Neuroblasts of the Drosophila nervous system.

Type-I neuroblasts (type-I NBs) (top panel) and type-II (type-II NBs) (lower panel). Type-I NB undergo stem-cell-like divisions with each round producing a ganglion mother cell (GMC) that divides one more time to produce two neurons or glia cells. In Drosophila, all type-I NBs express dpn and ase whereas GMCs express only ase. Type-II NBs express dpn and pnt but not ase. They also undergo repeated divisions, with each round producing an intermediate progenitor cell (INP) which also is a proliferating progenitor and expresses ase. INPs undergo a maturation process during which pnt is still expressed initially, but not dpn. Mature INPs express dpn, ase and fez/erm whereas GMCs express ase and fez/erm. Type-II NB lineages are only found in the anterior brain, they produce central complex neurons and glia [30,31].

Magic mushrooms (fez-mm-eGFP) reporter line.

A-C) eGFP-expression in larval instar brains, D) EGFP antibody labelling in combination with Tc-fez/erm RNA in situ in the embryonic head, A-D) Dapi labelling of nuclei. Scalebars in A and D are 50 µm. A) Full projection of whole brain scan (4th instar larvae): fez-mm-eGPF expression marks many cells of the mushroom bodies (white arrowheads) and of the larval optic lobe (purple arrowheads). Note that we use the terms dorsal and ventral with respect to the neuraxis of the larva. See [10] for orientation. B) Central part of the whole brain scan (projection of substack, same brain as in A): mm-eGFP marks the peduncles of the mushroom bodies (white arrowheads) and some central complex ensheathing cells (probably glia cells; yellow arrowhead). C) Projection of the dorsal part of same brain as in A shows calyces and peduncles of mushroom bodies (white arrowheads), and optic neuropile (purple arrowheads). Panel D) Extensive co-localization of fez-mm-eGFP with Tc-fez/erm-RNA in white encircled area. Projection of all planes where expression was detected, embryonic stage NS11.

Developmental occurrence and progression of Tc-pnt-clusters and associated fez-mm-eGFP cells (type-II NB lineages).

A-C) Anti-GFP antibody staining reflecting fez-mm-eGFP expression in combination with Tc-pnt RNA in situ hybridisation and dapi labelling of nuclei (A, C) or FM1-43 membrane staining (B), single planes from confocal z-stacks of right head lobes. Scalebars are 25 µm. A’-C’) Schematic drawing of the head lobe at the respective stages; clusters out of focus depicted by grey circles. D) Schematic depiction of the localization of type-II NBs during Tribolium head morphogenesis. A) At stage NS11 seven Tc-pnt-clusters (white arrows) are found in an anterior-medial horseshoe-like arrangement with fez-mm-eGFP cells in the centre. Asterisk indicates the position of one Tc-pnt-cluster which is outside the focal plane of this image. B) At stage NS13 the seven anterior-medial clusters have been moved to the medial margin of the developing brain by morphogenetic movements of the head lobes (white arrows). The clusters are situated in different dorso-ventral levels shown in the different images B-I to B-III. Two additional posterior Tc-pnt-clusters have appeared (yellow arrows). Blue arrows point to fez-mm-eGFP positive cells that are not part of the type-II NB lineages. B-I: ventral level B-II mid-level, B-III: dorsal level. C) At stage NS14 a group of six clusters are arranged in one plane anterior-medially in the brain lobe (white arrows) (C-I). Anti-PH3 labelling marks mitotic cells within the lineages (white signal) and clusters out of the focal plane are indicated by grey circles in C’-I. C-II One cluster of the anterior median group (white arrow) is in a deeper plane, as well as the two posterior clusters (yellow arrows). Blue arrowheads point to lateral fez-mm-eGFP expression not associated with type-II NBs. A’-C’) AMG =anterior medial group (blue), PG =posterior group (pink). Grey circles in B’-C’ indicate position of type-II NBs in different focal planes. D) Tc-pnt-expressing cells identified as type-II NBs first appear bilaterally in the anterior most part of the embryonic head (stage NS11). During head morphogenesis this anterior tissue folds over such that the type-II NBs end up in a more medial position. Two more type-II NBs emerge posteriorly (stage 13). At the end of embryogenesis (stage NS14) the type-II NBs can be divided in an anterior medial group (AMG (blue); including type-II NBs 1-7, numbering starting from anterior) and a posterior group (PG (pink), including type-II NBs 8-9).

Cell- and nuclear diameter of type-II NBs compared to a control group.

Stages NS13-14. Left: diameter of Tc-pnt-pos. type-II NBs (average diameter=12.98 µm; n=43) compared to control group (average diameter=6.75, n=1579). Right: average nuclear diameter of type-II NBs (=8,73 µm, n=17) compared to average nuclear diameter of control group (= 5.21, n=1080). Differences between groups are significant (t-test; *** ≙ P < 0.001).

Differential expression of the neural markers Tc-pnt, Tc-fez/erm, Tc-dpn and Tc-ase in type-II NBs, INPs and GMCs.

A, B, D) Anti-GFP staining visualizing fez-mm-eGFP expression in combination with HCR-labelling of further factors. C, E) eGFP antibody staining in combination with RNA in situ hybridisation. A-E) Dapi staining of nuclei. All panels are single planes from confocal z-stacks. Panel A) Stage NS12, expression of Tc-pnt and Tc-dpn in type-II NBs (yellow arrowhead) with adjacent fez-mm-eGFP positive INPs. Panel B) Stage NS13, white line marks one lineage. Expression of Tc-dpn is absent in the youngest INPs Tc-pnt+ (pink arrowhead) but present in more mature INPs (blue arrowheads), white line marks one lineage. Type-II NB not in focus of this image. Panel C) Stage NS14, expression of Tc-dpn in type-II NBs (yellow arrowheads), and INPs (blue arrowheads). Asterisk marks mitosis in a fez-mm-eGFP cell visible through the condensation of chromatin stained by dapi. Panel D) Stage NS13, Tc-ase- and Tc-pnt-expression in type-II NBs and INPs, white line marks one lineage. Yellow arrowhead: type-II NBs (Tc-pnt+), pink arrowheads: immature INP (Tc-pnt+), blue arrowhead: young INP (Tc-fez+/ Tc-ase+/ Tc-pnt+). Panel E) Stage NS13, expression of Tc-ase in fez-mm-eGFP positive cells, location of type-II NBs and young INPs is outside the focus.

Expression of Tc-pros in fez-mm-eGFP cells.

A-B) Anti-GFP antibody staining in combination with Tc-pros RNA in situ hybridization. Membranes stained with FM 1-43. Single planes from confocal z-stacks. Panel A) Stage NS13, two type-II NBs lineages producing INPs and GMCs in opposing directions. Expression of Tc-pros in mature INPs and GMCs. Cluster of type-II NBs (pink arrowheads) and immature INPs (yellow arrowheads). Young INPs express Tc-fez/erm but not Tc-pros (blue arrowheads). Panel B) Stage NS13, detail of Tc-pros-negative type-II NBs (pink arrowhead) and immature INPs (yellow arrowheads) and young INP (blue arrowhead). Note that distinction between type-II NB and immature INPs in A) and B) is only made based on position.

Schematic drawing of expression different markers in type-II NB lineages.

A) Subclassification into type-II NBs (Tc-pnt+), immature INPs (Tc-pnt+), young INPs (Tc-pnt+, Tc-fez/erm+, Tc-ase+) and mature INPs and GMCs (Tc-fez/erm+, Tc-ase+) (based on staining shown in Fig. 5). B) Type-II NB, young and mature INPs express Tc-dpn, Tc-pros and Tc-fez whereas GMCs express only Tc-fez and Tc-pros. (based on staining shown in Fig. 5 and 6). Note that of the selected markers immature INPs express Tc-pnt only.

Expression of the transgenic central complex reporter skh/eGFP in relation to type-II NBs lineages.

A-D) Antibody labelling of eGFP, which is expressed in the pattern of skh [49] in combination with HCR visualizing Tc-fez/erm and Tc-pnt, dapi labelling of nuclei, single planes of confocal z-stacks, all stage NS14. A) Left head lobe, anterior group of Tc-pnt-positive type-II NBs, Tc-fez/erm-expressing INPs/GMCs and skh/eGFP positive postmitotic cells. B) Left head lobe, posterior group of type-II NBs lineages. Tc-pnt+ type-II NB or young/immature INPs (white arrows), Tc-fez/erm+ INPs/GMCs (orange arrowheads) and skh/eGFPpostmitotic cells (yellow arrowheads). C) Detail of one lineage including type-II NB cluster (Tc-pnt+, white arrows), INPs/GMCs (fez+, orange arrowheads) and skh/eGFPcells at the end of the lineage (yellow arrowheads). D) Detail of transition from Tc-fez/erm+ cells (orange arrowheads) to skh/eGFPcells reveals a small area of co-expression of both factors (yellow arrowhead). E) Schematic drawing of type-II NBs lineages showing relative positions and markers of type-II NBs, INPs/GMCs and postmitotic central complex forming cells. F) Schematic overview of the arrangement of the type-II NBs lineages including skh/eGFP+ central complex forming cells in the head lobe of stage NS 14 (young/immature INPs not shown for simplicity). No data on skh-GFP expression in lineage of type-II NB 7 (marked in grey) is available.

Tc-fez/erm positive cell number (NB type II, INPs and GMCs)

Young and mature INPs (Tc-fez and Tc-dpn positive)

Size comparison of Tribolium and Drosophila type-II NBs lineages.

Anterior and median lineages from Tribolium stages 13 and 14 (quantified in this work) compared to Drosophila stages 15 and 16 (pooled data from [29]). A) Lineage sizes comprising type-II NBs, INPs and GMCs (Tribolium n=4; Drosophila n=8). B) Number of Tc-dpn-positive mature INPs (Tribolium n=5; Drosophila n=8). T-test p-value significance level (t-test; *** ≙ P < 0.001, ** ≙ P < 0.01).

Expression of Tc-otd in relation to the fez-mm-eGFP lineages.

A/B) RNA in situ hybridisation for Tc-otd in combination with eGFP antibody staining and dapi labelling of nuclei, A and B show projections of different levels of NS14 embryos, right head lobe. A) Tc-otd is neither expressed in type-II NBs nor the in the fez-mm-eGFP positive INPs of the anterior medial group (both positioned within encircled area) but it is expressed in the directly surrounding tissue. B) Likewise, Tc-otd is not expressed in type-II NBs or fez-mm-eGFP cells of the posterior group (encircled area).

Expression of Tc-six4 and Tc-six3 in type-II NBs and INPs.

A-C) RNA in situ hybridisations of Tc-six4 and Tc-six3 in combination with GFP antibody staining reflecting fez-mm-eGFP expression, dapi labelling of nuclei, left head lobes shown. Panel A) Stage NS13; Tc-six4 is expressed in an area encompassing type-II NBs (positioned in encircled area) and mm-eGFP expressing INPs. Panel B) Stage NS14; Tc-six4 is now restricted to type-II NBs and their youngest progeny that do not express fez-mm-eGFP (yellow arrowheads). Panel C) Stage NS14; Tc-six3 specifically marks the four anterior-most lineages. It is expressed in the type-II NBs at the base of the lineages (yellow arrowheads) and in the fez-mm-eGFP positive INPs. It is absent from the remaining lineages of the anterior medial group and is also not seen in the posterior group (not shown).

Conserved aspects of gene expression in type-II NB lineages of the holometabolan models Drosophila and Tribolium.

Overview on the different cell types within one lineage (type-II NBs, INPs, GMCs and CX neurons), their mitotic activity, and summary of the expression of key factors in the respective cell types. INPs are divided into immature, young, and mature INPs with each subtype having a unique expression profile. Note that each lineage can have several mature INPs. We strongly assume that glia are also produced from these lineages in Tribolium, as they are in Drosophila [30]. * In the Tribolium embryo Tc-six4 is only expressed in the anterior medial lineages, and its expression is overlapping with Tc-fez/erm at an earlier stage (NS12) whereas it is restricted to type-II NBs and immature INPs, mutually exclusive with Tc-fez/erm, at a later embryonic stage (NS14). Drosophila six4 was found in all 8 larval lineages [43]. The scheme is based on this work and [30,39,40,43,49]. NB= neuroblast; imm. INP = immature intermediate progenitor; mat. INP= mature intermediate progenitor; CX=central complex; N=neuron; Skh=shaking hands (enhancer trap) [49].