Examples of twin-spot MARCM clones used in an analysis of MB lineage are shown: two-cell + multi-cellular Nb clones (a-i) and two single-cell clones (j-u) in adult (a-r) and wandering larval (s-u) brains. Mitotic recombination was induced by FLP activity in MB Nbs (a-i) or GMCs (j-u) at different developmental stages, including 1^st instar larval (a-c, j-l and s-u), mid 3^rd instar larval (d-f and m-o) and mid-late pupal (g-i and p-r) stages. GAL4-OK107 (a-i and m-r) and MB247-GAL4 (j-l and s-u) were used to visualize twin-spot MARCM clones. In the two-cell + multi-cellular Nb clones: two γ MB neurons (a) are associated with a MB clone (b) highlighting all MB lobes (γ, α, α′, β and β′ lobes); two α′/β′ MB neurons (d) are associated with a MB clone (e) revealing α, α′, β and β′ lobes; two α/β MB neurons (g) are associated with a MB clone (h) containing only α and β lobes. In the two single-cell clones: a green γ MB neuron (j and s) is associated with the other magenta γ MB neuron (k and t); a green α′/β′ MB neuron (m) is associated with the other magenta α′/β′ MB neuron (n); a green α/β MB neurons (p) is associated with the other magenta α/β MB neuron (q). These analyses allow us to distinguish two-cell clones from double inductions in separate GMCs (j-u). The green and magenta merged images are shown in c, f, i, l, o, r and u.

The pan-neuronal driver, GAL4-C155, was used to survey proliferation patterns of Drosophila larval CNS lineages. Twin-spot MARCM clones were induced at a low frequency to simplify the lineage analysis at Drosophila central brain (a-i, l, o and r) and VNC (j, k, m, n, p and q). Like GAL4-OK107, GAL4-C155 can faithfully label two-cell + multi-cellular Nb clones (a, d and g) in MB lineages, which validates the use of GAL4-C155 to probe the overall proliferation patterns of Drosophila CNS lineages. Three types of twin-spot MARCM clones were classified based on the cell number of their associated sister clones. The first class, like MB clones, consists of two-cell + multi-cellular Nb clones (b, e, h, j, m and p) that indicate the familiar production of two progeny by a GMC division. The second class of clones has single-cell + multi-cellular Nb clones (c, f, i, k, n and q) that resemble lineages in which only one daughter cell from the GMC division survives. The lineage identity of [p] and [q] resembles the previously described VNC lineage 13 and lineage 20, respectively 27. The third class is represented by 8-12 cells + large multi-cellular Nb clones (l, o and r) and resemble the recently described PAN lineages in which each transit-amplifying precursor produces multiple GMCs through a limited run of self-renewing asymmetric division.

Six central complex neurons (a), nc82 staining for revealing relative positions to these neurons (a-b) and a schematic drawing of these neurons (c). Twin-spot MARCM analyses on this central complex sublineage reveals birth-order of these neurons (d-h and o). The first four neurons (d-g) extend into the noduli with the projections to discrete subcompartments, including the dorsal noduli (d), anterior ventral noduli (e), posterior ventral noduli (f) and middle noduli (g). The last two neurons (h and o) innervate the ellipsoid body. Neuronal morphology alternates between narrow and elaborate neurites (d, f and h vs. e, g and o) in the lateral accessory lobe. A third-born chinmo neuron (3rd^4th; i), determined by its reference wild-type Nb clone (p), has the morphology of wild-type fourth-born neuron. This 3rd^4th born neuron innervates the middle noduli that normally is innervated by the 4th born neuron (arrowheads in i, p and w). The neurite pattern in the lateral accessory lobe of this 3rd^4th born neuron is also transformed from narrow to elaborate (triple arrows in f, g and i). In contrast, the reverse twin-spot MARCM analysis shows that the second-born wild-type single-cell clone is associated with a chinmo Nb clone (j, q and x). Compared to the projection to the posterior ventral noduli in the wild-type Nb clone (arrow in l), the same morphology is not present in the chinmo Nb clone (arrow in q), confirming the transformation of the third-born neuron to the wild-type fourth-born neuron morphology. Asterisk indicates cell body position.

Comparison of expected clone patterns from various mitotic recombination-based mosaic systems [conventional mosaics (a), MARCM (b), and twin-spot MARCM (c)] to actual twin-spot MARCM clones in a 3^rd instar wing disc (d). (a) In conventional mosaic analysis, clones of interest with the homozygous genotype are invisible (shown in black). The reference clones (dark magenta) are intermingled with the background (light magenta). (b) In MARCM, clones of interest with the homozygous genotype (shown in green) stand out from the unlabeled background (black) and the reference clones (arrows). (c and d) In twin-spot MARCM, clones of interest and the reference clones with homozygous genotypes are visible in green and magenta, respectively, and stand out clearly from the unlabeled background (black). (e) In the twin-spot MARCM system, “suppressor 1” (which inhibits the expression of “reporter 1”) and “reporter 2” are placed distal to one FRT site, while “suppressor 2” (which inhibits the expression of “reporter 2”) and “reporter 1” are placed distal to the other FRT site. After induction of FLP to activate FRT-mediated mitotic recombination, one daughter cell (green) inherits “suppressor 2” and “reporter 1”, while the other (magenta) gets “suppressor 1” and “reporter 2”. A lineage undergoing symmetric (f) or asymmetric (g) proliferation will produce two multi-cellular clones (f) or a two-cell + multi-cellular clone (g). Two single-cell clones (g) appear, one in green and the other magenta, when mitotic recombination occurs during last mitoses (as in a GMC). The scale bar in this and all other figures equals 20 μm.