BIOLOGICAL OVERVIEW

Single-minded is required for the developmental specification of the ventral midline. This is an organizing locus that appears as a result of gastrulation. Prior to gastrulation, there develop two anterior-to-posterior rows of single cells, one on either side of the embryo. All cells in both rows express sim. These rows form a border between the presumptive neuroectoderm (on the dorsal side) and the presumptive mesoderm (ventral side). Also known as the midline of the central nervous system, the ventral midline is formed once the inverting process of gastrulation is complete, and the two widely separated rows of single cells now abut one another, forming two adjacent lines of cells. The presumptive mesoderm that had composed the ventral side of the embryo is now inverted, as though it were the lining of a purse zipped shut by the ventral midline.

Without single-minded expression, genes usually activated in the ventral midline remain silent, midline neural and glial cells do not form, and the midline cannot fulfill its function. Because of its strategic importance, single-minded is classified as a selector gene, responsible for specifying the fate of a developmental tissue, in this case, the ventral midline.

How is expression of a gene so narrowly and precisely confined to a single band of cells on either side of the embryo? Two critical processes make this possible. One is the establishment of dorsal-ventral polarity through the influnce of the dorsal gene. Dorsal regulates transcription factors twist and snail. Snail protein represses sim in the ventral cell sheet destined to become mesoderm (Kasai, 1992). It is thought that Twist activates sim.

The second essential process is lateral inhibition, controlled by the neurogenic genes. In this case both Notch and neuralized are involved in sim regulation (Martin-Bermudo, 1995). Thus Notch is responsible for lateral inhibition, a trait exhibited when single cells are selected from groups of cells to carry forward a differentiation event. Cells adjoining the selected cell are inhibited in the process. Notch is thought to be involved in the selection of the single band of cells that will determine mesectodermal fate. Structuring as fine and complex as this, at as early a developmental stage as the blastoderm, points to the incredible power of developmental regulatory systems to specify regional organization.

Spitz group genes and commissureless are involved in development of the brain commissure that interconnects the two brain hemispheres and longitudinal pathways that connect the brain to the ventral nerve cord.Early in neurogenesis two bilaterally symmetrical cephalic neurogenicregions form. Initially, they are separated from each other and from the ventral nerve cord. Axons that project towardsthe midline in close association with an interhemispheric cellular bridge pioneer the commissure. A chain of longitudinalglial cells pioneer the descending pathway to the subesophageal ganglion. Both the commissure and descending pathway are dependent on cells of the ventral (or CNS) midline. Knock out mutations of thecommissureless gene result in a markedreduction of the brain commissure. Mutation of the single-minded gene and in other spitz groupgenes result in the absence or aberrantprojection of longitudinal pathways (Therianos, 1995).

The CNS midline cells coordinate proper cellcycle progression and identity determinationof the Drosophila ventral neuroectoderm

Defects in the sim mutant are characterizedby the loss of the gene expression required for the proper formation of the ventral neurons and epidermis, and by a decrease in the spacing of longitudinal and commissural axon tracks. Molecular and cellular mechanisms for these defectswere analyzed to elucidate the precise role of the CNS midline cells in proper patterning of the ventral neuroectoderm during embryonic neurogenesis. These analyses have shown that the ventral neuroectoderm in the sim mutant fails to carry out its proper formation and characteristic cell division cycle. This results in the loss of the dividing neuroectodermal cellsthat are located ventral to the CNS midline. The CNS midline cells are also required for the cell cycle-independent expression of the neural and epidermal markers. This indicates that the CNS midline cells are essential for theestablishment and maintenance of the ventral epidermal and neuronal cell lineage by cell-cell interaction. Nevertheless, the CNS midline cells do not cause extensive cell death in the ventral neuroectoderm. This study indicates that theCNS midline cells play important roles in the coordination of the proper cell cycle progression and the correct identity determination of the adjacent ventral neuroectoderm along the dorsoventral axis (Chang, 2000).

sim is required for the proper developmentof ventral epidermis. This was demonstrated by the fact that theexpression of the ventral ectodermal markers, enhancertrap line BP28 and otd and pnt genes, is missing in thesim mutant. Thus, this study focuses onthe role of the CNS midline cells in the formation andidentity determination of the ventral NBs during earlyneurogenesis. Initial NB formation and identity determinationdepend on the function of the achaete-scute(ac-sc) complex of proneural genes to provide a group ofneuroectodermal cells with the competence to become aNB. To investigate whether the CNS midline cells affect theexpression of a proneural gene that is essential for theinitial NB formation and identity determination, acexpression pattern was analyzed by in situ hybridization.In wild-type stage 9 embryos, ac is expressed in the MP2and S1 NBs 3-5, 7-1, and 7-4 in each hemisegment. In sim embryos, ac expression is absent in more than90% of the examined hemisegments. This result indicatesthat the CNS midline cells are required for theexpression of the proneural genes in the medial andlateral S1 NBs from the initial stage of neurogenesis (Chang, 2000).

The hkb gene is a useful marker for NBsdelaminating at the S2-S5 stages of neurogenesis. hkb, expressedin the broad area of the ventral neuroectoderm, was used todetermine whether the CNS midline cells affect the formationand identity determination of many NBs delaminatingat later S2-S5 stages after the initial round of neurogenesishas begun. The hkb expression starts in the neuroectodermof medial NB 2-2 and intermediate NB 4-2 at the middle ofstage 9. At stage 10, hkb is expressed in the S3 NBs 2-2 and4-2 and in the neuroectodermal clusters of NBs 2-4, 4-4, and5-4 and finally in the S5 NBs 2-1, 2-2, 2-4, 4-2, 4-3,4-4, 5-4, 5-5, and 7-3 at late stage 11. In sim embryos at stage 10, hkb expression in NBs 2-2 and 4-2 and in the neuroectodermal clusters of NBs 2-4, 4-4, and 5-4 is absent in 94% of hemisegments in simembryos (Chang, 2000).

To precisely analyze the expression pattern of the NBmarkers for the well-defined NBs in the sim mutant and todetermine the range of effect by the CNS midline cells onthe formation and identity determination of the ventralNBs, the expression patterns of odd-skipped (odd) and eagle(eag) during the S4-S5 stages of neurogenesis were examined.Odd is expressed in the MP2s at stage 11. In sim embryos, 17% of hemisegments do not express the odd gene. eagexpression first appears in the lateral S4 NBs 2-4 and 3-3 atearly stage 11 and then in the S5 NBs 6-4 and 7-3 at latestage 11. In sim embryosat late stage 11, eag expression in NBs 2-4, 7-3, and 6-4 isabsent in 65%, 55%, and 25% of the examined hemisegments, respectively (Chang, 2000).

Taken together, these results demonstratethat the CNS midline cells are required for the properexpression of the genes that are necessary for the formationand identity determination of the ventral S1-S5 NBsand ectodermal cells. These data indicate that the absence of NB and ectodermal marker expression in the simmutant may reflect the defects in NB formation anddivision, identity change, or cell death in the ventralneuroectoderm (Chang, 2000).

The sim mutant shows severe defects in the proper patterningof the ventral neuroectoderm, even though sim isexpressed mainly in the midline cells. These defects includethe absence of the ventral ectodermal and neuralmarker expression and the fusion of the longitudinal andcommissural connectives of the ventral nerve cord. The lack of the NB and ventral ectodermal marker expression in the sim mutant may originate from (1)defective formation and division; (2) incorrect identitydetermination, or (3) massive cell death of the ventralneuroectodermal cells during early neurogenesis. Thus, inorder to elucidate the molecular and cellular basis of howthe CNS midline cells, specified by the sim gene, arerequired for the proper patterning of the ventral neuroectoderm,the contribution of the above three possibilities toproper patterning of the ventral neuroectoderm was investigated (Chang, 2000).

To investigate whether the absence of NB marker expressionis in part due to improper NB formation, panneural NBmarkers dpn and scrt were used to examine NB formation. scrt and dpn expressing NBs start to form in threecolumns at stage 9 and give rise to a total of 10-11 S1 NBsat early stage 10. In stage 10 sim embryos,S1 NBs in the three columns of the ventral neuroectoderm are absent, at least in random positions, in 35% of the hemisegments examined. Thisanalysis demonstrates that the CNS midline cells are essentialfor the proper formation of the ventral NBs in thethree columns of ventral neuroectoderm. This result indicatesthat the absence of the NB marker expression is in part due to the defects in the formation of a correct number of the NBs in the ventral neuroectoderm (Chang, 2000).

To investigate whether the defects of the cell divisioncycle are responsible for the absence of dpn- and scrt-positiveNBs in sim embryos, the mitotic cell division pattern of theventral neuroectoderm was analyzed by staining with themitosis markers anti-Cyclin B3 and anti-phosphohistone H3 antibodies. The cyclin E gene was used to examine transition from the G1to the S phase. In wild-type embryos at stage 8, Cyclin B3 is detected inthe eight longitudinal columns of the ventral neuroectodermalcells per hemisegment. In contrast, the mesectodermalcells have no Cyclin B3 since they have alreadydivided. Later at stage 11, a group of Cyclin B3-expressingcells is located medial to the midline. In sim embryos, Cyclin B3 expression is reduced to a width ofthree to four cells in some segments of the ventral neuroectodermat stage 8. Later in sim embryos at stage11, a cluster of four to six Cyclin B3-expressing cells in themedial neuroectoderm show severely reduced Cyclin B3expression. This analysis suggests that the cycle 14mitotic cell division is defective, especially during the NBformation in sim embryos. This defect may cause the loss ofthe dpn and scrt positive NBs, which results in the absenceof the ventral neuroectodermal marker gene expression (Chang, 2000).

The expression of cyclin E is detected in the mesoderm, themesectoderm, and the striped neuroectoderm at stage 8.Later it remains only in the striped ventral neuroectodermat stage 9. The striped expression of cyclin E in theventral neuroectoderm, and in the mesoderm, is greatlyreduced in the sim mutant. Meanwhile, the expressionof cyclin E in the three columns of NBs in eachhemisegment of wild-type embryos at stage 10 is fused anddisorganized in sim mutant embryos, which may be in part due to the defects in the midline cell development. This result suggests that the ventral neuroectodermhas the defects in promoting continuous NBdivision, which results in premature NB differentiation andthe reduction of the NB number in the sim mutant (Chang, 2000).

In order to confirm that the CNS midline cells arerequired for proliferation of the ventral neuroectoderm, amitosis marker, the anti-phosphohistone H3 antibody, whichis known to recognize phosphorylated histone H3 duringmitosis, was used to directly examinethe mitotic cell division pattern of the ventral neuroectoderm.The overall mitosis pattern of the ventral neuroectodermduring early neurogenesis is basically complementaryto that of the Cyclin B3 expression. In the wild-typeembryos at stage 8, mitotic cells are detected in the midlineas well as in the head and lateral epidermis. In theearly stage 9 embryos, mitotic cells are observed in thelateral neuroectoderm (N domain). Mitotic cellsexpand ventrally into the intermediate and medial neuroectoderm,resulting in a segmentally repeated pattern in theposterior part of each parasegment at stage 10. Atstage 11, mitotic cells are detected in the entire ventralneuroectoderm. In the sim embryos throughoutthe entire neurogenesis process, mitotic cells are much reducedboth in the midline and in the ventral neuroectoderm. In the sim embryos, mitotic cells are absent in 56% of hemisegments at stage 9 and in 82% of hemisegments at stage 10 (Chang, 2000).

In order to show that the CNS midline cells controlproliferation of the ventral neuroectoderm by activation of stg, stg expression was analyzed in both wild-typeand sim mutant embryos. The stg expression profile almostcompletely matches that of phosphohistone H3 expression. stgexpression in the medial, intermediate, and lateral neuroectodermof wild-type embryos is abolished in sim embryos at stage 10. This indicates that the CNS midline cells promote mitosis of the ventral neuroectoderm by activation of stg expression through cell signaling (Chang, 2000).

To precisely identify the specific NB lineages that showthe mitotic defects in the sim mutant, double labeling ofthe neuroectodermal cells with the NB markers and themitosis marker anti-phosphohistone H3 antibody was carriedout. Initially, a panneural marker, the anti-Hb antibody,was employed to examine the general NB division pattern.In wild-type embryos at stage 10, several Hb-expressingNBs show mitotic activity. In sim embryos, 56% ofthe NBs lose Hb expression and among these NBs, morethan 95% Hb-expressing NBs have defects in both Hbexpression and mitotic activity. Some NBs such as theMP2s retain Hb expression but lose their mitotic activity. Next, a specific NB marker, eagle, was used to examine the relationship between NB-specific expression and mitotic activity. In wild-type embryos at stage 11, lateral S5 NBs 2-4, 3-3, 6-4, and 7-3 in each hemisegment showeagle expression and mitotic activity. Twenty-fivepercent of NB 2-4 and 23% of NB 7-3 exhibitexpression of both eagle and phosphohistone H3. Amongthese NBs, 93% of NB 2-4 and 97% of NB 7-3 lose theireagle expression and mitotic activity, although some NBs7-3 retain residual mitotic activity. This resultclearly demonstrates that Hb expression in most dividing NBs and eagle expression in the S5 NBs 2-4 and 7-3are coupled with mitotic activity. The CNS midline cellsmay be responsible for promoting NB division that isrequired for the general and specific NB marker expressionin the individual NBs. Taken together, these results indicate that the defectivemitotic cell division of the ventral neuroectoderm in thesim mutant is one of the major reasons for the loss of theventral neuroectodermal cells and NB-specific marker gene expression (Chang, 2000).

To separate cell cycle-independent regulation of the simgene from the effect on proper cell division in the ventralneuroectoderm, the stg mutant was employed in order toblock cell division. The stgmutant is arrested at the G2 phase of cycle 14 since zygotic stg controls the G2/M transition at cell cycle 14.Therefore, analysis of the ventral neuroectodermal markergene expression in stg and sim;stg double mutantsallows one to determine whether sim regulates the cellcycle-independent expression of the genes that determinethe identity of the ventral neural and ectodermal cells.The expression of neural (ac, castor, en) and ectodermal(BP28, otd, pnt) markers was analyzed in sim,stg, and sim;stg double mutants. ac gene is expressed infour ventral neuroectodermal clusters in each hemisegmentand is successively maintained only in a single NB that isselected from each cluster: MP2, 3-5, 7-1, and 7-4.The expression of ac in S1 NBs is absent in 90% of theexamined hemisegments of the sim and of sim;stgdouble mutant embryos. It is not,however, affected in stg embryos. This observationsuggests that the CNS midline provides the ventralneuroectodermal cells with the extrinsic signal(s) that isrequired for the initial establishment of the ventral neuroectodermalcell fate (Chang, 2000).

Castor is expressed in the S3-S5 NBs 1-2, 2-1,3-2, 3-3, 3-4, 4-1, 5-1, 5-2, 5-3, 6-1, 7-1, 7-2, and 7-4 of thewild-type embryos at stage 11. In sim embryos,its expression is absent in the medial NBs 1-2, 2-1, 4-1,and 5-1. Castor expression in most of the intermediate and lateral NBs is more severely reduced in the stg mutant than in the sim mutantembryos. This indicates that mitosis is requiredfor the proper expression of Castor in the individualdivided NBs. It is maintained in more than 95% ofthe NBs 2-1, 3-4, 4-1, and 6-1 of the stg mutant embryos. In sim;stg double mutant embryos, the expression of Castor disappears in all the medialNBs 2-1 and 4-1. This result indicates that theCNS midline cells are required for the identity determinationof the medial NBs 2-1 and 4-1. It is also demonstratedthat mitotic cell division is essential for theproper expression of Castor in order to establish theidentity of the NBs 1-2 and 5-1, which undergo severalrounds of cell division before Castor expression (Chang, 2000).

The expression pattern of En was examined in the sim, stg, and sim;stg double mutants in order to elucidate theeffect of the sim gene on the formation of lateral neurons.The number of the lateral En-positive 10-12 cells of thewild-type embryos is severely reduced to 2-3 cellsin more than 88% of hemisegments of the sim;stg mutant, while it is reduced to 5-7 cells in the stg mutant. This result indicates that the CNS midlinecells are also required for the proper generation of theEn-positive neurons (Chang, 2000).

The enhancer trap lineBP28 and otd and pnt genes were used as ventral ectodermal markers. The expression of the BP28 enhancer trap line and otdand pnt genes is abolished in the sim mutant. Beta-galactosidaseexpression of BP28 is missing in the ventralectodermal cells of sim and sim:stg mutants. The cell number of the ventral ectoderm is reduced to half in the stg mutant since these cells cannot divide. otd is expressed in two stripes of longitudinalcolumns of ventral ectoderm in the wild-type embryosat stage 11. It is absent in the simand sim;stg mutants except in a few NBs.However, it is reduced approximately by half in the stgmutant. The expression of another ectodermalmarker, pnt, disappears completely in the ventral regionof the sim, stg, and sim;stg mutants (Chang, 2000).

These results show that the CNS midline cells provide theventral neuroectodermal cells with the extrinsic signal(s)that is required for their unique identity, which is established both by cell cycle progression and by cell cycle-independent determinants (Chang, 2000).

This analysis has demonstrated that theexpression of neural (ac, castor/ming, en) and epidermal(BP28, otd) markers in the ventral neuroectodermal cellsof the stg mutant disappears in the sim;stg doublemutant. This indicates that the CNS midline cells alsocontribute to the establishment of NB identity by inducing the cell cycle-independent expression of NB, neural, and ectodermal marker genes by cell-cell interaction between the CNS midline and the ventral neuroectodermal (Chang, 2000).

In conclusion, this analysis demonstrates that the absenceof neural and epidermal marker gene expression, andthe loss of the ventral neuroectodermal cells in simmutant embryos, originates mainly from the defects in thecharacteristic cell cycle progression and in the correctidentity determination of the ventral neuroectodermalcells. Nevertheless, it appears that cell death does notmake a major contribution to the defects of the sim mutantduring the NB formation. This result indicates that theCNS midline cells are essential for the formation anddivision of a proper number of NBs and ectodermal cells andfor the expression of specific sets of the genes that providethe ventral neuroectoderm with a unique cell cycle-independentidentity through cell-cell interactions betweenthe CNS midline cells and the ventral neuroectoderm (Chang, 2000).

The following model of how the CNS midline cells are involved in proper patterning of the ventral neuroectoderm is proposed. CNS midline cellscould induce the ventral neuroectoderm via the Egfrsignaling pathway involving the spitz class, argos, Egfr, andvein genes. The secreted Spi, Vn, or the other unknownsignal(s) derived from the CNS midline cells inducethe proper patterning of the ventral neuroectoderm byactivation of the Egfr signaling pathway and the dorsoventralidentity genes during the proneural cluster formation.They could promote NB formation by providing theproneural genes with a extrinsic activation signal(s) toproduce a bias that helps the neuroectodermal cells withinthe equivalent proneural clusters commit to a NB fateagainst the lateral inhibition by the Notch/Delta signaling.Activated Egfr signaling by the CNS midline cells couldtrigger the NB division cycle to generate a sufficient numberof the NBs in the ventral neuroectoderm. Finally,stepwise activation of the Egfr signaling pathway in theindividual NBs helps each attain its unique NB identity.Each NB with its own developmental history establishes aunique genetic hierarchy, which regulates the expression ofa specific sets of genes and the timing of cell division in theNB lineage. To test the validity of this model, it remains tobe determined how the CNS midline cells influence theventral neuroectodermal patterning via Egfr signaling oradditional novel signaling by coordination of the cell cycleprogression and identity determination before gastrulationand during the neuroectodermal cluster formation along thedorsoventral axis (Chang, 2000).

GENE STRUCTURE

Exons - eight

PROTEIN STRUCTURE

Amino Acids - 673

Structural Domains

Both single-minded and period contain an amino acid motif known as PAS (Huang, 1993). The PAS repeat consists of two 51 amino acid repeats separated by 115 amino acids in SIM, and 99 amino acids in PER. The bHLH in SIM is found at the N-terminal. SIM also has an alanine-alanine-glutamine repeat region, a proline rich region, and a C-terminal glutamine rich region (Nambu, 1991).

Genetic experimentssuggest that Single-minded can function as a transcriptional activator. When regions of theSingle-minded protein are fused to the DNA binding domain of the mammalian transcriptionfactor Sp1, they activate transcription from a reporter gene linked to Sp1 binding sites.Three independent activation domains have been identified in the carboxy terminal region ofSingle-minded that include areas rich in serine, threonine, glutamine and proline residues. Germ linetransformation experiments indicate that the carboxy terminal activation domains, the PASdimerization domain, and the putative DNA binding basic domain of Single-minded are required forexpression of CNS midline genes in vivo. These results define in vivo a functional activationdomain within Single-minded and suggest a model in which Single-minded activates transcriptionthrough a direct interaction with promoter elements of CNS midline genes (Franks, 1994).

date revised: 30 January 2001

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