Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis

Abstract

bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an 'Orange' protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. We have studied the single Drosophila Hey orthologue. We show that it is primarily expressed in a subset of newly born neurons, which receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed us to study Notch signalling during the genesis of secondary neurons in the larval central nervous system.

Fig. 1.

Expression of Hey in the embryonic CNS. (A) Hey mRNA and (B) Hey protein patterns in stage 12 embryos. Both RNA and protein are detected in the CNS (most of the signal) and PNS (arrows). Anterior is left and ventral is down. (C) Ventral view of a stage 11 embryo stained for Pros (blue), Eve (green) and Hey (red). Hey-positive cells are a subset of the Pros-positive cells (GMCs and immature neurons). Eve marks a small subset of neurons and pericardial cells (arrows). (D) Sagittal view of stage 12 embryo stained for β-galactosidase (green) to image E(spl)-m8-lacZ expression in epithelial cells and Hey (red). Anterior is left and ventral is down. Note the lack of Hey staining in the superficial neuroepithelium (arrow). Weak β-galactosidase staining in the deeper neuronal layers (arrowhead) is probably perduring protein from earlier neuroepithelial expression. (E-H′) Ventral views of embryos, anterior is up. (E,F) Images of two different focal planes (E is more superficial) of a stage 15 embryo stained for Hey (green) and Elav (red). Elav marks neurons. Only a few Hey-positive cells (white arrows) are Elav-negative. (G,G′) Stage 10 embryo stained for Hey (green) and Asense (red, grey in G′). Asense marks GMCs and neuroblasts. Arrowheads in G′ indicate rare cases of GMCs that express Hey. (H,H′) Hey (green) and Repo (red, grey in H′) in a stage 15 embryo. Arrowheads in H′ mark examples of Repo-positive cells expressing Hey. Scale bars: 100 μm in A-C; 16 μm in D; 50 μm in E-H.

Fig. 2.

Hey expression in wild-type and mutant backgrounds. (A-E) AJ96-lacZ line stained for Hey (green) and β-gal (red) to mark the vMP2/dMP2 cells in wild-type (A-C) and mutant backgrounds (D,E). (A) Hey expression in the MP2 lineage starts at stage 10 within the undivided MP2 neuroblast. (B) In stage 12 embryos Hey is expressed in vMP2s, the anteriorly located AJ96-positive cells, but not in dMP2s. (C) In stage 15 embryos Hey expression is turned off. (D) In the numb genetic background, which induces transformation of dMP2 into vMP2, Hey is expressed in both AJ96-positive cells (stage 12). (E) The opposite (Hey absence) is observed in spdo embryos, in which vMP2 is transformed into dMP2 (stage12). Note the scarcity of Hey-positive cells (also in I) compared with equivalently staged wild-type (B) or numb (D) embryos. (F-I) Hey expression in Eve-positive lineages in wild-type (F,G) and mutant (H,I) backgrounds. (F) A stage 11 embryo montage showing deep focal planes with aCC/pCC and RP2/RP2sib pairs marked with Eve (red). Hey is expressed in pCC (arrow) but not aCC. RP2 does not express Hey in contrast to the smaller RP2sib (arrowhead) which is still Eve-positive at this stage. In some segments a Hey/Eve-positive U cell (asterisk) is evident. (G) A stage 15 embryo montage of superficial focal planes to visualize U and EL lineages. Eve marks the U and EL cells. Hey is expressed in the U cells (arrows) but not in the EL cells (arrowheads). (H) In a stage 15 numb embryo more cells within the U-cluster are labelled with Eve, as Usib is transformed into U. (I) Loss of Notch signalling in a spdo embryo (stage 11) results in two RP2s (arrowheads) and two aCCs (arrows) per hemisegment. Eve-positive U cells are transformed to Usib cells (Eve-negative or weakly positive near the aCC pairs). Persistent Eve-positive cells at the U position (asterisk) are either undivided GMCs or Usib cells that have not yet extinguished Eve expression. None of these cells express Hey. Hey expression is limited to a few midline cells and a cluster of lateral cells. (J-P) Ventral views of stage 15 embryos (J-L) and sagittal views of stage 12 embryos (M-P) of different genetic backgrounds stained for Hey. (J) Wild-type; (K) numb²; (L) spdo^c55; (M) wild-type; (N) Dl^rev10 Ser^RX106; (O) Df(3R)E(spl)^b32.2; (P) mam⁰⁴⁶¹⁵. Note the increased number of Hey-positive neurons in the E(spl) and numb embryos compared with that of wild type embryos. Conversely, there are fewer Hey-positive neurons in Dl Ser, mastermind and spdo embryos. Anterior is up (A-L) or to the left (M-P). Scale bars: 55 μm in A-L; 130 μm in M-P.

Fig. 3.

Expression of Hey in the larval CNS. (A,B,D) Single confocal sections of third instar larval brain hemispheres, anterior top, lateral left. (A′,B′,D′) are higher magnifications. (A,A′) Ase (red) marks neuroblasts (large nuclei) and GMCs (small nuclei). Hey (green) does not overlap with Ase. All Hey-positive cells are also positive for Elav (blue), a neuronal marker. (B,B′) GFP (green) highlights lineages marked 5 days before fixation. Ase (red) and Hey (blue) are visualized. In B′, five neuroblasts (NBs) are numbered; NB4 is not visible in this focal plane. Hey-positive (Ase-negative) cells exist in all five lineages, evident as green-blue nuclei (arrows) in lineages 1, 2, 4 and 5 in this focal plane. (C) Low magnification view (confocal projection) of a third larval instar CNS showing neuroblasts (Dpn, red), GMCs and secondary neurons (Pros, blue) and Hey (green). The abdominal ganglion (bracket) has ceased neurogenesis and is devoid of NBs, GMCs and young neurons, as well as Hey immunoreactivity, with the exception of a few midline cells. Note the different cellular organization of the optic lobe (pink arrow) versus central brain (white arrow). (D,D′) Repo (red) marks glia nuclei, which are predominantly found on the surface of the brain hemisphere (arrowheads) and are negative for Hey (green). A number (∼60) of glia (arrows) in the outer optic proliferation centre are found among the band of hundreds of Hey-positive cells, which are mostly neurons. K33-lacZ (blue) marks the NBs strongly (asterisks), but β-galactosidase perdures in GMCs and newly born neurons at lower levels. This section is deeper than the ones shown in A and B, thus containing fewer NBs in the central brain and more Hey-positive cells in the optic lobe. (E) Confocal projection of pupal brain, 2 days after pupariation. Posterior view, dorsal up. The majority of NBs have disappeared; only four mushroom body NBs are detectable by Ase staining (red). Each of these is accompanied by a cluster of Hey-positive cells (green), no other Hey-positive cells are detected. Prospero is detected in blue. (F,F′) Higher magnification of E. Single optical section showing two Ase-positive NBs accompanied by a few Hey-positive (green) nuclei. Two of the Hey-positive cells in each lineage are GMCs as they still express Ase (red). These are marked by red arrows in F′, which shows the Pros immunoreactivity (blue in F).

Fig. 4:

Analysis of Notch signalling in the larval CNS. (A) Schematic of MARCM clone (Lee and Luo, 2001) progression in the CNS. A single neuroblast (NB) lineage is shown, where the NB is depicted large, the GMCs are intermediate in size and the neurons are small circles. FLP recombinase is induced at time t₀ in a m/+ genetic background (m is any mutation). Mitotic recombination in the NB results in two progeny cells of different genotype after the next NB mitosis, at time t₁. The renewed NB has become m/m and has turned on GFP expression (green), whereas its new GMC progeny is +/+ (dark grey) and does not express GFP, the same as all the unrecombined m/+ cells. As the NB and GMCs continue to proliferate, more m/m GFP-positive (green) cells are produced (time t₂). (B-P′) All images shown are single confocal sections with examples of GFP-marked clones (green) stained for Hey (red) and other markers, as indicated in blue. GFP is nuclear in all panels, except in Notch clones (C,D,H), where a membrane-targeted GFP was used. (B) a neutral (wild-type) clone with several Hey-positive cells. (C) Two Notch^54l9 clones in the central brain are devoid of Hey-positive cells, whereas (D) a clone of the same genotype in a mushroom body lineage contains Hey-positive cells. (E) A Su(H)^Δ47 clone in the ventral nerve cord, also stained for Pros (blue), which persists in mutant cells, although Hey is lost. (F) Two neur¹ clones, also stained for Ase (blue), which is not affected by the mutation. (G) Two spdo^G104 clones marked as in F. (H-J) Examples of clones in the outer proliferation centre of the optic lobe. In all cases, Hey expression is lost from the clone, best seen as unstained patches in H′-J′ (Hey channel alone). In a Notch^54l9 clone (H), no Hey expression is detected in any mutant cells, whereas in Dl^rev10 Ser^RX106 (I) and neur¹ (J) clones, several mutant cells near the clone borders are Hey-positive (arrows in H′-J′). (K-P) MARCM clones in the central brain mutant for Dl^rev10 Ser^RX106 (K), Dl^rev10 (L), neur¹ (N) or spdo^G104 (O) lack Hey-positive cells; exceptions are marked by arrows in the Hey-only channel (K′-P′). Ser^RX106 (M) or mib1^EY9780 (P) clones contain Hey-positive cells (arrows). Scale bar: 10 μm.

Fig. 5.

Loss- and gain-of-function analysis of Hey. (A) Low magnification image of two embryos stained for Hey (red) and β-galactosidase (green), which reveals wg-lacZ from the balancer chromosome (Hey⁺) over which Hey^f06656 is kept. The homozygous mutant embryo (white arrowhead), identified by the absence of the wg-lacZ pattern, does not express Hey. (B-I) High magnification images. Anterior is up. (B) Eve expression pattern in a stage 15 Hey^f06656-homozygous embryo is similar to that in wild type embryos (see F; Fig. 2G). (C) Odd expression pattern (green) in a stage 15 Hey^f06656;AJ96-lacZ embryo. β-galactosidase (red) marks vMP2/dMP2 neurons. Besides MP1s (arrow), Odd is expressed only in dMP2s (arrowheads) as in wild-type embryos (see Fig. S1 in the supplementary material). (D-E) Pattern of UAS-GFP expression (red) driven by ftz.ng-Gal4³ versus Eve staining in blue. (D,D′) Montage of deep focal planes, showing aCC/pCC pairs (arrowhead) and RP2 (asterisk). GFP is detected in all three cells and additional neighbouring neurons, but not in the occasional U-cell that lies near the aCC/pCC pair and is also positive for Eve (blue arrows in D′, Eve channel). (E) Superficial focal plane montage showing the U (arrowhead) and EL (asterisk) clusters, both of which are devoid of GFP. (F) Eve expression pattern in a stage 16 wild-type embryo. Note the solo RP2 neurons (asterisk) between the clusters containing EL (lateral) and U/aCC/pCC (medial) neurons. (G) Eve staining of a stage 16 Hey-overexpressing embryo is similar to that of a numb mutant. RP2 cells are absent; two persisting ones are marked by asterisks. The EL and U clusters are not affected, as ftz.ng-Gal4³ is not expressed there. (H) In wild-type embryos, two dMP2 (lateral to midline) and two MP1 (midline) neurons per segment are marked with Odd. (I) In Hey-overexpressing embryos, Odd expression persists in the two MP1 neurons, but it is extinguished from most dMP2s. White arrows show two dMP2s that are still labelled with Odd. Scale bar: 25 μm.