Development 143: doi:10.1242/dev.133546: Supplementary information
Supplementary Materials
Supplementary Material and Methods
Fly strains
The following fly strains were used: Oregon R (wild type); AJ96-lacZ (Klaes et al. 1994) and vnd-lacZ
(Chu et al., 1998) (from Christian Klämbt); CQ2-Gal4 (Landgraf et al., 2003a) and eveRRK-Gal4
(Fujioka et al., 2003) (from Matthias Landgraf); Df(3L)H99 (White et al., 1994), Dfd16, NGT40-Gal4,
UAS-mCD8::GFP, UAS-nGFP (all from Bloomington Stock Center); gooseberry-distal-lacZ (from
Fernando Diaz-Benjumea); H15-lacZ (Brook and Cohen, 1996) (from Laura Nilson); msh68 (Isshiki et
al., 1997) (from Takako Isshiki); Mz97-Gal4 (Ito et al., 1995); sca-Gal4 (Ellis et al., 1994);
unplugged-lacZ, huckebein-lacZ, castor-lacZ, mirror-lacZ, seven-up-lacZ and wingless-lacZ (Doe,
1992) (all from Chris Doe); UAS-ind (von Ohlen et al., 2007) (from Tonia von Ohlen).
Antibodies
The following primary antibodies were used: mouse-anti-Achaete (1:2) (Skeath and Caroll, 1991),
mouse-anti-Antennapedia 8C11 (1:20) (Condie et al., 1991), mouse-anti-Dachshund 2-3 (1:250)
(Mardon et al., 1994), mouse-anti-Invected (1:2) (Patel et al., 1989), mouse-anti-Prospero (1:10),
mouse-anti-Sex combs reduced 6H4.1 (1:20) (Glicksman and Brower, 1988), mouse-anti-Wingless
4D4 (1:15) (all from Developmental Studies Hybridoma Bank); rat-anti-Giant (1:200), guinea pig-
al., 1998) (all from Ralf Pflanz); chicken-anti-Beta-Gal (#ab9361) (1:1000) (Abcam); rabbit-antiCastor (1:500) (Kambadur et al., 1998) (from Ward Odenwald); sheep-anti-Cousin of atonal (1:1000)
(zurLage and Jarman, 2010) (from Andrew Jarman); mouse-anti-Collier (1:100) (Dubois et al., 2007)
(from Michèle Crozatier); guinea pig-anti-Dbx (1:1500) (Lacin et al., 2009) (from James Skeath);
rabbit-anti-Death caspase-1 (#9578) (1:50) (Cell Signaling Technology); guinea-pig-anti-Deadpan
(1:1000) (Homem et al., 2013) (from Jürgen Knoblich); rabbit-anti-Deformed (#sc-98593) (1:20),
rabbit-anti-Engrailed (#sc-28640) (1:100) (both from Santa Cruz Biotechnology); rabbit-anti-Eagle
(1:500) (Dittrich et al., 1997), guinea pig-anti-Hunchback (1:1000) (Mettler et al., 2006) (both from
Joachim Urban); rat-anti-Empty spiracles (1:1000) (Walldorf and Gehring, 1992) and rabbit-anti-
Development • Supplementary information
anti-Knirps (1:400), guinea pig-anti-Odd-skipped (1:200), guinea pig-anti-Runt (1:300) (Kosman et
Development 143: doi:10.1242/dev.133546: Supplementary information
Eyeless (1:1000) (Kammermeier et al., 2001) (both from Uwe Walldorf); rabbit-anti-Even skipped
(1:1000) (Frasch et al., 1987) (from Manfred Frasch); chicken-anti-GFP (#AB16901) (1:500)
(Millipore); rat-anti-Gooseberry-distal (1:2), rat-anti-Gooseberry-proximal (1:2) (Zhang et al., 1994)
(both from Robert Holmgren); rabbit-anti-Intermediate neuroblasts defective (1:5000) (Von Ohlen and
Moses, 2009) (from Tonia von Ohlen); mouse-anti-Ladybird-early (1:2) (Jagla et al., 1997) (from
Krzysztof Jagla); guinea pig-anti-Midline (1:500) (Fregoso Lomas et al., 2013) (from Laura Nilson);
rabbit-anti-Miranda (1:100) (Betschinger et al., 2006) (from Jürgen Knoblich); rabbit-anti-Muscle
segment homeobox (1:500) (from Matthew Scott); rat anti-Muscle segment homeobox (Moses et al.,
2011) (from Ze’ev Paroush); guinea pig-anti-Orthodenticle (1:500) (Xie et al., 2007) (from Tiffany
Cook); rabbit-anti-Pox neuro (1:75) (Bopp et al., 1989) (from Markus Noll); guinea pig-anti-Reversedpolarity (1:5000) (from Benjamin Altenhein); rabbit-anti-Ventral nervous system defective (1:2000)
(Shao et al., 2002) (from Marshall Nirenberg); mouse-anti-Worniu (1:1000) (Cai et al., 2001) (from
Yu Cai); sheep-anti-DIG alkaline-phosphatase conjugated (#11093274910) (1:1000), anti-DIG-POD
(#11207733910) (1:500) (both Roche Diagnostics).
The secondary antibodies were either biotinylated, conjugated with alkaline-phosphatase, or
DyLight™, Cyanine (all Jackson Immunoresearch) and Alexa (Life technologies) fluorescent dyes (all
diluted 1:500). Tyramide signal amplification (TSA biotin system; #NEL700A001KT; PerkinElmer)
was used in non-fluorescent stainings for anti-En, anti-Repo and anti-Beta-Gal antibodies following
Development • Supplementary information
the manufacturer’s protocol.
Development 143: doi:10.1242/dev.133546: Supplementary information
Supplemental Figures
Figure S1. Identification of NBs in the gnathal neuromeres.
Composite confocal images of flat preparations (ventral view; anterior to the top) of stage 11 embryos.
(A-B) Expression of cousin of atonal (cato), specifically expressed in all sensory organ precursor cells
(SOPs) at stage 11 (Goulding et al., 2000), (A) in combination with Deadpan (Dpn) or (B) Worniu
additionally detected in most SOPs, partly lying in close vicinity to NBs (yellow arrows).
Development • Supplementary information
(Wor). Note that expression of Wor is more restricted to NBs than Dpn, and that Dpn expression is
Figure S2.1. Identification of NBs in the gnathal neuromeres – expression of gap genes.
Composite confocal images of flat preparations (ventral view; anterior to the top) of stage 11 embryos
stained against different combinations of molecular markers as illustrated. Individual NBs, which can
be identified by marker staining(s) and position are indicated (by lettering). (A-H) Expression of the
gap genes empty spiracles (Ems protein), orthodenticle (Otd protein), huckebein (hkb, as revealed by
hkb-lacZ), giant (Gt protein), buttonhead (btd mRNA), sloppy paired-1(slp-1 mRNA), and knirps (kni
mRNA) at the level of gnathal NBs at late stage 11. (A, A´) Stainings against Ems, Cato and Wor
Development • Supplementary information
Development 143: doi:10.1242/dev.133546: Supplementary information
Development 143: doi:10.1242/dev.133546: Supplementary information
show that the Ems-positive NBs 3-3, 3-5, 4-4 (see also Hartmann et al. 2000) lie adjacent to SOPs. In
MN we recognized only two Ems-positive NBs, as NB3-3 is missing. (B, B´) Otd/En/Dpn staining.
Otd is segmentally expressed in four ventral NBs (NB2-1, NB3-1, NB5-1 and NB6-1) in each thoracic
hemineuromere (not shown; see Fig. 3A for prothorax), which all are found in LB and MX, while in
MN Otd-expression is detected only in NB3-1 and NB6-1, but not in NB2-1, and NB5-1 is absent (in
B Otd-negative ventral NBs are indicated in yellow). (C, C´) hkb-lacZ/En/Dpn staining. hkb-lacZ is
detected in 10 NBs in the prothoracic hemineuromere (NBs 1-1, 2-1, 2-2, 2-4, 4-2, 4-3, 4-4, 5-4, 5-5,
7-3; see also Fig. 3A and previous descriptions of hkb expression in NBs of the VNC, Broadus et al.,
1995; McDonald and Doe,1997). It can be identified in eight corresponding NBs in LB, while NBs 2-4
and 5-4 are missing; note that labial and maxillary NB1-1 are not in focus. In MX, hkb-lacZ is
detectable in only six NBs, and not expressed in existing NB1-1; NBs 2-4, 5-4 are missing. Also in
MN, hkb-lacZ is detected in only six NBs, not in existing NB5-5 and the missing NBs 1-1, 2-4, 5-4.
(D, D´) Gt/Dpn/Ladybird early (Lbe) staining at early stage 10 (est10) focussing on the neuroectoderm
(D´) and the NB layer (D). (D) In MN, NB5-6 (Urbach and Technau, 2003b; De Graeve et al., 2004)
and atypically NB5-5 express Lbe (see also Fig. 2 B,B´), whereas only NB5-5 co-expresses Gt. (E)
Gt/En/Dpn staining at midstage 11 (mst11). In MN, NB5-5 develops from a distinct Gt/Lbe-positive
proneural cluster (encircled with white stippled line in D´). Gt-positive neuroectoderm also gives rise
to MP2a,b,c (F) btd/svp-lacZ staining. In prothorax, btd mRNA is expressed in four NBs per
hemineuromere (NBs 4-1, 4-2, 5-6, 7-3; see Fig.3A). In all gnathal neuromeres it is found in three of
those NBs, but not in NB5-6. (G) slp1/svp-lacZ staining. In all gnathal neuromeres (and prothorax, see
Fig. 3A), slp1 mRNA is segmentally expressed in row 4 and 5 NBs (similar to previous description of
btd expression in NBs of the VNC (Bhat et al., 2000)), and in T1, LB and MX additionally in NB3-5.
(H) kni/svp-lacZ staining. kni mRNA is expressed in NBs 2-5 and 5-1 in T1 (see Fig. 1), LB and MX.
In LB, exceptionally, kni is also expressed in NB2-1. In MN kni is not expressed as NB2-4 and NB5-1
Development • Supplementary information
are lacking.
Figure S2.2. Identification of NBs in the gnathal neuromeres – expression of segment polarity
and Hox genes.
Composite confocal images of flat preparations (ventral view; anterior to the top) of stage 11 embryos
stained against different combinations of molecular markers as illustrated. Individual NBs, which can
be identified by marker staining(s) and position are indicated (by lettering). (A-F) Expression of
segment polarity genes gooseberry-distal (gsb-d; as revealed by gsb-lacZ), wingless (Wg protein),
H15 (revealed by H15-lacZ), mid (Mid protein), the homeotic genes Sex comb reduced (Scr protein),
Antennapedia (Antp protein), and cap and collar (cnc mRNA). (A, A´) Gsb-d-lacZ in T1 is expressed
in ten NBs per hemineuromere, including all row 5 and 6 NBs, as well as NB 7-1 (see Fig. 3 and
Broadus et al., 1995; Zhang et al., 1994). In LB, Gsb-d-lacZ is detected in corresponding NBs, except
Development • Supplementary information
Development 143: doi:10.1242/dev.133546: Supplementary information
Development 143: doi:10.1242/dev.133546: Supplementary information
in missing NB5-4. In MX it is detected in eight NBs, except in the missing NBs 5-4, 5-5. In MN, it is
detected in only seven of those NBs, among which is NB5-5, but not in lacking NBs 5-1, 5-4, 6-4. (B,
B´) Wg is expressed in all row 5 NBs (Doe, 1992). Accordingly, in LB it is expressed in all row 5 NBs
(except in lacking NB5-5), and exceptionally in NBs 4-3 and 4-4. In MX and MN, Wg is detected in
four row 5 NBs each (in MX not in missing NBs 5-4 and 5-5; in MN not in missing NBs 5-1 and 5-4).
(C, C´) H15-lacZ (similar to Mid expression; see also Büscher et al, 2006; and Figs. 2,3) is expressed
in NBs 1-2, 3-4, 7-2, 7-3 and 7-4 in T1, LB and MX. In MX and MN it is additionally weakly detected
in NB6-2, and in MN additionally in NB2-2 but not in lacking NB1-2. The number of (eight) H15lacZ-positive NBs is thus highest in MN. (D, D´) Mid is expressed in four NBs per hemineuromere
(Büscher et al., 2006); however, we detected Mid in NB3-4 instead of NB2-5 (NBs 1-2,3-4,7-2,7-4).
In MN, Mid is expressed in only three NBs (NBs 3-5,7-2,7-4). (E, E´) Scr is expressed in NBs within
the posterior compartment of MX (all row 6 and 7 NBs) and anterior compartment of LB. (F, F´) Antp
is expressed in all NBs within the posterior compartment of the LB (and additionally in thoracic NBs,
not shown). (G) The expression domain of cnc, a functional suppressor of Dfd (McGinnis et al., 1998),
at the level of the outer neuroectoderm; the posterior limit of the cnc domain corresponds to the
segment boundary between MN and MX. (G´) However, whereas all NBs developing from the
Development • Supplementary information
anterior compartment of the MN express cnc (as indicated), the posterior En-positive NBs do not.
Figure S2.3. Identification of NBs in the gnathal neuromeres – expression of DV genes.
Composite confocal images of flat preparations (ventral view; anterior to the top) of stage 11 embryos
stained against different combinations of molecular markers as illustrated. Individual NBs, which can
be identified by marker staining(s) and position are indicated (by lettering). (A-D) Expression of DV
genes ventral nervous system defective (Vnd protein), Dichaete (D protein), Nkx6 (mRNA) and muscle
segment homeobox (Msh; Drop – FlyBase). (A, A´) Vnd is expressed in all (10) ventral NBs and the
intermediate NB7-2 in the VNC (Chu et al., 1998; Shao et al., 2002) (see also Fig. 3C for T1); Vnd is
Development • Supplementary information
Development 143: doi:10.1242/dev.133546: Supplementary information
Development 143: doi:10.1242/dev.133546: Supplementary information
also expressed in MP2 (not shown). The same set of Vnd-positive NB can be found in LB and MX
(except the missing MP2), and in MN (except the missing NBs 1-1,1-2) in which Vnd is additionally
detected in NB2-2. (B, B´) Dichaete is dynamically expressed in many NBs. Although prominently
expressed in ventral and intermediate neuroectoderm, Dichaete is often detected at low levels in NB
subsets. At late stage 11, only few Dichaete-positive NBs were found in T1 and LB, including some
dorsal NBs (see also Zhao and Skeath, 2002): NBs 3-2, 4-1, 4-3, 5-6, 6-4, 7-3, 7-4 (except for NB4-3
in LB); in MX, it is additionally detected in NB 1-2. By contrast, almost all NBs in MN express
Dichaete. (C, C´) Nkx6 is expressed in subsets of ventral and intermediate NBs, in T1 in NBs 2-2, 3-1,
3-2 and 4-2 (see also Fig. 3E, and Uhler et al., 2002). In all gnathal neuromeres, Nkx6 is detected in
further ventral and intermediate NBs, although at lower levels: in LB additionally in NBs 5-2, 6-1, 7-1,
and in MX additionally in NBs 1-2, 2-1, 4-1, 5-1, 5-2, 6-1, 7-1. In MN six Nkx6-expressing NBs were
detected. Outlines of NBs (as given by Dpn stainings in C) have been indicated with white stippled
lines in (C´). (D, D´) Msh expression is confined to subsets of dorsal NBs (see also Isshiki et al.,
1997). In T1 it is detected in eight NBs (NBs 2-4, 2-5, 3-4, 4-3, 5-4, 6-4, 7-4, LGB; see Fig. 3E), in LB
in seven (NB 2-4 missing) and in MX in six (NBs 2-4 and 5-4 missing). Only five Msh-positive NBs
are detected in MN (additionally expressed in NB3-5, 5-6, but not in the missing NBs 2-5, 5-4, 6-4,
Development • Supplementary information
LGB).
Figure S2.4. Identification of NBs in the gnathal neuromeres - expression of temporal and other
genes
Composite confocal images of flat preparations (ventral view; anterior to the top) of stage 11 embryos
stained against different combinations of molecular markers as illustrated. Individual NBs, which can
be identified by marker staining(s) and position are indicated (by lettering). (A-G) Expression of
temporal genes hunchback (Hb protein), pou domain 1 (Pdm1, Nubbin – FlyBase; protein), castor
(Cas protein), and of other NB identity genes Dbx (protein), unplugged (unpg, revealed by unpg-lacZ),
charybdee (chrb, mRNA), CenG1A (mRNA), Aj96-lacZ, odd-skipped (Odd protein) and pox neuro
(Poxn protein)
Development • Supplementary information
Development 143: doi:10.1242/dev.133546: Supplementary information
Development 143: doi:10.1242/dev.133546: Supplementary information
(A-C´) All temporal genes are very dynamically expressed in NBs, including those in the gnathal
neuromeres. However, at late stage 11 we recognized NB subsets expressing significant levels of Hb,
Pdm1 or Cas. (A, A´) In T1 Hb is expressed in seven NBs (NBs 2-3, 2-4, 4-3, 4-4, 5-4, 7-3, MP2) that,
except MP2, develop late (see Fig. 1; 3D). A subset of those Hb-positive NBs is found in LB (except
the missing NBs 2-3, 2-4), MX and MN (in both except the missing NBs 2-3, 2-4, 5-4). In MN,
additional NBs express Hb (NBs 3-5, 4-2, 5-5, MP2a,b, MP2c (not in focus)). (B, B´) Pdm1 is at late
stage 11 transiently found in many gnathal NBs with different levels of expression: it is reproducibly
detected in about 2/3 of NBs in T1 (see Fig. 3D and Kambadur et al., 1998), LB and MX, and almost
all NBs in MN. (C, C´) At late stage,11 Cas is transiently expressed in more than half of the NB in T1
(see Fig. 3D; see also Doe, 1992), in LB (except in existing NB2-2) and in MX (except in existing
NBs 2-1 and 2-2). In MN some Cas-positive NBs are missing (NBs 3-3,5-1) and others additionally
express Cas (NBs 3-1, 4-2). (D, D´) Dbx is expressed in small subsets of gnathal NBs not before stage
11 (see also Lacin et al., 2009). At that stage Dbx is detected in LB (and in T1, see Fig. 3E) only in
NB3-2, in MX additionally in NBs 5-2,5-3,7-1, and in MN only in those three NBs, as NB3-2 is
missing (in the specimen shown, Dbx expression is not yet initiated in mandibular NB5-2). (E, E´) We
detected unpg-lacZ in five NBs (NBs 4-1, 5-3, 5-5, 6-2, 7-2) per prothoracic hemineuromere (see Fig.
3D; see also Doe, 1992), and, except for NB5-5, in the corresponding NBs in LB, MX and MN. (F,
F´) Pox neuro (Poxn) is detected in thoracic NB2-4, but not in gnathal segments where NB2-4 is
lacking. Poxn is also expressed in sensory organ precursors (SOP) (Dambly-Chaudiere et al., 1992), in
close vicinity to NBs. (G, H) Odd (G) and AJ96-lacZ (H) are by stage 11 expressed in MP2 (and after
division also retained in the two daughter cells) in T1 (see Fig. 3D; for Odd see also Doe, 1992) and
LB. In MX MP2 is missing (G) In MX and LB Odd is detected in midline progenitor MP1. (H)
Similar to Odd, expression of Aj96-lacZ is not detected in mandibular MP2-like NBs. (I) chrb mRNA
is at stage 11 very dynamically expressed in a subset of mandibular NBs (indicated with black
gnathal segments. (J) CenG1A mRNA is by stage 11 dynamically expressed in subsets of gnathal
NBs: in three to five mandibular NBs (consistently in NBs 4-3,5-6,7-1), in two to three maxillary NBs
(consistently in NBs 4-2, 7-1), and in up to seven labial NBs (among which usually are NBs 4-1, 4-2,
4-3, 4-4, and often NB3-5).
Development • Supplementary information
lettering), including NBs 3-5, 4-3, 4-4, 5-3, 5-5, 5-6 and often NB7-4, but not detected in NBs of other
Figure S2.5. Identification of NBs in the gnathal neuromeres – expression of sublineage markers.
(A-D) Composite confocal images. Expression of sublineage markers for characteristic progeny cells
of identified NBs in wt (A-B,D) and Df(3L)H99 mutant embryo (C). Flat preparations of stage 15
embryos (in A-C dorsal view, anterior to the top; in D lateral view, anterior to the left). Individual
daughter cells identified by marker labeling and position are indicated (by lettering). (A-A´´) Antibody
staining against Eve, En, and GFP that detects the expression of membrane-bound CD8::GFP (mGFP)
in the eveRRK-Gal4 pattern. Eve expression is found in NB1-1-derived aCC and pCC (Broadus et al.,
1995), NB3-3-derived EL-neurons (Schmidt et al., 1997), NB4-2-derived RP2 (Bossing et al., 1996),
NB7-1-derived U-neurons (Bossing et al., 1996). eveRRK-Gal4 is expressed only in the NB1-1-derived
aCC and pCC and NB4-2-derived RP2. Eve/eveRRK>mGFP-expressing daughter cells of all four NBs
are found in T1, LB and MX (U-neurons out of focus). At that stage, RP2s are largely Eve-negative
and show signs of programmed cell death (PCD, white arrows in A), or are missing (yellow arrow) in
Development • Supplementary information
Development 143: doi:10.1242/dev.133546: Supplementary information
Development 143: doi:10.1242/dev.133546: Supplementary information
LB and MX. In MN, however, we could never detect RP2, although NB4-2 is existing (see also Suppl.
Table 3). Furthermore, aCC/pCC and the EL-neurons are missing in MN, corresponding with our
finding that mandibular NBs 1-1 and 3-3 are missing (arrowheads point to Eve-positive cells that are
of unknown origin). (B-B´´) Antibody staining against Eve, En, and GFP that detects the expression of
nuclear GFP (nGFP) in the CQ2-Gal4 pattern. CQ2-Gal4 drives expression in the NB7-1-derived Uneurons (Landgraf et al., 2003) and is found in the respective cells in all gnathal neuromeres. (C)
Antibody staining against Eve and En in Df(3L)H99 at stage 15. Note that the NB4-2-derived Evepositive RP2 is present in MX and LB, indicating that it normally undergoes PCD in both neuromeres
(compare A). RP2 is never observed in MN of the PCD-deficient mutant, supporting that it is normally
not formed in the mandibular NB4-2 lineage (see also Suppl. Table 3). (D) Antibody staining against
Repo, En, and GFP that detects the expression of membrane-bound CD8::GFP (mGFP) in the Mz97Gal4 pattern. Due to the lack of a marker specifically expressed in the gnathal NB1-3, we tested Mz97Gal4, reported to be expressed in NB1-3 derived peripheral glial cells in the abdominal segments (von
Hilchen et al., 2008). We observe Mz97>mGFP expression in subsets of peripheral glial cells at distal
positions along the labial nerve (LBN; white arrowheads) which may hint at the existence of labial
NB1-3. As the maxillary/mandibular nerves are fused (Campos-Ortega and Hartenstein, 1997), the
situation is unclear in MX and MN. A few peripheral glia were detected in distal positions also along
this nerve (MXN; white arrowheads) which may stem from a maxillary NB1-3. However, since Mz97Gal4 is also expressed in daughter cells of abdominal sensory organ precursors (SOPs; von Hilchen et
al., 2008), we cannot rule out the possibility that Mz97>mGFP-positive cells near the gnathal nerves
Development • Supplementary information
may stem from SOPs, which are abundant in all gnathal segments.
Development 143: doi:10.1242/dev.133546: Supplementary information
Figure S2.6. Identification of MP2s and further NBs developing from the enlarged
Ac-expressing domain in the anterior compartment of MD – expression of MP2-specific marker
Ac, Pros (nuclear) and Hb.
(A-C) Midstage 11 (mst11). About 8 to 9 Ac-positive NBs (rows 3,4,5 and MP2s) emerge from the
mandibular Ac-domain (A-B´´). MP2s express Ac (B´), nuclear Pros (B´´), and Hb (C). (D-E´´) Pros
is cortically localized (arrows) in mitotic NBs (shown for maxillary NB5-2), but not in dividing MP2s
Development • Supplementary information
(shown for MP2c).
Development 143: doi:10.1242/dev.133546: Supplementary information
Supplementary Tables
Marker gene
gnathal
NBs
thoracic
NBs
brain
NBs
achaete (ac)
Antennapedia (Antp)
asense (ase)
AJ96*
mRNA
protein
protein
lacZ
transcription factor
transcription factor
transcription factor
-
+
+
+
+
+
+
+
+
+
+
-
buttonhead (btd)*
cap and collar (cnc)
castor (cas)*
charybde (chrb)
Centaurin gamma 1A (CenG1A)
mRNA
mRNA
protein, lacZ
mRNA
mRNA
transcription factor
transcription factor
transcription factor
(RTP801-like) ?
GTPase
+
+
+
+
+
+
+
+
+
+
+
+
+
collier (col, knot)
dachshund (dac)
dbx*
deadpan (dpn)
Deformed (Dfd)
dichaete (d)*
protein
protein
protein
protein
protein
mRNA, protein
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
+
+
+
+
+
+
+
+
+
+
+
+
+
+
eagle (eg)*
empty spiracles (ems)*
engrailed (en)*
eyeless (ey)*
giant (gt)
gooseberry distal (gsb-d)*
H15 (nmr1)*
protein
protein
protein, lacZ
protein
protein
protein, lacZ
protein, lacZ
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
hunchback (hb)*
huckebein (hkb)*
intermediate neuroblast defective
(ind)*
knirps (kni)*
ladybird early (lbe)*
midline (mid, nmr2)*
mirror (mirr)*
muscle specific homoebox (msh,
drop)*
Nkx6*
odd-skipped (odd)*
protein
lacZ, mRNA
protein, mRNA
transcription factor
transcription factor
transcription factor
+
+
+
+
+
+
+
+
+
protein
protein
protein
lacZ
protein,
mRNA
mRNA
protein
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
transcription factor
transcription factor
+
+
+
+
+
-
orthodenticle (otd)*
pou domain-1 (pdm-1, nub)*
pox neuro (pox n)*
prospero(pros)*
reversed polarity (repo)*
runt*
seven up (svp)*
Sex comb reduced (Scr)
sloppy paired 1 (slp1)*
unplugged (unpg)*
ventral nervous system defective
(vnd)*
wingless (wg)*
worniu (wor)
protein
protein
protein
protein
protein
protein
lacZ
protein
protein, mRNA
mRNA, lacZ
protein,
mRNA, lacZ
protein, lacZ
protein
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
transcription factor
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
secreted ligand
transcription factor
+
+
+
+
+
+
Expressed in brain NB subsets but
not in gnathal/ thoracic NBs
branchless (bnl)
drumstick (drm)
earmuff (erm)
eyes absent (eya)
mRNA
mRNA
mRNA
protein
secreted ligand
transcription factor
transcription factor
phosphatase
-
-
+
+
+
+
Development • Supplementary information
Table. S1 Expression of molecular markers analysed in embryonic NBs of the gnathal and
thoracic segments, and brain.
Development 143: doi:10.1242/dev.133546: Supplementary information
hikaru genki (hig)
Homeobox 9 (Hb9)
optix (opt, Six3)
optomotor blind (omb)
pyramus (pyr)
retinal homeobox (rx)
mRNA
protein
mRNA
protein
mRNA
protein
ECM protein
transcription factor
transcription factor
transcription factor
secreted ligand
transcription factor
-
-
+
+
+
+
+
+
scarecrow (scro)
single minded (sim)
target of paired box neuro (tap)
thisbe (ths)
thor (4E-PB)
tropospondin (tsp)
twin of eyeless (toy)
visual system homeobox 1 ortholog
(vsx1)
mRNA
protein, lacZ
mRNA
mRNA
mRNA
mRNA
protein
protein
transcription factor
transcription factor
transcription factor
secreted ligand
translation factor
ECM protein
transcription factor
transcription factor
-
-
+
+
+
+
+
+
+
+
protein
Gal4
Gal4
Gal4
protein
transcription factor
transcription factor
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
na.
(+)
Sublineage marker genes for
identified NB lineages
even-skipped (eve)
CQ2
eveRRK
Mz97
reversed polarity (repo)
Development • Supplementary information
* indicates `segmentally conserved´ genes expressed in repeated subsets of gnathal and thoracic NBs
at stage 11; + indicates marker gene expression in NBs, (+) indicates marker gene expression in
sublineage components of an identified NB. Data on marker gene expression in brain NBs is from
Urbach et al., 2003a, Urbach and Technau, 2003a,b; Urbach, 2007; Kunz et al., 2012; and data not
shown. na., not analysed.
Development 143: doi:10.1242/dev.133546: Supplementary information
Table S2. Comparison of marker gene expression, neuroectodermal origin and developmental
time point of identified embryonic NBs in the prothoracic (T1) and gnathal (LB, MX, MN)
neuromeres, and in the trito- (TC) and deutocerebrum (DC).
l´sc *1
ind
gsbd-lacZ
slp1
cas
pdm1
svp-lacZ
ey
unpg-lacZ
runt
wg
dbx
Nkx6
AJ96-lacZ
btd
eg
ems
en
H15-lacZ
hb
hkb
lbe
mid
mirr-lacZ
msh
odd
otd
pros
repo
runt
vnd
LB
MX
MN
TC
DC
3-5
(lst8)
3-5
(lst8)
3-5
(lst8)
3-5
(est10)
Td6
(est10)
Dd8
(lst8)
+
+
+
+
+2
+
-
+
+
+
+
+2
+
-
+
+
+
+
+2
+
-
+
+
~
~
~
-
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
-
5-3
(lst8)
5-3
(lst8)
5-3
(lst8)
5-3
(est10)
Td2
(est10)
Dd1
(est9)
+
+
+
+
+
~
+
+
+
+
+
-
+
+
~
+
+
~
+
+
+
+
~
-
+
+
~
+
+
~
+
+
+
~
~
+
-
+
+
+
+
+
~
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
~
+
+
+
+
+
-
5-6
5-6
5-6
5-6
Td4
Dd7
Unique marker gene code.
Similar AP/DV origin: dorsal
msh+ NE, anterior adjacent to
gsb-lacZ+ domain.
Similar formation time point
(except MN3-5/Td6); first NBs
in TC/DC, Dd8 and NB3-5 first
ems+ NBs;
*1 develop from proneural
cluster expressing the same
proneural gene
+2 msh+ by st9/10
Unique marker gene code.
Similar AP/DV origin:
intermediate ind+ NE, anterior
adjacent to en+ domain.
Similar formation time point
(except MN5-3/Td2);
*1 develop from proneural
cluster expressing the same
proneural gene
Development • Supplementary information
ac/sc *1
ems
svp-lacZ
pdm1
msh
slp1
hkb
mid
unpg-lacZ
AJ96-lacZ
btd
cas
dbx
eg
en
ey
gsbd-lacZ
H15-lacZ
hb
ind
lbe
mirr-lacZ
Nkx6
odd
otd
poxn
pros
repo
runt
vnd
wg
T1
Development 143: doi:10.1242/dev.133546: Supplementary information
(lst8)
+
+
+
+
~
~2
+
+1
+
~
-
(lst8)
+
+
+
+
~
~2
+
+1
+
+
-
(lst8)
+
+
+
+
+
~2
+
+1
+
+
-
vnd
en
mid
H15-lacZ
cas
pdm1
mirr-lacZ
svp-lacZ
Nkx6
AJ96-lacZ
btd
dbx
eg
ems
ey
gsbd-lacZ
hb
hkb
ind
lbe
msh
odd
otd
poxn
pros
repo
runt
slp1
unpg-lacZ
wg
1-2
(est10)
+
+
+
+
~
~
+
+
-
1-2
(est10)
+
+
+
+
~
~
+
+
-
1-2
(est10)
+
+
+
+
~
~
+
+
-
(st9)
+
+
+
+
~
+
+
~
+
+
-
X
(est10)
+
+
+
+
+
+
+
+
+
-
(est9)
+
+
+
+
+
+
+
+
+
+
+
+
-
Tv4
(est11)
+
+
+
+
+
+
+
-
Dv8
(est10)
+
+
+
+
+
+
+
+
-
Unique marker gene code.
Similar AP/DV origin: dorsal
msh+ NE, within wg+/gsbdlacZ+ domains.
Similar formation time point
(except Td7);
*1 develop from proneural
cluster expressing the same
proneural gene
+1 msh+ by st9/10;
+2 cas+ by early st12
Unique marker gene code.
Similar AP/DV origin: ventral
vnd+ NE, within segmental en+
domain.
Similar formation time point
(except Tv4).
Development • Supplementary information
l´sc *1
wg
gsbd-lacZ
lbe
pdm1
cas
svp-lacZ
msh
slp1
D
hb
hkb
unpg-lacZ
AJ96-lacZ
btd
dbx
eg
ems
en
ey
H15-lacZ
ind
mid
mirr-lacZ
Nkx6
odd
otd
poxn
pros
repo
runt
vnd
Development 143: doi:10.1242/dev.133546: Supplementary information
6-1
(st9)
+
+
+
+
+
+
~
-
6-1
(st9)
+
+
+
+
+
+
~
-
6-1
(st9)
+
+
+
+
+
+
~
~
-
Tv4 (est11)
Tv5 (lst11)
+/+
+/+
+/-/+
-/-/+/+
-/+
-
vnd
en
cas
pdm1
mirr-lacZ
svp-lacZ
gsbd-lacZ
Nkx6
dbx
AJ96-lacZ
btd
eg
ems
ey
H15-lacZ
hb
hkb
ind
lbe
mid
msh
odd
otd
poxn
pros
repo
runt
slp1
unpg-lacZ
wg
7-1
(lst8)
+
+
+
~
+
+
+
-
7-1
(lst8)
+
+
+
~
+
+
+
~
-
7-1
(lst8)
+
+
+
+
+
+
+
~
~
-
7-1
(lst8)
+
+
+
~
+
+
+
~
~
-
Tv4 (est11)
Tv5 (lst11)
+/+
+/+
+/-/+
-/+
-/-/+/+
-
en
gsbd-lacZ
ind
6-2
(st9)
+
+
+
6-2
(st9)
+
+
+
6-2
(st9)
+
+
+
6-2
(st9)
+
+
+
Td3
(est10)
+
+
+
X
Unique marker gene code of
NB6-1 is most closely mirrored
by Tv5 (and Tv4).
Similar AP/DV origin: ventral
vnd+ NE, within segmental en+
domain.
X
Unique marker gene code of
NB7-1 is most closely mirrored
by Tv5 (and Tv4).
Similar AP/DV origin: ventral
vnd+ NE, within segmental en+
domain.
Dd5
(lst9)
+
+
-*
Unique marker gene code of
NB6-2 is closely mirrored by
Td3 and Dd5.
Development • Supplementary information
vnd
en
cas
mirr-lacZ
otd
gsbd-lacZ
Nkx6
pdm1
AJ96-lacZ
btd
dbx
eg
ems
ey
H15-lacZ
hb
hkb
ind
lbe
mid
msh
odd
poxn
pros
repo
runt
svp-lacZ
slp1
unpg-lacZ
wg
6-1
(st9)
+
+
+
+
+
+
-
Development 143: doi:10.1242/dev.133546: Supplementary information
+
+
+
-
+
+
+
-
~
+
+
~
-
~
+
~
-
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
-
en
ind
mid
H15-lacZ
cas
unpg-lacZ
pdm-1
svp-lacZ
gsbd-lacZ
hb
hkb
msh
runt
vnd
AJ96-lacZ
btd
dbx
eg
ems
ey
lbe
mirr-lacZ
Nkx6
odd
otd
poxn
pros
repo
slp1
wg
7-2
(st9 )
+
~
~
+
+
+
~
+
-
7-2
(st9)
+
~
~
+
+
+
~
+
-
7-2
(st9)
+
~
~
+
+
+
+
+
-
7-2
(st9)
+
~
~
+
+
+
+
+
-
Td3
(est10)
+
+
+
+
+
+
+
-
Dd5
(lst9)
+
-*
+
+
+
+
+
+
+
+
+
+
+
-
l´sc *1
ind
ey
cas
pdm1
Nkx6
runt
svp-lacZ
3-2
(st9)
+
~
+
+
+
+
+
+
3-2
(st9)
+
~
+
+
+
+
+
+
3-2
(st9)
+
~
+
+
+
+
+
+
Td1
(est10)
+
+
+
+
+
~
-
Dv6
(est9)
+
+
+
+
+
+
+
+
X
Similar AP/DV origin:
intermediate ind+ NE, within
segmental en+ domain.
Similar formation time point.
* Dd5 coexpresses msh/vnd
indicating an intermediate
identity
Unique marker gene code of
NB7-2 is most closely mirrored
by Td3 and Dd5.
Similar AP/DV origin:
intermediate ind+ NE, within
segmental en+ domain.
Similar formation time point.
* Dd5 coexpresses msh/vnd
indicating an intermediate
identity
Unique marker gene code of
NB3-2 is most closely mirrored
by Td1 and Dv6.
Similar AP/DV origin:
intermediate ind+ NE; posterior
adjacent to segmental border.
Development • Supplementary information
pdm1
unpg-lacZ
svp-lacZ
cas
H15-lacZ
hb
hkb
msh
runt
vnd
btd
AJ96-lacZ
btd
eg
ems
ey
lbe
mid
mirr-lacZ
Nkx6
odd
otd
poxn
pros
repo
slp1
wg
Development 143: doi:10.1242/dev.133546: Supplementary information
l´sc *1
ind
ey
pdm1
Nkx6
hkb-lacZ
slp1
btd
svp-lacZ
cas
hb
mirr-lacZ
otd
runt
unpg-lacZ
AJ96-lacZ
dbx
eg
ems
en
gsbd-lacZ
H15-lacZ
lbe
mid
msh
odd
poxn
pros
repo
vnd
wg
~
-
~
-
~
-
4-2
(st9)
+/+
~
+
~
+
+
~
+
+
-
4-2
(st9)
+/+
~
+
~
~
+
~
+
+
-
4-2
(st9)
+/+
~
+
~
+
+
~
+
+
-
4-2
(est11)
?
+
+
~
~
+
+
+
~
-
-
+
+
+
+
+
+
-
Td1
(est10)
+
+
+
+
-
Dv6
(est9)
+
+
+
~
+
+
+
+
+
+
+
+
+
-
Similar formation time point
(except Td1);
*1 develop from proneural
cluster expressing the same
proneural gene
Unique marker gene code of
NB4-2 is most closely mirrored
by Td1 and Dv6.
Similar AP/DV origin:
intermediate ind+ NE;
NB3-2, Td1, Dv6 posterior
adjacent to segmental border.
Similar formation time point
(except MD4-2, Td1);
*1 develop from proneural
cluster expressing the same
proneural gene
Homology between NBs in prothoracic/gnathal neuromeres and TC:
T1
LB
MX
MN
TC
X
Nkx6
otd
pdm1
vnd
runt
svp-lacZ
3-1
(est10)
+
~
~
+
+
+
3-1
(est10)
+
~
~
+
+
+
3-1
(est10)
+
~
~
+
+
+
3-1
(st11)
~
~
~
+
~
-
Tv1
(st11)
+
+
+
-
X
Unique marker gene code.
Similar AP/DV origin: ventral
vnd+ NE, posterior to segmental
mirr-lacZ domain.
Development • Supplementary information
dbx
hb
hkb
mirr-lacZ
otd
slp1
unpg-lacZ
AJ96-lacZ
btd
eg
ems
en
gsbd-lacZ
H15-lacZ
lbe
mid
msh
odd
poxn
pros
repo
vnd
wg
Development 143: doi:10.1242/dev.133546: Supplementary information
-
-
-
-
+
-
msh
mirr-lacZ
H15-lacZ
svp-lacZ
cas
pdm1
mid
hkb
AJ96-lacZ
btd
dbx
eg
ems
en
ey
gsbd-lacZ
hb
ind
lbe
Nkx6
odd
otd
poxn
pros
repo
runt
slp1
unpg-lacZ
vnd
wg
3-4
(est11)
+
+
+
+
+
~
+
-
3-4
(est11)
+
+
+
+
+
+
+
-
3-4
(est11)
+
+
+
+
+
+
+
-
3-4
(est11)
+
+
~
+
~
+
-
Td8
(est11)
+
+
+
+
+
+
-
vnd
btd
cas
pdm1
unpg-lacZ
svp-lacZ
slp1
dac
Nkx6
hb
dbx
4-1
(est10)
+
+
+
+
+
+
+
-
4-1
(est10)
+
+
+
+
+
+
+
-
4-1
(est10)
+
+
+
+
+
+
+
+
-
4-1
(est10)
+
+
+
+
~
+
-
Tv2
(lst9)
+
+
+
+
+
+
+
X
Unique marker gene code.
Similar AP/DV origin: dorsal
msh+ NE, posterior adjacent to
segmental border.
Similar formation time point.
X
Unique marker gene code.
Similar AP/DV origin: ventral
vnd+ NE, anterior adjacent to
segmental gsbd-lacZ+ domain.
Similar formation time point.
Development • Supplementary information
hkb
AJ96-lacZ
btd
cas
dbx
eg
ems
en
ey
gsbd-lacZ
H15-lacZ
hb
ind
lbe
mid
mirr-lacZ
msh
odd
poxn
pros
repo
slp1
unpg-lacZ
wg
Development 143: doi:10.1242/dev.133546: Supplementary information
-
-
-
-
l´sc
vnd
gsbd-lacZ
slp1
cas
wg
Nkx6
svp-lacZ
pdm1
runt
dbx
mid
AJ96-lacZ
btd
eg
ems
en
ey
H15-lacZ
hb
hkb
ind
lbe
mirr-lacZ
msh
odd
otd
poxn
pros
repo
unpg-lacZ
5-2
(lst8)
+
+
+
~
+
+
+
~
-
5-2
(lst8)
+
+
+
+
+
~
~
+
~
~
-
5-2
(lst8)
+
+
+
+
+
~
~
+
~
+
-
5-2
(lst8)
+
+
+
+
+
+
+
~
-
Tv3
(lst11)
+
+
+
+
+
+
+
-
en
msh
mid
H15-lacZ
pdm-1
svp-lacZ
cas
hb
AJ96-lacZ
btd
dbx
eg
ems
ey
7-4
(lst8)
+
+
+
+
~
+
+
-
7-4
(lst8)
+
+
+
+
~
+
+
-
7-4
(lst8)
+
+
~
+
+
+
+
-
7-4
(lst8)
+
+
~
+
+
+
~
-
Td5
(lst11)
+
+
+
+
+
+
-
*1
X
Unique marker gene code.
Similar AP/DV origin: ventral
vnd+ NE, anterior adjacent to
segmental en+ domain.
Similar formation time point
(except for Tv3);
*1 develop from proneural
cluster expressing the same
proneural gene
X
Unique marker gene code.
Similar AP/DV origin: dorsal
msh+ NE, within segmental en+
domain.
Similar formation time point
(except for Td5)
Development • Supplementary information
-
AJ96-lacZ
eg
ems
en
ey
gsbd-lacZ
H15-lacZ
hkb
ind
lbe
mid
mirr-lacZ
msh
odd
otd
poxn
pros
repo
runt
wg
Development 143: doi:10.1242/dev.133546: Supplementary information
-
-
-
msh
repo
mirr-lacZ
hb
slp1
mid
AJ96-lacZ
btd
cas
dbx
eg
ems
en
ey
gsbd-lacZ
H15-lacZ
hkb
ind
lbe
Nkx6
odd
otd
pdm-1
poxn
pros
runt
svp-lacZ
unpg-lacZ
vnd
wg
LGB
(est10)
+
+
+
-
LGB
(est10)
+
+
+
-
LGB
(est10)
+
+
+
-
X
Td7
(est10)
+
+
+
+
+
+
-
X
Unique marker gene code.
Similar AP/DV origin: dorsal
msh+ NE, posterior adjacent to
segmental en+ domain.
Similar formation time point.
Homology between NBs in prothoracic/gnathal neuromeres and DC:
T1
LB
MX
MN
X
DC
ems
cas
runt
pdm1
eg
hkb
mirr-lacZ
msh
otd
svp-lacZ
unpg-lacZ
AJ96-lacZ
btd
dbx
en
ey
3-3
(est10)
+
+
+
+
+
-
3-3
(est10)
+
+
+
+
+
-
3-3
(est10)
+
+
~
+
+
-
X
X
Dd3 (lst8)
Dd6 (lst9)
+/+
-/+
+/+/+
-/+/+
-/+
+/+
+/+
+/+/+
-/-/-/-/-/-
Unique marker gene code of
NB3-3 is most closely mirrored
by Dd3/Dd6.
Similar AP/DV origin: dorsal
msh+ NE, posterior adjacent to
segmental en+ domain.
Similar formation time point
(except for Dd3).
Development • Supplementary information
gsbd-lacZ
hkb
ind
lbe
mirr-lacZ
Nkx6
odd
otd
poxn
pros
repo
runt
slp1
unpg-lacZ
vnd
wg
Development 143: doi:10.1242/dev.133546: Supplementary information
-
-
-
ems
ey
slp1
hkb-lacZ
svp-lacZ
hb
cas
mirr-lacZ
msh
pdm1
unpg-lacZ
wg
AJ96-lacZ
btd
dbx
eg
en
gsbd-lacZ
H15-lacZ
ind
lbe
mid
Nkx6
odd
otd
poxn
pros
repo
runt
vnd
4-4
(est11)
+
+
+
+
+
+
-
4-4
(est11)
+
+
+
+
+
+
+
-
4-4
(est11)
+
+
+
+
+
+
-
-/-/-/-/-/-/-/-/-/-/-/-/-/-/4-4
(lst11)
+
+
+
~
+
~
-
X
Dd4
(lst11)
+
+
+
+
+
+
+
+
+
+
-
Unique marker gene code.
Similar AP/DV origin: dorsal
msh+ NE, posterior adjacent to
segmental gsb-lacZ+ domain.
Similar formation time point.
Homology between NBs in prothoracic and gnathal neuromeres:
T1
LB
MX
MN
X
X
hkb-lacZ
mirr-lacZ
vnd
pdm1
otd
runt
cas
kni
Nkx6
AJ96-lacZ
btd
eg
ems
en
ey
gsbd-lacZ
H15-lacZ
hb
2-1
(lst11)
+
+
+
~
+
+
+
-
2-1
(lst11)
+
+
+
~
+
+
+
+
-
2-1
(lst11)
+
+
~
~
+
+
-
2-1
(lst11)
+
+
+
~
~
-
X
X
Unique marker gene code.
Similar AP/DV origin: ventral
vnd+ NE, within mirr-lacZ+
domain.
Similar formation time point.
Development • Supplementary information
gsbd-lacZ
H15-lacZ
hb
ind
lbe
mid
Nkx6
odd
poxn
pros
repo
slp1
vnd
wg
Development 143: doi:10.1242/dev.133546: Supplementary information
-
-
-
-
hkb-lacZ
runt
mirr-lacZ
Nkx6
svp-lacZ
H15-lacZ
vnd
D
cas
pdm1
AJ96-lacZ
btd
dbx
eg
ems
en
ey
gsbd-lacZ
hb
ind
lbe
mid
msh
odd
otd
poxn
pros
repo
slp1
unpg-lacZ
wg
2-2
(est10)
+
+
+
+
+
-
2-2
(est10)
+
+
+
+
+
-
2-2
(est11)
+
+
+
+
+
-
2-2
(est11)
+
~
+
~
+
~
+
+
~
+
-
X
X
X
runt
mirr-lacZ
D
hb
pdm1
AJ96-lacZ
btd
cas
dbx
eg
ems
en
ey
gsbd-lacZ
H15-lacZ
hkb
ind
lbe
mid
msh
Nkx6
2-3
(est11)
~
+
~
+
~
-
X
X
Unique marker gene code.
Similar AP/DV origin: ventral
vnd+ NE, adjacent anterior to
mirr-lacZ+ domain.
X
X
Development • Supplementary information
ind
lbe
mid
msh
Nkx6
odd
poxn
pros
repo
svp-lacZ
slp1
unpg-lacZ
wg
Development 143: doi:10.1242/dev.133546: Supplementary information
-
mirr-lacZ
msh
eg
poxn
svp-lacZ
hb
hkb-lacZ
AJ96-lacZ
btd
cas
dbx
ems
en
ey
gsbd-lacZ
H15-lacZ
ind
lbe
mid
Nkx6
odd
otd
pdm-1
pros
repo
runt
slp1
unpg-lacZ
vnd
wg
2-4
(est11)
+
+
+
+
+
+
+
-
kni
msh
svp-lacZ
pdm1
AJ96-lacZ
btd
cas
dbx
eg
ems
en
ey
gsbd-lacZ
H15-lacZ
hb
hkb
ind
lbe
mid
mirr-lacZ
Nkx6
odd
otd
poxn
2-5
(lst8)
+
+
+
~
-
X
X
2-5
(lst8)
+
+
+
+
-
2-5
(lst8)
+
+
+
-
X
X
X
X
X
X
Unique marker gene code.
Unique marker gene code.
Similar AP/DV origin: dorsal
msh+ NE, at anterior segmental
border.
Similar formation time point.
Development • Supplementary information
odd
otd
poxn
pros
repo
svp-lacZ
slp1
unpg-lacZ
vnd
wg
Development 143: doi:10.1242/dev.133546: Supplementary information
-
-
-
slp1
hkb-lacZ
msh
D
ey
svp-lacZ
hb
pdm1
wg
AJ96-lacZ
btd
cas
dbx
eg
ems
en
gsbd-lacZ
H15-lacZ
ind
lbe
mid
mirr-lacZ
Nkx6
odd
otd
poxn
pros
repo
runt
unpg-lacZ
vnd
4-3
(lst11)
+
+
+
~
+
~
+
-
4-3
(lst11)
+
+
+
+
~
+
~
-
4-3
(lst11)
+
+
+
+
+
~
+
-
otd
kni
slp1
wg
gsbd-lacZ
vnd
svp-lacZ
cas
pdm1
Nkx6
AJ96-lacZ
btd
dbx
eg
ems
en
ey
H15-lacZ
hb
hkb
ind
lbe
mid
mirr-lacZ
msh
odd
poxn
5-1
(lst11)
+
+
+
+
+
+
+
+
+
-
5-1
(lst11)
+
+
+
+
+
+
+
+
+
-
5-1
(lst11)
+
+
+
+
+
+
+
+
+
~
-
4-3
(lst11)
+
+
+
+
+
~
+
~
X
X
X
Unique marker gene code.
Similar AP/DV origin: dorsal
msh+ NE, anterior adjacent to
segmental gsb-lacZ+ domain.
Similar formation time point.
X
X
Unique marker gene code.
Similar AP/DV origin: ventral
vnd+ NE, within segmental gsblacZ+ domain.
Similar formation time point.
Development • Supplementary information
pros
repo
runt
slp1
unpg-lacZ
vnd
wg
Development 143: doi:10.1242/dev.133546: Supplementary information
-
-
hkb-lacZ
slp1
wg
msh
svp-lacZ
hb
gsbd-lacZ
AJ96-lacZ
btd
cas
dbx
eg
ems
en
ey
H15-lacZ
ind
lbe
mid
mirr-lacZ
Nkx6
odd
otd
pdm-1
poxn
pros
repo
runt
unpg-lacZ
vnd
5-4
(est10)
+
+
+
+
+
~
+
-
5-4
(est11)
+
+
+
+
+
~
+
-
slp1
wg
gsbd-lacZ
svp-lacZ
unpg-lacZ
hkb-lacZ
lbe
hb
pdm1
AJ96-lacZ
btd
cas
dbx
eg
ems
en
ey
H15-lacZ
ind
mid
mirr-lacZ
msh
Nkx6
odd
otd
poxn
pros
repo
runt
vnd
5-5
(est11)
+
+
+
+
~
~
-
X
X
X
X
X
Unique marker gene code.
Similar AP/DV origin: dorsal
msh+ NE, within wg+ domain.
X
5-5
(est11)
+
+
+
+
+
~
-
X
X
Unique marker gene code.
Similar AP/DV origin: dorsal to
ind+ domain, within wg+
domain.
Similar formation time point.
Development • Supplementary information
pros
repo
runt
unpg-lacZ
Development 143: doi:10.1242/dev.133546: Supplementary information
6-4
(est10)
+
+
+
+
+
~
-
6-4
(est11)
+
+
+
+
+
+
-
hkb-lacZ
btd
en
ind
D
ey
eg
H15-lacZ
hb
svp-lacZ
pdm1
AJ96-lacZ
cas
dbx
ems
gsbd-lacZ
lbe
mid
mirr-lacZ
msh
Nkx6
odd
otd
poxn
pros
repo
runt
slp1
unpg-lacZ
vnd
wg
7-3
(est11)
+
+
+
+
~
+
+
+
+
~
~
-
7-3
(est11)
+
+
+
+
~
+
+
+
+
~
~
-
7-3
(est11)
+
+
+
+
~
+
+
+
+
~
~
-
MP2
(lst8)
+
MP2
(st9)
+
ac
X
X
X
X
Unique marker gene code.
Similar AP/DV origin: dorsal
msh+ NE, within en+ domain.
7-3
(lst11)
~
+
+
+
~
+
+
+
~
~
~
MP2a,b,c
(est10)
+
X
X
Unique marker gene code.
Similar AP/DV origin:
intermediate ind+ NE, within
en+ domain.
Similar formation time point.
X
X
Unique marker gene code.
Development • Supplementary information
en
gsbd-lacZ
msh
D
eg
svp-lacZ
pdm1
AJ96-lacZ
btd
cas
dbx
ems
ey
H15-lacZ
hb
hkb
ind
lbe
mid
mirr-lacZ
Nkx6
odd
otd
poxn
pros
repo
runt
slp1
unpg-lacZ
vnd
wg
6-4
(est11)
+
+
+
+
+
~
-
Development 143: doi:10.1242/dev.133546: Supplementary information
+
+
+
+
+
-
+
+
+
+
+
-
+
+ (b,c)
+
+
- (c)
+
-
Similar AP/DV origin: within
vnd+ domain,
anterior to slp1+ domain.
Expression of `segmentally conserved´ genes (marked with * in Suppl. Table1) in NBs of the trunk
and brain at stage 11. Indicated in red color is the marker gene code which is `unique´ for each
individual NB in T1 (taken as a reference) and for serial homologous NBs in the gnathal neuromeres,
and for many corresponding NBs in the tritocerebrum (TC) and/or deutocerebrum (DC). In few cases
the unique marker gene code of two thoracic NBs mirrors closely the molecular signature of one NB
in each the tritocerebrum and deutocerebrum; for example, the specific molecular profile of
deutocerebral Dv6, tritocerebral Td1 (and mandibular NB4-2) closely matches that of NBs 3-2/4-2 in
T1, LB and MX. "X" indicates that a corresponding NB is missing in the respective neuromere. "+"
indicates normal expression, "~" weak expression. Origin of a NB refers to its position of delamination
from the neuroectoderm (NE) along the anterioposterior (AP) and dorsoventral (DV) axis. The
formation time point of individual NBs is indicated in brackets. Data on proneural gene expression in
thoracic NBs refer to Skeath et al. (1994), in brain NBs to Urbach et al. (2003), and in gnathal NBs to
this study.
Development • Supplementary information
pros
vnd
hb
odd
Aj96-lacZ
gsbd-lacZ
btd
cas
dbx
eg
ems
en
ey
H15-lacZ
hkb
ind
lbe
mid
mirr-lacZ
msh
Nkx6
odd
otd
pdm-1
poxn
repo
runt
svp-lacZ
slp1
unpg-lacZ
wg
Development 143: doi:10.1242/dev.133546: Supplementary information
Table S3. Presence of NB4-2 derived RP2 motorneuron in wt and Df(3L)H99.
MN
wt
stages 11/12/13
0%
wt
stages 15/16
0%
Df(3L)H99
stages 15/16
0%*
MX
100%
27%
100%
LB
100%
41%
100%
T1
100%
100%
100%
Development • Supplementary information
n=12,12,16 hemisegments for stages 11,12,13, respectively; n=22 hemisegments for stages 15,16, each
in wt and Df(3L)H99 mutants. * In Df(3L)H99 mutant MN an ectopic Eve-positive cell is sometimes
observed. We exclude that it represents an ectopic RP2, as Eve-positive RP2 is not observed at any
stage in MN of wild type embryos.
Development 143: doi:10.1242/dev.133546: Supplementary information
Probe
ind
Template
(Source)
cDNA
(S.Marcellini)
cDNA
(S.Marcellini)
EST-clone
RE59335
(DGRC)
cDNA (E.Bier)
msh
cDNA (E.Bier)
hkb
EST clone
RE60512
(DGRC)
cDNA (E.Bier)
ac
sc
l’sc
vnd
Nkx6
Restriction
enzyme
XhoI
Polymerase
T3
5’-primer
3’-primer
-
-
pGEM TEasy
pFLC1
NcoI
SP6
-
-
PstI
T3
-
-
pBS
SKII(+)
pBS
SKII(+)
pFLC1
XhoI
T7
-
-
HindIII
T7
-
-
BglII
T3
-
-
pBS
SKII(+)
pFLC1
SacI
T7
-
-
SphI
T3
pGem TEasy
BamHI
SP6
CAATAGTG
CGGCTACC
AATG
GATTATTAC
GGCAATGC
GGTGG-
AGAACCGTGG
TGTTGTTGCT
Vector
pBS SK(-)
cnc
EST clone
RE18506
(DGRC)
PCR product
btd
PCR product
-
-
T7
cenG1A
PCR product
-
-
SP6
CGA TCT
GAA GCG
TTG CTC C
slp1
PCR product
-
-
SP6
AGT CCC
GAG CAG
CGG TTG
TAATACGACT
CACTATAGGG
AGACCACTTTC
TCTCATTCTAT
ATGGTATGGC
ATTTAGGTGA
CACTATAGAA
GAGGGT ATG
GAA CGT CCA
CTG C
ATTTAGGTGA
CACTATAGAA
GAGGCT GGG
CTT GGT TCT
GCG
Probes were synthesized from linearized plasmids containing cDNA (ac, sc, ind, msh, vnd), EST
clones (hkb, l’sc, Nkx6) or an exon sequence amplified via PCR (cnc). All other probes were
synthesized using a purified PCR product containing a SP6 or T7 RNA polymerase promoter
(underlined sequence) reverse complementarily attached 3’ to the amplified exon sequence via the
3’primer. T3, T7 or SP6 Polymerase and DIG-RNA Labeling Mix (all Roche Diagnostics) were used
for probe synthesis according to the manufacturers protocol.
Development • Supplementary information
Table S4. Templates used for in situ-probe synthesis.
Development 143: doi:10.1242/dev.133546: Supplementary information
Supplementary References
Betschinger, J., Mechtler, K. and Knoblich, J. A. (2006). Asymmetric segregation of the tumor
suppressor brat regulates self-renewal in Drosophila neural stem cells. Cell 124, 1241-1253.
Bhat, K. M., van Beers, E. H. and Bhat, P. (2000). Sloppy paired acts as the downstream target of
wingless in the Drosophila CNS and interaction between sloppy paired and gooseberry inhibits
sloppy paired during neurogenesis. Development 127, 655-665.
Bopp, D., Jamet, E., Baumgartner, S., Burri, M. and Noll, M. (1989). Isolation of two tissuespecific Drosophila paired box genes, Pox meso and Pox neuro. EMBO J 8, 3447-3457.
Bossing, T., Udolph, G., Doe, C. Q. and Technau, G. M. (1996). The embryonic central nervous
system lineages of Drosophila melanogaster. I. Neuroblast lineages derived from the ventral half of
the neuroectoderm. Dev Biol 179, 41-64.
Broadus, J., Skeath, J. B., Spana, E. P., Bossing, T., Technau, G. and Doe, C. Q. (1995). New
neuroblast markers and the origin of the aCC/pCC neurons in the Drosophila central nervous
system. Mech Dev 53, 393-402.
Brook, W. J. and Cohen, S. M. (1996). Antagonistic interactions between wingless and
decapentaplegic responsible for dorsal-ventral pattern in the Drosophila Leg. Science 273, 13731377.
Büscher, M., Hing, F. S. and Chia, W. (2002). Formation of neuroblasts in the embryonic central
nervous system of Drosophila melanogaster is controlled by SoxNeuro. Development 129, 41934203.
Büscher, M., Tio, M., Tear, G., Overton, P.M., Brook, W.J. and Chia, W. (2006). Functions of the
segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis. Dev Biol. 292,
418-429.
Cai, Y., Chia, W. and Yang, X. (2001). A family of snail-related zinc finger proteins regulates two
distinct and parallel mechanisms that mediate Drosophila neuroblast asymmetric divisions. EMBO
J 20, 1704-1714.
Campos-Ortega, J. A. and Hartenstein, V. (1997). The embryonic development of Drosophila
melanogaster. 2nd ed.
Condie, J. M., Mustard, J. A. and Brower, D. L. (1991). Generation of anti-Antennapedia
monoclonal antibodies and Antennapedia protein expression in imaginal discs. Drosophila
Information Service 70.
Dambly-Chaudiere, C., Jamet, E., Burri, M., Bopp, D., Basler, K., Hafen, E., Dumont, N.,
Spielmann, P., Ghysen, A. and Noll, M. (1992). The paired box gene pox neuro: a determinant of
poly-innervated sense organs in Drosophila. Cell 69, 159-172.
De Graeve, F., Jagla, T., Daponte, J. P., Rickert, C., Dastugue, B., Urban, J. and Jagla, K.
(2004). The ladybird homeobox genes are essential for the specification of a subpopulation of
neural cells. Dev Biol 270, 122-134.
Dittrich, R., Bossing, T., Gould, A. P., Technau, G. M. and Urban, J. (1997). The differentiation of
the serotonergic neurons in the Drosophila ventral nerve cord depends on the combined function of
the zinc finger proteins Eagle and Huckebein. Development 124, 2515-2525.
Doe, C. Q. (1992). Molecular markers for identified neuroblasts and ganglion mother cells in the
Drosophila central nervous system. Development 116, 855-863.
Dubois, L., Enriquez, J., Daburon, V., Crozet, F., Lebreton, G., Crozatier, M. and Vincent, A.
(2007). Collier transcription in a single Drosophila muscle lineage: the combinatorial control of
muscle identity. Development 134, 4347-4355.
Development • Supplementary information
Chu, H., Parras, C., White, K. and Jimenez, F. (1998). Formation and specification of ventral
neuroblasts is controlled by vnd in Drosophila neurogenesis. Genes Dev 12, 3613-3624.
Development 143: doi:10.1242/dev.133546: Supplementary information
Ellis, M.C., Weber, U., Wiersdorff, V., Mlodzik, M. (1994). Confrontation of scabrous expressing
and non-expressing cells is essential for normal ommatidial spacing in the Drosophila
eye. Development 120, 1959-1969.
Fujioka, M., Lear, B.C., Landgraf, M., Yusibova, G.L., Zhou, J., Riley, K.M., Patel, N.H. and
Jaynes, J.B. (2003) Even-skipped, acting as a repressor, regulates axonal projections in
Drosophila. Development 130, 5385-5400.
Frasch, M., Hoey, T., Rushlow, C., Doyle, H. and Levine, M. (1987). Characterization and
localization of the even-skipped protein of Drosophila. EMBO J 6, 749-59.
Fregoso Lomas, M., Hails, F., Lachance, J. F. and Nilson, L. A. (2013). Response to the dorsal
anterior gradient of EGFR signaling in Drosophila oogenesis is prepatterned by earlier posterior
EGFR activation. Cell Rep 4, 791-802.
Glicksman, M. A. and Brower, D. L. (1988). Expression of the Sex combs reduced protein in
Drosophila larvae. Dev Biol 127, 113-138.
Goulding, S.E., White, N.M. and Jarman, A.P., 2000. cato encodes a basic helix-loop-helix
transcription factor implicated in the correct differentiation of Drosophila sense organs. Dev Biol
221, 120-131.
Hartmann, B., Hirth, F., Walldorf, U. and Reichert, H. (2000). Expression, regulation and function
of the homeobox gene empty spiracles in brain and ventral nerve cord development of Drosophila.
Mech Dev 90, 143-153.
Homem, C. C., Reichardt, I., Berger, C., Lendl, T. and Knoblich, J. A. (2013). Long-term live cell
imaging and automated 4D analysis of drosophila neuroblast lineages. PLoS One 8, e79588.
Isshiki, T., Takeichi, M. and Nose, A. (1997). The role of the msh homeobox gene during Drosophila
neurogenesis: implication for the dorsoventral specification of the neuroectoderm. Development
124, 3099-3109.
Ito, K., Urban, J. and Technau, G. M. (1995). Distribution, classification, and development of
Drosophila glial cells in the late embryonic and early larval ventral nerve cord. Rouxs Arch. Dev.
Biol. 204, 284-307.
Kambadur, R., Koizumi, K., Stivers, C., Nagle, J., Poole, S. J. and Odenwald, W. F. (1998).
Regulation of POU genes by castor and hunchback establishes layered compartments in the
Drosophila CNS. Genes Dev 12, 246-260.
Kammermeier, L., Leemans, R., Hirth, F., Flister, S., Wenger, U., Walldorf, U., Gehring, W. J.
and Reichert, H. (2001). Differential expression and function of the Drosophila Pax6 genes
eyeless and twin of eyeless in embryonic central nervous system development. Mech Dev 103, 7178.
Klaes, A., Menne, T., Stollewerk, A., Scholz, H. and Klambt, C. (1994). The Ets transcription
factors encoded by the Drosophila gene pointed direct glial cell differentiation in the embryonic
CNS. Cell 78, 149-160.
Kosman, D., Small, S. and Reinitz, J. (1998). Rapid preparation of a panel of polyclonal antibodies
to Drosophila segmentation proteins. Dev Genes Evol 208, 290-294.
Kunz, T., Kraft, K.F., Technau, G.M., Urbach, R., 2012. Origin of Drosophila mushroom body
neuroblasts and generation of divergent embryonic lineages. Development 139, 2510-2522.
Lacin, H., Zhu, Y., Wilson, B. A. and Skeath, J. B. (2009). dbx mediates neuronal specification and
differentiation through cross-repressive, lineage-specific interactions with eve and hb9.
Development 136, 3257-3266.
Development • Supplementary information
Jagla, K., Jagla, T., Heitzler, P., Dretzen, G., Bellard, F. and Bellard, M. (1997). ladybird, a
tandem of homeobox genes that maintain late wingless expression in terminal and dorsal epidermis
of the Drosophila embryo. Development 124, 91-100.
Development 143: doi:10.1242/dev.133546: Supplementary information
Landgraf, M., Jeffrey, V., Fujioka, M., Jaynes, J. B. and Bate, M. (2003). Embryonic Origins of a
Motor System: Motor Dendrites Form a Myotopic Map in Drosophila. PLoS Biol 1, 221-230.
Mardon, G., Solomon, N. M. and Rubin, G. M. (1994). dachshund encodes a nuclear protein
required for normal eye and leg development in Drosophila. Development 120, 3473-3486.
McDonald, J. A. and Doe, C. Q. (1997). Establishing neuroblast-specific gene expression in the
Drosophila CNS: huckebein is activated by Wingless and Hedgehog and repressed by Engrailed
and Gooseberry. Development 124, 1079-1087.
McGinnis, N., Ragnhildstveit, E., Veraksa, A. and McGinnis, W. (1998). A cap 'n' collar protein
isoform contains a selective Hox repressor function. Development 125, 4553-4564.
Mettler, U., Vogler, G. and Urban, J. (2006). Timing of identity: spatiotemporal regulation of
hunchback in neuroblast lineages of Drosophila by Seven-up and Prospero. Development 133, 429437.
Moses, C., Helman, A., Paroush, Z. and Von Ohlen, T. (2011). Phosphorylation of Ind by MAP
kinase enhances Ind-dependent transcriptional repression. Dev Biol 360, 208-215.
Patel, N. H., Schafer, B., Goodman, C. S. and Holmgren, R. (1989). The role of segment polarity
genes during Drosophila neurogenesis. Genes Dev 3, 890-904.
Schmidt, H., Rickert, C., Bossing, T., Vef, O., Urban, J. and Technau, G. M. (1997). The
embryonic central nervous system lineages of Drosophila melanogaster. II. Neuroblast lineages
derived from the dorsal part of the neuroectoderm. Dev Biol 189, 186-204.
Shao, X., Koizumi, K., Nosworthy, N., Tan, D. P., Odenwald, W. and Nirenberg, M. (2002).
Regulatory DNA required for vnd/NK-2 homeobox gene expression pattern in neuroblasts. Proc
Natl Acad Sci U S A 99, 113-117.
Skeath, J. B. and Carroll, S. B. (1991). Regulation of achaete-scute gene expression and sensory
organ pattern formation in the Drosophila wing. Genes Dev 5, 984-995.
Skeath, J. B., Panganiban, G. F. and Carroll, S. B. (1994). The ventral nervous system defective
gene controls proneural gene expression at two distinct steps during neuroblast formation in
Drosophila. Development 120, 1517-1524.
Uhler, J., Garbern, J., Yang, L., Kamholz, J. and Mellerick, D. M. (2002). Nk6, a novel
Drosophila homeobox gene regulated by vnd. Mech Dev 116, 105-116.
Urbach, R., Technau, G.M., 2003a. Segment polarity and DV patterning gene expression reveals
segmental organization of the Drosophila brain. Development 130, 3607-3620.
Urbach, R., Technau, G.M., 2003b. Molecular markers for identified neuroblasts in the developing
brain of Drosophila. Development 130, 3621-3637.
Urbach, R., Schnabel, R. and Technau, G. M. (2003). The pattern of neuroblast formation, mitotic
domains and proneural gene expression during early brain development in Drosophila.
Development 130, 3589-3606.
von Hilchen, C. M., Beckervordersandforth, R. M., Rickert, C., Technau, G. M. and Altenhein,
B. (2008). Identity, origin, and migration of peripheral glial cells in the Drosophila embryo. Mech
Dev 125, 337-352.
Von Ohlen, T., Syu, L. J. and Mellerick, D. M. (2007). Conserved properties of the Drosophila
homeodomain protein, Ind. Mech Dev 124, 925-934.
Von Ohlen, T. L. and Moses, C. (2009). Identification of Ind transcription activation and repression
domains required for dorsoventral patterning of the CNS. Mech Dev 126, 552-562.
Walldorf, U. and Gehring, W. J. (1992). Empty spiracles, a gap gene containing a homeobox
involved in Drosophila head development. EMBO J 11, 2247-2259.
Development • Supplementary information
Urbach, R., 2007. A procephalic territory in Drosophila exhibiting similarities and dissimilarities
compared to the vertebrate midbrain/hindbrain boundary region. Neural Dev 2, 23.
Development 143: doi:10.1242/dev.133546: Supplementary information
White, K., Grether, M. E., Abrams, J. M., Young, L., Farrell, K. and Steller, H. (1994). Genetic
control of programmed cell death in Drosophila. Science 264, 677-683.
Xie, B., Charlton-Perkins, M., McDonald, E., Gebelein, B. and Cook, T. (2007). Senseless
functions as a molecular switch for color photoreceptor differentiation in Drosophila. Development
134, 4243-4253.
Zhao, G. and Skeath, J.B. (2002). The Sox-domain containing gene Dichaete/fish-hook acts in
concert with vnd and ind to regulate cell fate in the Drosophila neuroectoderm. Development 129,
1165-1174.
Zhang, Y., Ungar, A., Fresquez, C. and Holmgren, R. (1994). Ectopic expression of either the
Drosophila gooseberry-distal or proximal gene causes alterations of cell fate in the epidermis and
central nervous system. Development 120, 1151-1161.
Development • Supplementary information
zur Lage, P. I. and Jarman, A. P. (2010). The function and regulation of the bHLH gene, cato, in
Drosophila neurogenesis. BMC Dev Biol 10, 34.