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doi: 10.1242/10.1242/dev.00528

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The pattern of neuroblast formation, mitotic domains and proneural gene expression during early brain development in Drosophila

¹ Institut für Genetik, Universität Mainz, D-55099 Mainz, Germany
² Institut für Genetik, TU Braunschweig, D-38106 Braunschweig, Germany

View larger version (121K): [in a new window]   Fig. 3. Identification of individual brain NBs at different embryonic stages. (A) Deadpan (Dpn) expression in the procephalic neuroectoderm by mid stage 8 (mst8) at two different focal planes (flat preparation; anterior is towards top, dorsal is leftwards). Dpn protein is found in two small spots in the ocular (arrowheads in A), and in a stripe-like domain in the antennal ectoderm (black arrows) as well as in the first brain NBs (Pcv7, 9), in a focal plane about 10 µm deeper (A'). Red arrows indicate same positions in the two optic foci, revealing that Pcv7 derives from a small cluster of Dpn-positive ectodermal cells. (B-H) svp-lacZ (brown)/Engrailed (blue) double stainings in flat preparations (B,D,F,H; ventral views, anterior (a) is towards the top and dorsal (d) is leftwards) and wholemounts (C,E,G; lateral views, anterior is towards the top and dorsal is towards the left). Individual NBs are identifiable by marker expression, morphology (size), and position relative to each other. Compare with semi-schematic presentation in Fig. 2. (B) Early stage 9 (est9); svp is weakly expressed in a subset of NBs. Ectodermal progenitor cells immediately prior to NB formation are marked by brackets (untypically, in this embryo Dv4 develops before Dd7). (CH) For comparison, whole-mount and flat preparation of the same individuals are shown. (C,D) Late stage 9 (lst9). Note the higher optic resolution and easier identification of NBs in the flat preparation. (E,F) Mid stage 10 (mst10; F, the most dorsal Svp-positive Pcd18 was removed accidentally during preparation). (G,H) Late stage 11 (lst11). a, d: anterior, dorsal; as, is: antennal and intercalary en stripe, respectively; cl: en expression in the clypeolabrum; hs: en head spot; CL: clypeolabrum; FG: foregut.

View larger version (98K): [in a new window]   Fig. 1. Survey of the spatial organisation of the embryonic head and brain neuroblasts at stage 11. Semi-schematic drawing showing a lateral view of a (A) wholemount and (B) a ventral view of a flat preparation, in which the head capsule has been dorsally opened. The pregnathal (light grey) and gnathal (dark grey) head region is indicated in relation to the engrailed expression pattern (dark blue). (C) Left half of a head flat preparation, including the complete pregnathal NB pattern. NBs are named according to their assignment to the trito- (T), deuto- (D) and protocerebrum (P), based on the reconstruction of segmental borders [indicated by red lines; for details see Urbach and Technau (Urbach and Technau, 2003a)]. Protocerebral NBs are subdivided into an anterior (Pa), central (Pc) and posterior (Pp) group (indicated by blue broken lines), roughly reflecting their origin from distinct mitotic domains (see Fig. 5 and Table 1). Each of the protocerebral groups, as well as the deuto- and tritocerebral NBs, is further subdivided into a dorsal (d) and a ventral (v) subgroup (indicated by yellow line) based on vnd expression [except NB Dd5, which co-expresses msh and is therefore attributed to the dorsal deutocerebrum (Urbach and Technau, 2003a)]. Within each subgroup, NBs are numbered from anterior to posterior and from ventral to dorsal. a, d, p, v: anterior, dorsal, posterior, ventral. as, is, las, mds, mxs: antennal, intercalary, labial, mandibular and maxillary en stripe, respectively. cl: en expression in the clypeolabrum. hs: en head spot. AN, IC, LA, MD, MX, PT, 1.AB: antennal, intercalary, labial, mandibular, maxillary, prothoracic and first abdominal segments, respectively. CF: cephalic furrow. CL: clypeolabrum. dML: dorsal midline. FG: foregut. OA: Bolwig organ/optic lobe anlagen. vML: ventral midline.

View larger version (100K): [in a new window]   Fig. 2. Spatial and temporal development of the embryonic brain NB pattern. (A-G) Semi-schematic representations of ventral views on the left half of head flat preparations, double stained for svp-lacZ and Engrailed (en-lacZ or anti-Invected antibody) at (A) mid/late stage 8 (m/lst8), (B) early stage 9 (est9), (C) late stage 9 (lst9), (D) early stage 10 (est10), (E) mid stage 10 (mst10), (F) early stage 11 (est11) and (G) late stage 11 (lst11). Expression of svp-lacZ and engrailed (en) in NBs is indicated in blue and red, respectively; newly formed NBs at each stage are in yellow; red hatching marks en expression in the peripheral ectoderm. Anterior (a) is towards the top and dorsal (d) is towards the left. Note, most of the depicted stages correspond to those described as phases `S1-S5' for the pattern of NB formation in the trunk (Doe, 1992): m/lst8 corresponds to early S1; est9 to S1; lst9 to S2; e/mst10 to S3; est11 to S4; and lst11 to S5 phase. In addition to the pregnathal segments, the evolving NB pattern is also shown for the mandibular segment (MD; asterisks indicate mandibular NBs). At early/mid stage 9 (B), when svp expression initiates, it is detected in a simple pattern, including about seven protocerebral and all deutocerebral NBs. At late stage 9 (C), two En-positive deutocerebral NBs (Dd5 and Dd13) derive from the en antennal stripe (as), and one protocerebral NB (Ppd5) from the en head spot (hs). New NBs form at the borders of the developing NB array, but in addition, individual NBs become integrated at various positions into the pre-existing NB pattern (D-G). By late stage 11 (G), Inv (but not en-lacZ) is weakly detected in the anteriormost procephalon (dh); the faint Inv expression in about 10 NBs deriving from the dh is not indicated (see Urbach and Technau, 2003a). Note that the formation of NBs in the intercalary (IC) and (anterior) mandibular segment (MD) is significantly delayed. Formation of tritocerebral NBs starts at stage 10. (H) Fully developed NB array (lst11) with the stage of formation indicated for each cell (see key). Most of the early (stage 8/early stage 9) NBs occupy central parts of the protocerebral primordium at different D/V positions. a, d: anterior, dorsal; as, is, mds: antennal, intercalary and mandibular en stripe, respectively; cl: en expression in the clypeolabrum; dh: en expression in the dorsal hemispheres; hs: en head spot; AN, IC, MD, MX: antennal, intercalary, mandibular and maxillary segment, respectively; CL: clypeolabrum; FG: foregut; ML: ventral midline; OA: Bolwig organ/optic lobe anlagen.

View larger version (119K): [in a new window]   Fig. 4. Expression of markers for glial cells and sensory precursors. (A-C) svp-lacZ (brown)/Repo (blue) double stainings between late stage 10 and late stage 11 (as indicated). Identified Svp-positive NBs are designated. By late stage 10, a cluster of two or three small protocerebral cells becomes Repo positive (white arrowheads in A,C; slightly different stages and focal planes). These are the first cells expressing Repo in the CNS (slightly before the longitudinal glioblast in the ventral nerve cord) (Halter et al., 1995), and might belong to the glia cluster `VPSG' described previously (Hartenstein et al., 1998). Considering their relatively small size, they could represent progeny cells of closely associated early NBs (e.g. Pcv6, Pcv7 or Pcv 9, which might act as neuroglioblasts as they are Repo negative). Slightly later, Repo is found in three further proto- and two tritocerebral cells (A). Two of the protocerebral cells (black arrows) are located ventrally in close vicinity to Pad2 (E, not in focus in A), and one more dorsally (white arrow) (possibly belonging to `VPSG' and `DPSG', respectively) (Hartenstein et al., 1998). By late stage 11, about eight additional Repo-positive cells can be detected in the protocerebrum at various positions along the DV axis (B). Considering their small size and distribution, they could be progeny cells of at least two central protocerebral NBs. Furthermore, at this stage three further Repo-expressing cells appear in the dorsal tritocerebrum at the border between the intercalary and mandibular segment (close to Td6, Td8 and Dd9; yellow arrowheads in B). The red arrowhead indicates the first deutocerebral cell expressing Repo. In two cases, glial precursors could be identified: Td7 (orange arrowhead in A,B,D) and Td4. (D) Td4 is a neuroglioblast; Ladybird early (lbe)/Repo double staining revealed a glial component (blue arrowhead) of the Lbe-positive Td4 lineage; black arrowheads mark other Lbe-positive daughters of Td4. (E) Because in all other cases it is not possible to link the Repo-labelled cells to identified precursors, their distribution relative to the NB pattern is marked by blue hatching in the semi-schematic presentation. (F,G) Atonal (blue)/A37-lacZ (brown) double staining at late stage 11. (F) In the pregnathal head A37-lacZ is found at strong levels in the ectoderm of the antennal appendage (AN), in a ventral ectodermal cell cluster near the foregut anlage (FG), and in ectodermal spots in the labral appendage (LR; violet and green arrowheads). Note that A37-lacZ is also detected at significantly lower levels in other parts of the procephalic and truncal neuroectoderm (black arrows), which is believed to be not indicative for sensory cells. Ato is co-expressed in subsets of strongly A37-lacZ positive cells (violet arrowheads); moreover, Ato is found in a dorsal cell cluster (blue arrowhead), including NB Ppd19 (E), in the primordial Bolwig organ cells of the optic anlage (OA), and in the labral appendage (black arrowheads). Dashed lines contour the outline of the mandibular, antennal and labral appendages; the dotted line contours the outline of the ventral midline (ML). (G) Close-up of the region framed in F at the level of NBs. Indicated are five A37-lacZ/Ato co-expressing antennal NBs; considering their position at the basis of the antennal appendage, Dd9, Dd11, Dd12, Dd13 are putative precursors of the Dorsal organ, the ventral Dv1 (and Dv3, which is not in focus) of the hypopharyngeal/latero-hypopharyngeal organ. Expression of A37-lacZ and Ato in stem cells is summarized in E. a, d: anterior, dorsal; AN, MD, MX: antennal, mandibular and maxillary segment, respectively; CL: clypeolabrum; FG: foregut; ML: ventral midline; OA: Bolwig organ/optic lobe anlagen.

View larger version (66K): [in a new window]   Fig. 7. Expression of proneural genes in the procephalic neuroectoderm and brain NBs. Semi-schematic presentation of the expression patterns of achaete (ac), scute (sc), lethal of scute (l'sc) and atonal (ato) (from stage 8 to 11) at the level of the procephalic ectoderm (A,C,E,G; positions of underlying NBs are marked by broken lines) and at the level of individual brain NBs (B,D,F,H) (see key for neuroblasts, NB and neuroectoderm, NE; high (+) and low () levels of expression are indicated by colour intensities). In the peripheral ectoderm (A,C,E,G) single cells are outlined when expression domains comprise less than eight cells. Additionally, the expression of engrailed (en) in the peripheral ectoderm is highlighted. Orientation as in Fig. 2. By stage 7/8, L'sc protein is detected in a large domain covering the central neuroectoderm (encompassing mitotic domain B and 9) from where first proto- and deutocerebral NBs develop (A,B). By stage 10, L'sc expression has expanded into other regions of the procephalic neuroectoderm (E), but by late stage 11 becomes confined to several smaller ectodermal domains, and especially to the dorsomedial neuroectoderm from where anteriormost protocerebral NBs emerge (G,H). Of all investigated proneural genes only L'sc is found in the intercalary NE (E,G) and about seven deriving tritocerebral NBs (F,H). Ac expression is detected by stage 8 in a small dorsal ocular and antennal group of neuroectodermal cells (from which Pcd21 and Dd8 derive). Furthermore, Ac is expressed in a large domain of the central procephalic neuroectoderm where four or five NBs, which co-express L'sc derive from (A,B). During stage 9 Ac is found in several large domains at different sites of the procephalic neuroectoderm (C), from which, by stage 10, about nine additional, weakly Ac-positive NBs emerge (D,F). During stage 10, Ac expression decreases in the peripheral ectoderm and is, by stage 11, confined to two most dorsal NBs (Pcd16 and Pcd19; E-H). sc is not expressed before stage 10, when it is found in about six small patches in the antennal and preantennal ectoderm, which (except for a patch in the clypeolabral ectoderm) give rise to single, or small groups of, NBs. Furthermore, it is strongly expressed in a domain of the anteriormost procephalic neuroectoderm and a corresponding group of about seven protocerebral NBs (E-H). Ato protein is expressed in a cluster of about six ocular ectodermal cells (A) that presumably represent the `proneural cluster' from which Ppd19 derives (D). Dv3 expresses Ato only after formation (compare B with D) in contrast to the adjacent Ato-positive Dv1 (H), which develops from the Ato-expressing proneural cluster in the ventral antennal ectoderm (C,E). By stage 11 Ato is additionally expressed in primordial cells of the optic anlagen (OA in G). a, d: anterior, dorsal; as, is: antennal and intercalary en stripe, respectively; hs: en head spot; AN, MD, MX: antennal, mandibular and maxillary segment, respectively; CL: clypeolabrum; FG: foregut; ML: ventral midline; OA: Bolwig organ/optic lobe anlagen.

View larger version (109K): [in a new window]   Fig. 5. Procephalic mitotic domains and the origin of brain NBs. (A) Projection of the spatial arrangement of mitotic domains (Foe, 1989) onto a schematic flat preparation at stage 8 [anterior (a) is towards the top, dorsal (d) is towards the left] based on 4D microscopic data (shown in C) as well as anti-alpha-Tubulin antibody staining (see Fig. 8). Different colours mark the mitotic domains 1, 2, 5, 9, 20 and B. The earliest set of NBs derives from mitotic domains B and 9. (B) Schematic flat preparation at late stage 11. Mitotic domains 1, 2, 5, 9 and B (and perhaps 20) contribute NBs to the embryonic brain (see also Table 1). Coloured hatched lines mark subpopulations of NBs that derive from the respective domains (compare A). (C) 4D microscopic analysis. Left panel depicts Nomarski pictures taken from an in vivo timelapse sequence of the same embryo between stages 6 and 10 (st6, stage 6; est8, early stage 8; est9, early stage 9; st10, stage 10; lateral views of the procephalic ectoderm in the same focal plane; anterior is towards the left, dorsal is towards the top; red, stippled lines mark the cephalic furrow). Right panel demonstrates computer models of the pictures on the left side (see Materials and Methods); each ectodermal cell is represented by a dot, the centre of which corresponds to the centre of the nucleus. According to their typical mitotic behaviour (e.g. time point of mitosis; orientation of mitotic spindle), ectodermal cells can be grouped into distinct mitotic domains (Foe, 1989). Colour code indicates cells belonging to the same mitotic domain. The movements and fate of each blastodermal cell can be traced through ongoing development (here shown until stage 10). Posterior black dots at stage 6 indicate cells that subsequently move into the cephalic furrow and thus out of the focal planes (not shown in following stages). Ventral dark grey dots indicate cells belonging to other mitotic domains that were not traced because they move out of focal planes. Note the slight rotation of the head ectoderm: dorsal cells move towards posterior positions and posterior cells towards more ventral positions. Importantly, despite these movements and the delamination of NBs, the relative positions among mitotic domains and among cells within each domain do not change in principal. a, d: anterior, dorsal; AN, MD: antennal and mandibular segment, respectively; FG: foregut; OA: Bolwig organ/optic lobe anlagen.

View larger version (119K): [in a new window]   Fig. 8. Brain NBs develop from neighbouring neuroectodermal cells (in mitotic domain B). (A-C) Anti -Tubulin/anti Dpn double staining of a late stage 8 flat preparation; anterior (a) is towards the top and dorsal (d) towards the left. (A) Anti--Tubulin (brown) highlights the outline of ectodermal cells and indicates cells in mitosis (e.g. some posterior cells of domain 5) (Foe, 1989). Position of mitotic domains 1, 5, 9 and B is indicated. Dpn protein (blue) is expressed in ectodermal domains; rectangle encloses most of the ocular Dpn expression that is part of mitotic domain B and gives rise to first brain NBs (compare with Fig. 2A and Fig. 5). At that stage, domain B cells are normally distinguished from domain 1 and 5 cells (most of which have already undergone mitosis) by their larger size. (B,C) Higher magnification of the region framed in A at two different focal planes: the apical level of the peripheral ectoderm (B) and about 20 µm deeper at the level of NBs (C). Red dots in B indicate transient apical cellular processes of three adjacent cells in the centre of domain B. In C, individual Dpn-positive NBs deriving from ocular Dpn domains are assigned. The adjacent apical cellular processes in B belong to the neighbouring NBs marked by red inscription in C. (D-G) 4D microscopic analysis indicates that the population of stage 8 protocerebral NBs derives from adjacent neuroectodermal progenitor cells of mitotic domain B. The origin of identified late stage 8 NBs (D,E) is traced back to the neuroectoderm at stage 6 (st6; F,G) using z-stack timelapse recordings (see Materials and Methods). (D,F) In vivo Nomarski pictures (lateral view; anterior is towards the left and dorsal is towards the top) at the level of NBs (D, focal plane at about 25 µm depth) and peripheral ectoderm (F; focal plane at about 8 µm depth). (E,G) Corresponding 3D computer models. Nuclei of identified NBs (as assigned in E) and of their corresponding neuroectodermal precursors (F,G) are indicated by the same colour code. Nuclei of other ectodermal cells are dark blue (E,G). The borders of mitotic domains 1, 2, 5, 9 and B are outlined in G. a, d, m: anterior, dorsal, medial.

View larger version (119K): [in a new window]   Fig. 6. Modes of brain NB formation differ between mitotic domains. (A) In mitotic domain B, NBs form by basally orientated delamination from the neuroectoderm (NE). The scheme on the left highlights, in a lateral view, the arrangement of mitotic domains (at stage 7) that contribute to the embryonic brain. (B) NE cells in domain 9 move apically (red arrow in Ba; see also D) and subsequentially reintegrate into the NE layer to delaminate as NBs (Ba1) or remain in the ectoderm to develop as epidermoblasts (EB in Ba2). Other cells in domain 9 divide perpendicular to the ectodermal surface (as indicated by the mitotic spindle; Bb); one daughter cell moves apically but later reintegrates into the NE as an epidermoblast, the other is deposited basally to become a NB. (C) In domains 1 and 5, NE cells divide parallel to the ectodermal surface; thereafter one of the two daughter cells usually stays in the peripheral ectoderm as an epidermoblast, the second delaminates as a NB. (D) Timelapse sequence focusing on part of the NE layer in mitotic domain 9 a few minutes after cephalic furrow formation. Sequence of pictures shows the apically directed movement of NE cells. First, the yellow NE cell leaves and reintegrates into the layer, followed by the red cell; each cell needs about 2 minutes to achieve this movement (time is depicted in pictures). Note the changing shape of apically moving cells (compare with Ba).