forms. However, during later stages, the cells that will give rise to the anus will die and thus prevent the external opening of the hindgut (Pielage, 2002).

What is the function of the ventral midline during genital disc development? In both female and male flies, the sexually dimorphic terminalia are formed by a common genital disc comprising three primordia. The female genital primordium is derived from the 8th abdominal segment, the male genital primordium from the 9th abdominal segment, and the anal primordium from the 10th and 11th abdominal segments. In both sexes, the anal primordium develops, whereas depending on the sex of the animal, either the female or the male primordium develops. The definition of the genital disc anlage does not appear to be affected in sim mutants, but the subsequent delamination from the ectoderm is abnormal. In wild type embryos, the genital disc anlage forms just posterior to the developing ventral nerve cord. Following germ band retraction, the ventral nerve cord retracts, and concomitantly, the genital disc anlage delaminates from the ectoderm. At present, these analyses do not allow for discrimination as to whether the genital disc phenotype found in mutant sim embryos is an indirect consequence of the nerve cord condensation defect or whether it is due to the loss of sim expression in the genital disc primordium (Pielage, 2002).

The temperature-sensitive mutation sim^J1-47 made possible an investigation to determine whether sim is required in larval or adult stages. Following the reduction of sim function, a number of interesting phenotypes emerged. The sterility phenotype displayed by the hypomorphic sim allele J1-47 as well as the amorphic mutation sim^H9 in trans to deficiencies affecting only one of the two promoters demonstrated that these phenotypic traits are indeed due to a reduction in the level of sim function. The sterility phenotype is likely to be a direct consequence of abnormal genital disc development during embryonic stages. Interestingly, these flies also showed abnormal courtship behavior, suggesting a requirement of sim in larval/adult neurogenesis. A similar conclusion has to be drawn by the walking defects of mutant sim flies. Mutant flies are only able to walk in circles. This phenotype could be due to a loss of motoneurons in the ventral nerve cord or it could be due to disruption of higher centers that coordinate walking (Pielage, 2002).

An extended analysis of the walking behavior in different structural brain mutants shows that the central complex (CX) between the protocerebral brain hemispheres serves as such a higher center. A hallmark of mutants affecting the CX is a slower mean and maximum walking speed and a decaying locomotor activity is also shown by sim^J1-47 /sim^H9 flies. In the majority of these mutants, the protocerebral bridges and their fan-shaped bodies are affected. The remaining flies with no gross morphological CX defects may well have defects that may be detectable only at the single cell level (Pielage, 2002).

Why do sim^J1-47/sim^H9 flies circle? Circling in only one direction is not the normal behavior of blind flies in the arena situation. They would show random-search behavior with a balanced frequency of left and right turns. It is assumed that most sim flies are not entirely blind since most of them reacted, at least weakly, to optomotor stimuli. sim flies are not the first example of circling flies. In the screen for locomotor mutants, the CX-defective mutant C31 was isolated that frequently walked in wavy lines. C31 function was subsequently studied in mosaics that were generated by using the gynandromorph technique. When one half of the body, including the head, is mutant, the flies are unable to walk straight and persistently turn toward the defective body side. However, unilateral mutant flies with an intact brain can walk straight, pointing toward the role of the CX in balancing left-right motocontrol. In support of the notion that the CX controls locomotive behavior is the finding that Pax-6/eyeless mutants cause gross morphological CX defects and, concomitantly, severe locomotor deficits (Pielage, 2002).

A mutation in the gene pirouette, which was identified in a screen for genes affecting auditory behavior, shows a similar walking phenotype as described for the hypomorphic sim mutation. Within the CNS, the optic lobes degenerate but no information about the development of the CX is available. No genetic interaction was detected between the two loci. To date, only few other mutations have been described that specifically affect the development of the CX. The transcription factor AP2 is not required during embryonic development; however, adult flies display severe disruptions in the CX. It is unknown whether AP2 mutations affect behavior similar to sim. Other mutants affecting the formation and connectivity of the CX have been described, but no information is available on walking abilities of the different mutant flies (Pielage, 2002).

Beside expression within the central complex, high levels of Sim expression were noted in the optic lobes, the lamina, and the medulla -- this is in agreement with the mutant phenotype. During larval development, the optic lobes undergo extensive rounds of cell proliferation to give rise to the mature neurons and glia. DNA replication and cell division occur at several discrete sites: the inner proliferative center (IPC), the outer proliferative center (OPC), and the laminar precursor center (LPC). Since sim expression is observed during the proliferative phase of optic lobe development, whether sim is expressed in proliferating cells or the postmitotic cells was addressed. The proliferative zones were visualized by expression of GFP from a PCNA-GFP transgenic strain. Proliferating cell nuclear antigen (PCNA) is encoded by the mus209 locus and is expressed in replicating cells. Since PCNA-GFP and Sim expression do not overlap, it appears that Sim is only expressed in postmitotic cells in the optic lobes (Pielage, 2002).

Double labeling experiments with glial and neuronal antigens indicate that, within the brain, Sim is expressed only in neuronal cells. The optic lobes of sim mutant flies show aberrant axonal projects, but the medullary and laminar neurons are present. This suggests that the role of sim in optic lobe development may be different from its role in controlling formation of the CNS midline cells in embryonic development (Pielage, 2002).

The highly ordered assembly of retinal axons and their synaptic partners is regulated by Hedgehog/Single-minded in the Drosophila visual system

During development of the Drosophila visual center, photoreceptor cells extend their axons (R axons) to the lamina ganglion layer, and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal Hedgehog (Hh). This inductive mechanism helps to establish an orderly arrangement of connections between the R axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding R axons. The bHLH-PAS transcription factor Single-minded (Sim) is induced by Hh in the lamina neurons and is required for the association of lamina neurons with R axons. In sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with R axons. As a result, lamina neurons are set aside from R axons. The data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks (Umetsu, 2006).

Most axons in the brain establish topographic maps in which the arrangement of synaptic connections maintains the relationships between neighboring cell bodies. A notable model of topographic map formation is the visual system, where the relay of visual information from the retina to the visual center must be arranged in a spatially ordered manner through the topographic connections of retinal axons with their midbrain target, which is the optic tectum (OT) in lower vertebrates and the superior colliculus (SC) in mammals. This topographic map is termed a retinotopic map. Many studies have shown that Ephrin protein family members, acting through their Eph receptors, play pivotal roles in the establishment of the retinotopic map. In the mouse and the chick, for example, the retinal ganglion cells (RGCs) extend their axons to the OT/SC, and the low-to-high anteroposterior gradient of ephrin A in the target limits the posterior extension of growth cones at various positions, dependent on the EphA level of each RGC (Umetsu, 2006).

The Drosophila visual system has also provided insight into topographic mapping. In Drosophila, the projections of photoreceptor neurons (R cells) themselves induce development of the corresponding postsynaptic neurons. The Drosophila visual system consists of the compound eyes and the three optic ganglia: the lamina, the medulla and the lobula complex. Each of the approximately 750 ommatidial units comprising the compound eye contain six outer photoreceptors (R1-R6) and two inner photoreceptors (R7, R8). R1-R6 cells send their axons to the first optic ganglion, the lamina, whereas R7 and R8 cells send axons through the lamina to the second ganglion, the medulla. R1-R6 cells in each ommatidium make stereotypic connections with particular lamina neurons. Synaptic units in the lamina are referred to as lamina cartridges. During the initial step of the assembly of a lamina cartridge, an arriving photoreceptor axon (R axon) fascicle forms a pre-cartridge ensemble, the 'lamina column', with a set of five lamina neurons. Formation of the ensemble results in a one-to-one correspondence of ommatidia to column units, and is fundamental to the subsequent establishment of intricate synaptic connections. Development of the lamina is tightly regulated by the projection of R axons. Failure in eye formation results in concurrent loss of the lamina, as in a normal brain, lamina neurogenesis is directly coupled to the arrival of R axons. Both R cell differentiation and ommatidial assembly progress in a posterior-to-anterior direction across the eye disc. Differentiated R cells begin to send their axons to the brain in the same sequential order, reflecting their position in the retina along the anteroposterior and the dorsoventral axes. Wnt signaling plays a role in regulating projections along the dorsoventral axis (Umetsu, 2006).

As the axons from each new row of ommatidial R cell clusters arrive in the lamina, a corresponding group of lamina precursor cells (LPCs) undergo a final division and initiate differentiation into lamina neurons. In the first step of their neurogenesis, direct contact with R axons triggers the transition of G1-phase LPCs into S phase. Both the G1-S transition and the initial specification into a lamina neuron are induced by Hedgehog (Hh), which is delivered by arriving R axons, and the next step in lamina differentiation is induced by the Spitz signaling molecule, which is also delivered by R axons. Hh expressed in R cells functions as a signal for photoreceptor development as well: secreted Hh induces anterior precursor cells to enter the pathway of R cell specification (Umetsu, 2006).

Thus, the retinotopic map along the anteroposterior axis of the lamina seems to be established autonomously and in a posterior-to-anterior order, as newly specified R cells send their axons to the lamina layer and make lamina columns. Each ommatidial unit sends a set of R axons as a single bundle to the lamina along the pre-existing fascicle that has been just projected. Then, the axon bundles are enveloped by the processes of newly induced lamina neurons. This step is key to forming the one-to-one associations between R axon bundles and their corresponding lamina neurons. This study shows that the activity of Single-minded (Sim) is required for developing lamina neurons to establish an association with the corresponding R axons and, hence, to form the lamina column. sim encodes a basic-helix-loop-helix-PAS (bHLH-PAS) transcription factor and is induced by Hh provided by the R axons. In sim mutant brains, the developing lamina neurons fail to associate with R axon bundles, resulting in a failure to establish connections between R axons and lamina neurons. It is inferred that sim programs developing lamina neurons to express a molecule(s) that is required for the association with R axons (Umetsu, 2006).

Retinotopic mapping in Drosophila provides unique insights into neuronal network formation not only because of its tight coupling to the control of development, but also because of the interactions between axons and neuronal cell bodies. The interactions observed stand in sharp contrast to what has been found for other models of axon guidance, where the growth cones of axons respond to a variety of attractive or repulsive guidance cues to navigate to their synaptic target cells. The cues include the netrins, Slits, semaphorins and ephrins, and the restricted expression pattern of these cues and the reactivity of growth cones play pivotal roles in the establishment of the proper synaptic connections. In this context, postsynaptic cells are seen as mere providers of guidance/adhesion molecules, passively awaiting the arrival of a growth cone. In other words, it is conceivable that presynaptic growth cones seek their targets dynamically, whereas postsynaptic cells remain static. Unlike the roles of presynaptic axons, the cellular behaviors of postsynaptic cells in the establishment of synaptic targeting are poorly understood. This study proposes another possible model for the establishment of topographic neuronal connections in which postsynaptic cells dynamically interact with presynaptic axons (Umetsu, 2006).

Thus, Sim, a target of Hh, is required for at least the first step of lamina column formation; namely, the incorporation of developing lamina neurons into the area where R axons project and lamina columns mature, an area referred to as the assembling domain. This model for Sim is based on four observations. First, sim²/sim^ry75 brains have a reduced number of lamina neurons in the assembling domain, leaving an abnormally large number of premature lamina neurons behind in the pre-assembling domain. Second, in clonal analysis, sim² clones fail to be recovered in the assembling domain (similar to smo¹ clones). Third, lamina neuron-specific inhibition of Sim function causes R axon bundles to be tightly packed and lamina neurons to be excluded from R axon bundles. And fourth, overexpression of sim in lamina neurons causes precocious incorporation of lamina neurons into the assembling domain (Umetsu, 2006).

In case of overexpression, neither expansion of the assembling domain nor increase in the number of lamina neurons relative to the number of R axon bundles was observed, even though lamina neurons prematurely incorporated into the assembling domain. This is probably because a reduced number of lamina neurons were generated. In fact, loss of E2F expression was observed at the lamina furrow in NP6099-GAL4 UAS-sim brains. The onset of incorporating lamina neurons into the assembling domain might be linked to an inhibition of cell proliferation. However, this is thought to be unlikely for two reasons: (1) lamina neurons did not show any extra E2F signal in the sim mutant brain in spite of an increase in unincorporated lamina neurons; and (2) lamina neurons ectopically expressing a cell cycle-braking factor, the Drosophila p21/p27 homolog dacapo (dap) cause the precocious incorporation of lamina neurons. Thus, a direct link between cell cycle regulation and the incorporation of lamina neurons is less plausible (Umetsu, 2006).

An alternative model, the 'time lag' model, is proposed. There appears to be a lag between the onset of sim expression and the onset of incorporation of lamina neurons. Differentiating lamina neurons are held temporarily in the pre-assembling domain and then the proper amount of lamina neurons are coordinately integrated into columns as more R axons are projected. Thus, it is speculated that a certain degree of accumulation of the Sim/dARNT heterodimer in nuclei is needed to exert cellular function. Consistent with this idea, graded accumulation of Sim is observed, with lower Sim levels in anterior (younger) lamina neuron nuclei and higher levels in posterior (older) lamina neuron nuclei. Overexpression of Sim in lamina neurons would thus cause higher levels of accumulation of the protein in young lamina neurons and facilitate their incorporation into the assembling domain. Interestingly, overexpression of the wild-type dARNT did not have any detectable effects, suggesting that Sim accumulation is a limiting factor for cell incorporation (Umetsu, 2006).

The mechanism of neuronal maturation and that of assembly of lamina neurons are independent, although both are under the control of Hh signaling. Disruption of sim did not affect the differentiation and proliferation of lamina neurons. Correspondingly, neither the incorporation of lamina neurons into the lamina column nor the expression of sim were affected by dac mutation. The cellular function required for assembling the column or the function of Sim at the cellular level is still not known. Electron microscopic observations by have revealed an intriguing behavior of lamina neurons at the early pupal stage; large processes extending from lamina neurons engulf R1 and R6 axons of newly incoming R axon bundles. This may be the key step in lamina column formation and interaction between the R axons and lamina neurons. Sim may regulate genes required for process formation, interaction with R axons and/or events that follow shortly after, since lamina neurons seem to fail to make interactions with R axons from the beginning in the sim mutant background. Sim is expressed in the midline cells of the CNS throughout neurogenesis in the Drosophila embryo and is required for the proper differentiation of the midline cells into mature neurons and glial cells. Midline precursor cells undergo synchronized cell division and then transform into the bottle-shaped cells, in which the nuclei migrate internally and leave a cytoplasmic projection joined to the surface of the embryo. The sim mutant midline cells fail to delaminate from the epidermal cell layer. Cells do not make the normal bottle-like shape and, instead, they appear rounded. In addition, overexpression of sim can induce other cell types to exhibit midline morphology. sim may thus regulate the transcription of a set of genes required for morphological changes, which in turn are required for interaction between cells, both in the lamina and during embryonic CNS development (Umetsu, 2006).

Although sim expression is regulated by Hh signaling, this does not answer the question of whether sim function is sufficient to confer on cells the ability to be incorporated into the assembling domain. Whether smo mutant clones can be recovered in the assembling domain was examined by forcing sim expression in smo clones using the MARCM technique. However, smo mutant clones expressing sim were not recovered in the assembling domain. This suggests that additional factors are involved in lamina neuron assembly. Hh may also contribute to specification of the difference in affinity between lamina neurons and R axons and/or between anterior and posterior lamina neurons. In Drosophila wing discs, the Hh signal differentiates the affinity of anterior compartment cells from that of the posterior compartment cells, thereby maintaining the compartment border (Umetsu, 2006).

An active role is proposed for postsynaptic cells in making a topographic map of the Drosophila visual system. Targeted expression of the dominant-negative form of the Sim partner in the lamina neurons clearly showed a role for postsynaptic cells in assembling lamina columns. This presumably affects an early step of assembly. It is not known if Sim function is also required for later steps in more mature lamina neurons. The forced expression of the dominant-negative Sim partner in the posterior lamina neurons had no effect, although it may simply be that the level of expression of the dominant-negative form of dARNT was not sufficient to have an observable effect on Sim function. In the lamina column, the R axon bundle associates with a precisely arranged row of five lamina neurons. No mechanisms for the development and formation of this stereotypic structure have been revealed. Another signal might be provided from the R axons with lamina neurons, and/or intrinsic structures of the R axons might play a role in this architecture. An intriguing property of postsynaptic muscle cells for axonal targeting has been observed: the muscle cells bear numerous postsynaptic filopodia ('myopodia') during motoneuron targeting. They showed that postsynaptic cells actively contribute to synaptic matchmaking by direct, long-distance communication. Together with what has been learned about myopodia in neuromuscular synapse formation, the curent findings reveal an active role for postsynaptic cells for the establishment of precise neural networking (Umetsu, 2006).

Sim belongs to the family of bHLH-PAS transcription factors, whose members function in many developmental and physiological processes, including neurogenesis, tissue development, toxin metabolism, circadian rhythms, response to hypoxia, and hormone receptor function. bHLH-PAS proteins usually function as dimeric DNA-binding protein complexes. The most common functional unit is a heterodimer. These heterodimers consist of one partner that is broadly expressed, and another whose expression is regulated spatially, temporally or by the presence of inducers. Sim and the bHLH-PAS protein dARNT heterodimerize to bind to their responsive element, the CME (CNS midline enhancer element), to activate target gene transcription. In this complex, dARNT is the general dimerization partner and Sim is the tissue-specific partner (Umetsu, 2006).

The Drosophila Sim and mammalian Sim1 and Sim2 proteins are highly conserved in their amino-terminal halves, which contain a bHLH and a PAS domain. Murine Sim1 and Sim2 are also expressed in both proliferative and postmitotic zones of the central nervous system at different stages of neural development. These zones of expression include the longitudinal basal plate of the diencephalon (Sim1 and Sim2), the mesencephalon (Sim1), the zona limitans intrathalamica (Sim1 and Sim2) and the portion of the spinal cord that flanks the floor plate (Sim1). Sim2 maps to the region responsible for Down Syndrome (DS) on Chromosome 21. Interestingly, Sim2 is also expressed in non-neuronal tissues, including branchial arches and the developing limb, which are primordia of tissues and organs where DS abnormalities are frequently observed (Umetsu, 2006).

Given the important roles of sim in Drosophila development and the expression of Sim2 in cell types that are affected in DS individuals, it was proposed that Sim2 may play a causative role in DS. However, because of a lack of direct evidence and the existence of other candidate genes, this remains speculative. Cells expressing sim during Drosophila development and Sim2-positive cells affected in DS seem to be able to migrate. The conserved role of Sim may enable cells to migrate and/or interact with surrounding cells in the various tissues, including the central nervous system. It will thus be intriguing to search for extra cellular targets of Sim regulation with the hope of elucidating mechanisms that underlie the behavior of Sim-expressing cells (Umetsu, 2006).

Roles of single-minded in the left-right asymmetric development of the Drosophila embryonic gut

Many animals have genetically determined left-right (LR) asymmetry of their internal organs. The midline structure of vertebrate embryos has important roles in LR asymmetric development both as the signaling center for LR asymmetry and as a barrier to inappropriate LR signaling across the midline. However, in invertebrates, the functions of the midline in LR asymmetric development are unknown. To elucidate these roles, the involvement of single-minded (sim) was studied in the LR asymmetry of the Drosophila embryonic gut, which develops in a stereotypic, asymmetric manner. sim encodes a bHLH/PAS transcription factor that is required for the development of the ventral midline structure. This study reports that sim is expressed in the midline of the foregut and hindgut primordia. The handedness of the embryonic gut is affected in sim mutant embryos and in embryos overexpressing sim. However, midline-derived events, which involve Slit/Robo and EGFr signaling and direct the development of the tissues adjacent to the midline, did not affect the laterality of this organ, suggesting a crucial role for the midline itself in LR asymmetry. In the sim mutants, the midline structures of the embryonic anal pad were deformed. The mis-expression of sim in the anal-pad primordium induced LR defects. Different portions of the embryonic gut require sim functions at different times for normal LR asymmetry. These results suggest that the midline structures are involved in the LR asymmetric development of the Drosophila embryonic gut (Maeda, 2007).

Based on this analyses, two models are proposed that can account for the functions of sim in the LR asymmetric development in this organ: (1) in the midline cells of the embryonic gut, Sim, a transcription factor, may directly or indirectly regulate the expression of genes that are required for the gut's LR asymmetric development; (2) the gut midline cells, whose normal formation depends on Sim, may play a critical role in the LR patterning of this organ. Currently, it is difficult to distinguish between these two possibilities. As regards these two models, however, it was found that two midline-derived events, which are downstream of Slit and EGFr signaling, are not involved in the LR asymmetric development of the embryonic gut, at least ruling out a role for their downstream genes in the LR patterning of this organ. It was also found that the expression of Myo31DF was not affected in sim mutants. Therefore, this gene is not a downstream target of sim either. It is speculated that sim and some other genes play redundant roles in LR asymmetric development, because the laterality of each part of the embryonic gut was not randomized, even in a null mutant of sim (Maeda, 2007).

Each part of the embryonic gut requires sim functions at a distinct period of embryonic development. Curiously, it was found that the esophagus and hindgut require sim by stage 8. The sim transcript is first detected at stage 5, and the Sim protein is detected beginning at stage 6 by anti-Sim antibody staining. This early expression of sim seems to be sufficient for the LR asymmetric development of these two organs. In contrast, sim functions are required during stages 6-14 for the most-anterior midgut (the proventriculus and the first chamber of the midgut) and main-midgut to develop LR asymmetry. However, whether sim is required continuously or discontinuously during this period was not examined. Given that the inversion of laterality in each part of the embryonic gut was independent, sim function may also be required at different times by these different portions of the gut (Maeda, 2007).

In sim mutants, the tubular structure of the anal pad is deformed, although other parts of the gut seem to develop normally, suggesting the involvement of this region in LR asymmetric development. This anal-pad defect is probably associated with the defect of the midline structure in the developing anal pad. Interestingly, the mis-expression of sim in the anal-pad primordium is sufficient to induce the LR defects in the midgut and hindgut and extra midline cells in the anal-pad primordium. These results support the idea that the midline cells in the anal pad play a crucial role in the LR asymmetric development in these two organs (Maeda, 2007).

The embryonic hindgut consists of several distinct epithelial domains. The large intestine of the embryonic gut is composed of dorsal and ventral domains with a single row of the boundary cells between them. The results suggest that sim defines another specific region, which is the ventral midline region of the hindgut, in this organ. The mis-expression of sim in the hindgut primordium suppressed the expression of marker genes in the large intestine. Thus, sim seems to prevent the specification of the dorsal and ventral domains and of the boundary cells. However, under this condition, these epithelial cells did not transform into the midline cells: mis-expression of sim in the entire hindgut and anal-pad primordia induced extra midline cells only around the anal pad. Thus, sim expression seems to be sufficient to suppress the specification of the dorsal and ventral domains and of the boundary cells, but, in addition to Sim, some other factor is involved in the formation of extra midline cells (Maeda, 2007).

In vertebrates, the Lefty proteins, which are divergent TGF-β superfamily ligands, are expressed in the midline structure. In mice, Lefty1, expressed in the prospective floor plate (PFP), inhibits Nodal function, thereby providing a midline barrier that prevents the diffusion of asymmetric Nodal signals to the right side of the embryo. Interestingly, in many respects, the ventral midline cells of Drosophila are similar to the floor plate cells of vertebrates; both cell types are important sources of developmental signals. Therefore, a downstream gene product of sim may function as a midline barrier in Drosophila LR development. However, orthologs of the vertebrate nodal and lefty genes do not seem to be present in Drosophila. In vertebrates, Pitx2 is expressed in the left side of the embryo under the control of nodal and plays an essential role in LR asymmetric development. Although an ortholog of the Pitx gene has been identified in Drosophila, unlike in vertebrates, it is not expressed with LR asymmetry. Furthermore, mutation of the Drosophila Pitx gene does not cause laterality defects. In Drosophila, there is no report regarding LR asymmetric gene expression, so far, although myosin I family proteins and the actin cytoskeleton have a crucial role in the LR asymmetric development of the embryonic and adult internal organs (Hozumi, 2006; Spéder, 2006). Because the involvement of actin-based motor proteins in LR asymmetric development is not known in vertebrates, the mechanisms of LR patterning may be different between vertebrates and invertebrates. Thus, even if the midline cells of Drosophila have roles equivalent to those of the vertebrate midline, the signaling molecules involved in the LR asymmetric development of Drosophila may be different from those used in vertebrates (Maeda, 2007).

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date revised: 20 September 2012
 

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