Gene expression profiles uncover individual identities of gnathal neuroblasts and serial homologies in the embryonic CNS of Drosophila

Abstract

The numbers and types of progeny cells generated by neural stem cells in the developing CNS are adapted to its region-specific functional requirements. In Drosophila, segmental units of the CNS develop from well-defined patterns of neuroblasts. Here we constructed comprehensive neuroblast maps for the three gnathal head segments. Based on the spatiotemporal pattern of neuroblast formation and the expression profiles of 46 marker genes (41 transcription factors), each neuroblast can be uniquely identified. Compared with the thoracic ground state, neuroblast numbers are progressively reduced in labial, maxillary and mandibular segments due to smaller sizes of neuroectodermal anlagen and, partially, to suppression of neuroblast formation and induction of programmed cell death by the Hox gene Deformed Neuroblast patterns are further influenced by segmental modifications in dorsoventral and proneural gene expression. With the previously published neuroblast maps and those presented here for the gnathal region, all neuroectodermal neuroblasts building the CNS of the fly (ventral nerve cord and brain, except optic lobes) are now individually identified (in total 2×567 neuroblasts). This allows, for the first time, a comparison of the characteristics of segmental populations of stem cells and to screen for serially homologous neuroblasts throughout the CNS. We show that approximately half of the deutocerebral and all of the tritocerebral (posterior brain) and gnathal neuroblasts, but none of the protocerebral (anterior brain) neuroblasts, display serial homology to neuroblasts in thoracic/abdominal neuromeres. Modifications in the molecular signature of serially homologous neuroblasts are likely to determine the segment-specific characteristics of their lineages.

Keywords: Central nervous system; Deformed; Drosophila brain; Gene expression profile; Neuroblasts; Segmental patterning.

Fig. 1.

Spatiotemporal development of the NB pattern in the gnathal segments. (A-F) Semi-schematic representations of ventral views of the left half of the gnathal segments (MN, mandibular; MX, maxillary; LB, labial) and the prothoracic segment (T1), as framed in the inset in A. Typical NB arrangement is shown at (A) late stage 8 (lst8), (B) stage 9 (st9), (C) early stage 10 (est10), (D) early stage 11 (est11), (E) late stage 11 (lst11) and (F) early stage 12 (est12). The position, formation time point and expression of five marker genes (see color code) allows the identification of individual NBs. NB formation is significantly delayed in MN, similar to observations made in tritocerebrum (TC; NBs light gray) (Urbach et al., 2003). Existence of NB1-3 is unclear in MX and LB (see E and Fig. S2.5). (F) Dorsal labial NBs become separated by salivary gland anlagen (SGA). is, intercalary en stripe; MNB, median neuroblast; ML, ventral midline; LGB, longitudinal glioblast; TA, tracheal anlagen (see also following figures).

Fig. 2.

Mapping and identification of NBs in gnathal neuromeres. (A-G) Composite confocal images of flat preparations (ventral view) of late stage 11 embryos stained for different combinations of molecular markers as indicated. Subsets of NBs identified by marker staining(s) and position are labeled. (A′,A″,B′,B″,C′,C″,D′,D″,G′,G″) Left side. (A-A″) Ind⁺ NB3-2 is lacking in MN. (B-B″) Lbe is atypically expressed in mandibular NB5-5. (C-C″) Run is found in increasingly smaller NB subsets from LB to MN. Ey is expressed in a reduced NB subset in MN. (D-D″) mirr-lacZ is detected in reduced NB subsets in gnathal segments, and Ems in MN. (E,E′) Col is exclusively expressed in the four anteriormost mandibular NBs/hemisegment. (F,F′) Dac is exclusively expressed in MN, in four to five anterior NBs/hemisegment. (G-G″) Mid is expressed in a reduced NB subset in MN. Repo⁺ glial cells derive from NB6-4, 7-4 and LGB.

Fig. 3.

NB maps summarizing the expression of AP and DV patterning genes, temporal genes and other NB marker genes in gnathal neuromeres compared with the prothoracic neuromere. (A-E) Expression of subgroups of marker genes (as indicated by color code) in the full complement of gnathal NBs (left side) at late stage 11; sublineage markers for specific NBs in D are indicated by outer circles. (F) Gray columns represent the total number of NBs in the respective segments. Colored columns indicate numbers of NBs in which expression of the indicated genes is ‘ectopically′ detected (red) or lacking (blue), compared with T1. For example, in MN 14 NBs exhibit ‘ectopic’ expression of one or more of those genes (out of the ten indicated), whereas 13 NBs lack expression of one or more other genes (out of the 11 indicated). In some NBs, expression of certain genes is ‘ectopically’ detected and that of others is lacking (indicated by overlapping columns).

Fig. 4.

Serial homologous NBs in neuromeres of the trunk and brain. Assignment of potentially serially homologous NBs (same color) in neuromeres of brain (TC, tritocerebrum; DC, deutocerebrum; PC, protocerebrum), gnathal segments (MN, mandibular; MX, maxillary; LB, labial) and prothorax (T1; resembling the ground state), based on developmental time point, neuroectodermal origin (in AP and DV axes) and the specific molecular signature of individual NBs (as summarized in Table S2). A few NBs in TC and DC show serial homology to two NBs in neuromeres of ventral nerve cord (VNC). The brain NB map is according to Urbach et al. (2003).

Fig. 5.

Roles of Dfd, programmed cell death and the number of neuroectodermal progenitors in regulating NB numbers in gnathal segments. (A-D) Gnathal (MN, MX, LB) and prothoracic (T1) left hemisegments in wild type (wt) (A) or Dfd¹⁶ mutants (B-D). White dotted lines outline ectopic Eg⁺ NBs. (E,F) Lbe⁺ and Ey⁺ NBs are indicated. Note the ectopic Dpn⁺ NB5-4-like cell (e5-4) in F. (G,G′) Ectopic NB5-4 expresses row 5-specific Wg (G) and is positioned dorsally to Ind⁺ NB5-3 (G′). (H) Number of Dpn⁺ NBs in gnathal and prothoracic hemisegments in wild type (MN, 20.8±1.5; MX, 24.6±1.0; LB, 26.9±0.7; T1, 28.8±0.5; n=14 each), Df(3L)H99 (H99; MN, 21.2±1.1; MX, 25.7±1.4; LB, 27.5±2.0; T1, 29.2±1.4; n=17 each) and Dfd¹⁶ (MN, 21.9±0.9; MX, 27.8±1.8; n=16 each). ND, not determined (Dfd expression largely restricted to MN and MX). (I,J) Dfd expression in outer neuroectoderm (I) and within the NB layer (J). (I) Note the low level of Dfd in the neuroectoderm of MN compared with MX. Inset shows Dfd in anterodorsal labial neuroectodermal cells (arrow). (K-N′) At early stage 12 Dcp-1 signal is reduced in Dfd¹⁶ mutant mandibular and maxillary neuroectoderm. Solid white lines indicate the dorsal border of neuromeres. (M) The average number of Dcp-1⁺ neuroectodermal (NE) cells is diminished in Dfd¹⁶ MN and MX. wt, late stage 11 (lst11): MN, 2.6±1.8; MX, 5.1±3.1 (n=14 each). Dfd¹⁶ lst11: MN, 0.4±1.6; MX, 0.8±1.2 (n=28 each). wt, early stage 12 (est12): MN, 7.0±5.1; MX, 15.5±5.7 (n=29 each). Dfd¹⁶ est12: MN, 2.5±2.0; MX, 5.5±4.5 (n=32 each). (N,N′) Note the ectopic mandibular NB6-4 (e6-4) in Df(3L)H99. (O) Stage 9. Red lines indicate segmental borders, blue lines dorsal neuroectodermal border, black line the ventral midline. In LB, four spatial quadrants are indicated: ad, anterodorsal; av, anteroventral; pd, posterodorsal; pv, posteroventral. (P) Neuroectodermal cell numbers in gnathal and prothoracic hemisegments and in each quadrant of each hemisegment (color coded). n=7-11 (all quadrants). (A-G′,I-L,N,N′) Composite confocal images. (H,M,P) Data are mean±s.d. **P<0.01; ***P<0.0001; ns, not significant; two-tailed t-test.

Fig. 6.

Proneural gene expression in gnathal segments and its segment-specific modification by the activity of DV genes (ind, msh). (A) Stage 8, showing delayed onset of ac expression in MN. (B,C) During stage 9/10 ac is expressed in a large domain in MN. (D) Expression of sc is similar to that of ac. (E,F) At stage 11 l'sc expression is downregulated in MX and LB, but is prominent in anterior MN. Black lines (A,B,D,E) indicate the segmental border of MN. (G-L) Stage 10. Black dashed lines indicate segmental borders and white dotted frames indicate corresponding regions of intermediate neuroectoderm. Ectopic ind expression in intermediate mandibular neuroectoderm in msh⁶⁸ (H) or Ngt40>ind (K) embryos represses ac (J,K). (L) Summary of G-K (see main text for details). (M,N) Composite confocal images of Dpn and En labeling in wild-type (M) and Ngt40>ind (N) embryos at late stage 11. Upon ind overexpression many row 3, 4, 5 NBs and MP2s (arrows), which would normally develop from the mandibular ac expression domain (see Fig. S2.6A′,B′), are lacking.

Fig. 7.

Comparison of the NB pattern in gnathal, thoracic and abdominal segments. The scheme illustrates NBs that are formed (colored) or missing (X) in the left hemineuromere of mandibular (MN), maxillary (MX) or labial (LB) segment and terminal abdominal segments (A8, A9, A10; according to Birkholz et al., 2013a), as compared with the ground state pattern (according to Doe, 1992), which is repeated in all segments from T1 to A7.