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 neurogenic regions form. Initially, they are separated from each other and from the ventral nerve cord. Axons that project towards the midline in close association with an interhemispheric cellular bridge pioneer the commissure. A chain of longitudinal glial 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 the commissureless gene result in a marked reduction of the brain commissure. Mutation of the single-minded gene and in other spitz group genes result in the absence or aberrant projection of longitudinal pathways (Therianos, 1995).

The CNS midline cells coordinate proper cell cycle progression and identity determination of the Drosophila ventral neuroectoderm

Defects in the sim mutant are characterized by 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 defects were 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 cells that 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 the establishment 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 the CNS 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 development of ventral epidermis. This was demonstrated by the fact that the expression of the ventral ectodermal markers, enhancer trap line BP28 and otd and pnt genes, is missing in the sim mutant. Thus, this study focuses on the role of the CNS midline cells in the formation and identity determination of the ventral NBs during early neurogenesis. Initial NB formation and identity determination depend on the function of the achaete-scute (ac-sc) complex of proneural genes to provide a group of neuroectodermal cells with the competence to become a NB. To investigate whether the CNS midline cells affect the expression of a proneural gene that is essential for the initial NB formation and identity determination, ac expression pattern was analyzed by in situ hybridization. In wild-type stage 9 embryos, ac is expressed in the MP2 and S1 NBs 3-5, 7-1, and 7-4 in each hemisegment. In sim embryos, ac expression is absent in more than 90% of the examined hemisegments. This result indicates that the CNS midline cells are required for the expression of the proneural genes in the medial and lateral S1 NBs from the initial stage of neurogenesis (Chang, 2000).

The hkb gene is a useful marker for NBs delaminating at the S2-S5 stages of neurogenesis. hkb, expressed in the broad area of the ventral neuroectoderm, was used to determine whether the CNS midline cells affect the formation and identity determination of many NBs delaminating at later S2-S5 stages after the initial round of neurogenesis has begun. The hkb expression starts in the neuroectoderm of medial NB 2-2 and intermediate NB 4-2 at the middle of stage 9. At stage 10, hkb is expressed in the S3 NBs 2-2 and 4-2 and in the neuroectodermal clusters of NBs 2-4, 4-4, and 5-4 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 sim embryos (Chang, 2000).

To precisely analyze the expression pattern of the NB markers for the well-defined NBs in the sim mutant and to determine the range of effect by the CNS midline cells on the formation and identity determination of the ventral NBs, 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. eag expression first appears in the lateral S4 NBs 2-4 and 3-3 at early stage 11 and then in the S5 NBs 6-4 and 7-3 at late stage 11. In sim embryos at late stage 11, eag expression in NBs 2-4, 7-3, and 6-4 is absent in 65%, 55%, and 25% of the examined hemisegments, respectively (Chang, 2000).

Taken together, these results demonstrate that the CNS midline cells are required for the proper expression of the genes that are necessary for the formation and identity determination of the ventral S1-S5 NBs and ectodermal cells. These data indicate that the absence of NB and ectodermal marker expression in the sim mutant may reflect the defects in NB formation and division, identity change, or cell death in the ventral neuroectoderm (Chang, 2000).

The sim mutant shows severe defects in the proper patterning of the ventral neuroectoderm, even though sim is expressed mainly in the midline cells. These defects include the absence of the ventral ectodermal and neural marker expression and the fusion of the longitudinal and commissural 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 identity determination, or (3) massive cell death of the ventral neuroectodermal cells during early neurogenesis. Thus, in order to elucidate the molecular and cellular basis of how the CNS midline cells, specified by the sim gene, are required for the proper patterning of the ventral neuroectoderm, the contribution of the above three possibilities to proper patterning of the ventral neuroectoderm was investigated (Chang, 2000).

To investigate whether the absence of NB marker expression is in part due to improper NB formation, panneural NB markers dpn and scrt were used to examine NB formation. scrt and dpn expressing NBs start to form in three columns at stage 9 and give rise to a total of 10-11 S1 NBs at 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. This analysis demonstrates that the CNS midline cells are essential for the proper formation of the ventral NBs in the three columns of ventral neuroectoderm. This result indicates that 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 division cycle are responsible for the absence of dpn- and scrt-positive NBs in sim embryos, the mitotic cell division pattern of the ventral neuroectoderm was analyzed by staining with the mitosis markers anti-Cyclin B3 and anti-phosphohistone H3 antibodies. The cyclin E gene was used to examine transition from the G1 to the S phase. In wild-type embryos at stage 8, Cyclin B3 is detected in the eight longitudinal columns of the ventral neuroectodermal cells per hemisegment. In contrast, the mesectodermal cells have no Cyclin B3 since they have already divided. Later at stage 11, a group of Cyclin B3-expressing cells is located medial to the midline. In sim embryos, Cyclin B3 expression is reduced to a width of three to four cells in some segments of the ventral neuroectoderm at stage 8. Later in sim embryos at stage 11, a cluster of four to six Cyclin B3-expressing cells in the medial neuroectoderm show severely reduced Cyclin B3 expression. This analysis suggests that the cycle 14 mitotic cell division is defective, especially during the NB formation in sim embryos. This defect may cause the loss of the dpn and scrt positive NBs, which results in the absence of the ventral neuroectodermal marker gene expression (Chang, 2000).

The expression of cyclin E is detected in the mesoderm, the mesectoderm, and the striped neuroectoderm at stage 8. Later it remains only in the striped ventral neuroectoderm at stage 9. The striped expression of cyclin E in the ventral neuroectoderm, and in the mesoderm, is greatly reduced in the sim mutant. Meanwhile, the expression of cyclin E in the three columns of NBs in each hemisegment of wild-type embryos at stage 10 is fused and disorganized in sim mutant embryos, which may be in part due to the defects in the midline cell development. This result suggests that the ventral neuroectoderm has the defects in promoting continuous NB division, which results in premature NB differentiation and the reduction of the NB number in the sim mutant (Chang, 2000).

In order to confirm that the CNS midline cells are required for proliferation of the ventral neuroectoderm, a mitosis marker, the anti-phosphohistone H3 antibody, which is known to recognize phosphorylated histone H3 during mitosis, was used to directly examine the mitotic cell division pattern of the ventral neuroectoderm. The overall mitosis pattern of the ventral neuroectoderm during early neurogenesis is basically complementary to that of the Cyclin B3 expression. In the wild-type embryos at stage 8, mitotic cells are detected in the midline as well as in the head and lateral epidermis. In the early stage 9 embryos, mitotic cells are observed in the lateral neuroectoderm (N domain). Mitotic cells expand ventrally into the intermediate and medial neuroectoderm, resulting in a segmentally repeated pattern in the posterior part of each parasegment at stage 10. At stage 11, mitotic cells are detected in the entire ventral neuroectoderm. In the sim embryos throughout the entire neurogenesis process, mitotic cells are much reduced both 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 control proliferation of the ventral neuroectoderm by activation of stg, stg expression was analyzed in both wild-type and sim mutant embryos. The stg expression profile almost completely matches that of phosphohistone H3 expression. stg expression in the medial, intermediate, and lateral neuroectoderm of 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 show the mitotic defects in the sim mutant, double labeling of the neuroectodermal cells with the NB markers and the mitosis marker anti-phosphohistone H3 antibody was carried out. 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-expressing NBs show mitotic activity. In sim embryos, 56% of the NBs lose Hb expression and among these NBs, more than 95% Hb-expressing NBs have defects in both Hb expression and mitotic activity. Some NBs such as the MP2s 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 show eagle expression and mitotic activity. Twenty-five percent of NB 2-4 and 23% of NB 7-3 exhibit expression of both eagle and phosphohistone H3. Among these NBs, 93% of NB 2-4 and 97% of NB 7-3 lose their eagle expression and mitotic activity, although some NBs 7-3 retain residual mitotic activity. This result clearly demonstrates that Hb expression in most dividing NBs and eagle expression in the S5 NBs 2-4 and 7-3 are coupled with mitotic activity. The CNS midline cells may be responsible for promoting NB division that is required for the general and specific NB marker expression in the individual NBs. Taken together, these results indicate that the defective mitotic cell division of the ventral neuroectoderm in the sim mutant is one of the major reasons for the loss of the ventral neuroectodermal cells and NB-specific marker gene expression (Chang, 2000).

To separate cell cycle-independent regulation of the sim gene from the effect on proper cell division in the ventral neuroectoderm, the stg mutant was employed in order to block cell division. The stg mutant 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 marker gene expression in stg and sim;stg double mutants allows one to determine whether sim regulates the cell cycle-independent expression of the genes that determine the 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 in four ventral neuroectodermal clusters in each hemisegment and is successively maintained only in a single NB that is selected from each cluster: MP2, 3-5, 7-1, and 7-4. The expression of ac in S1 NBs is absent in 90% of the examined hemisegments of the sim and of sim;stg double mutant embryos. It is not, however, affected in stg embryos. This observation suggests that the CNS midline provides the ventral neuroectodermal cells with the extrinsic signal(s) that is required for the initial establishment of the ventral neuroectodermal cell 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 the wild-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 mutant embryos. This indicates that mitosis is required for the proper expression of Castor in the individual divided NBs. It is maintained in more than 95% of the 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 medial NBs 2-1 and 4-1. This result indicates that the CNS midline cells are required for the identity determination of the medial NBs 2-1 and 4-1. It is also demonstrated that mitotic cell division is essential for the proper expression of Castor in order to establish the identity of the NBs 1-2 and 5-1, which undergo several rounds 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 the effect of the sim gene on the formation of lateral neurons. The number of the lateral En-positive 10-12 cells of the wild-type embryos is severely reduced to 2-3 cells in 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 midline cells are also required for the proper generation of the En-positive neurons (Chang, 2000).

The enhancer trap line BP28 and otd and pnt genes were used as ventral ectodermal markers. The expression of the BP28 enhancer trap line and otd and pnt genes is abolished in the sim mutant. Beta-galactosidase expression of BP28 is missing in the ventral ectodermal 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 longitudinal columns of ventral ectoderm in the wild-type embryos at stage 11. It is absent in the sim and sim;stg mutants except in a few NBs. However, it is reduced approximately by half in the stg mutant. The expression of another ectodermal marker, pnt, disappears completely in the ventral region of the sim, stg, and sim;stg mutants (Chang, 2000).

These results show that the CNS midline cells provide the ventral 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 the expression of neural (ac, castor/ming, en) and epidermal (BP28, otd) markers in the ventral neuroectodermal cells of the stg mutant disappears in the sim;stg double mutant. This indicates that the CNS midline cells also contribute 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 absence of neural and epidermal marker gene expression, and the loss of the ventral neuroectodermal cells in sim mutant embryos, originates mainly from the defects in the characteristic cell cycle progression and in the correct identity determination of the ventral neuroectodermal cells. Nevertheless, it appears that cell death does not make a major contribution to the defects of the sim mutant during the NB formation. This result indicates that the CNS midline cells are essential for the formation and division of a proper number of NBs and ectodermal cells and for the expression of specific sets of the genes that provide the ventral neuroectoderm with a unique cell cycle-independent identity through cell-cell interactions between the 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 cells could induce the ventral neuroectoderm via the Egfr signaling pathway involving the spitz class, argos, Egfr, and vein genes. The secreted Spi, Vn, or the other unknown signal(s) derived from the CNS midline cells induce the proper patterning of the ventral neuroectoderm by activation of the Egfr signaling pathway and the dorsoventral identity genes during the proneural cluster formation. They could promote NB formation by providing the proneural genes with a extrinsic activation signal(s) to produce a bias that helps the neuroectodermal cells within the equivalent proneural clusters commit to a NB fate against the lateral inhibition by the Notch/Delta signaling. Activated Egfr signaling by the CNS midline cells could trigger the NB division cycle to generate a sufficient number of the NBs in the ventral neuroectoderm. Finally, stepwise activation of the Egfr signaling pathway in the individual NBs helps each attain its unique NB identity. Each NB with its own developmental history establishes a unique genetic hierarchy, which regulates the expression of a specific sets of genes and the timing of cell division in the NB lineage. To test the validity of this model, it remains to be determined how the CNS midline cells influence the ventral neuroectodermal patterning via Egfr signaling or additional novel signaling by coordination of the cell cycle progression and identity determination before gastrulation and during the neuroectodermal cluster formation along the dorsoventral 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 experiments suggest that Single-minded can function as a transcriptional activator. When regions of the Single-minded protein are fused to the DNA binding domain of the mammalian transcription factor 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 of Single-minded that include areas rich in serine, threonine, glutamine and proline residues. Germ line transformation experiments indicate that the carboxy terminal activation domains, the PAS dimerization domain, and the putative DNA binding basic domain of Single-minded are required for expression of CNS midline genes in vivo. These results define in vivo a functional activation domain within Single-minded and suggest a model in which Single-minded activates transcription through a direct interaction with promoter elements of CNS midline genes (Franks, 1994).

date revised: 5 December 2023

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