Segment-specific neuronal subtype specification by the integration of anteroposterior and temporal cues

Abstract

The generation of distinct neuronal subtypes at different axial levels relies upon both anteroposterior and temporal cues. However, the integration between these cues is poorly understood. In the Drosophila central nervous system, the segmentally repeated neuroblast 5-6 generates a unique group of neurons, the Apterous (Ap) cluster, only in thoracic segments. Recent studies have identified elaborate genetic pathways acting to control the generation of these neurons. These insights, combined with novel markers, provide a unique opportunity for addressing how anteroposterior and temporal cues are integrated to generate segment-specific neuronal subtypes. We find that Pbx/Meis, Hox, and temporal genes act in three different ways. Posteriorly, Pbx/Meis and posterior Hox genes block lineage progression within an early temporal window, by triggering cell cycle exit. Because Ap neurons are generated late in the thoracic 5-6 lineage, this prevents generation of Ap cluster cells in the abdomen. Thoracically, Pbx/Meis and anterior Hox genes integrate with late temporal genes to specify Ap clusters, via activation of a specific feed-forward loop. In brain segments, "Ap cluster cells" are present but lack both proper Hox and temporal coding. Only by simultaneously altering Hox and temporal gene activity in all segments can Ap clusters be generated throughout the neuroaxis. This study provides the first detailed analysis, to our knowledge, of an identified neuroblast lineage along the entire neuroaxis, and confirms the concept that lineal homologs of truncal neuroblasts exist throughout the developing brain. We furthermore provide the first insight into how Hox/Pbx/Meis anteroposterior and temporal cues are integrated within a defined lineage, to specify unique neuronal identities only in thoracic segments. This study reveals a surprisingly restricted, yet multifaceted, function of both anteroposterior and temporal cues with respect to lineage control and cell fate specification.

Figure 1. The Ap cluster is generated by thoracic neuroblast 5–6.

The transgenic reporters lbe(K)-lacZ and lbe(K)-Gal4 allow for visualization of the NB 5–6 lineage throughout the developing Drosophila CNS. (A) Expression of lbe(K)-lacZ at embryonic stage 11 in the NB 5–6 lineage in brain (B1–B3), subesophageal (S1–S3), thoracic (T1–T3), and abdominal segments (A1–A9). (B) At stage 14, the difference in size between NB 5-6A and NB 5-6T is becoming evident. To visualize the outline of the Drosophila CNS, the Deadpan and Prospero markers was used. (C) At stage 18 h AEL, coexpression of Eya, ap^lacZ, and lbe(K)-Gal4, reveal that the four Ap cluster neurons (Ap1–4) are generated within the NB 5-6T lineage. Boxes to the right depict a T3 Ap cluster (dashed box in left panel), with the expression of Eya, ap^lacZ, and lbe(K)-Gal4 in separate panels. (D) Within the Ap cluster, the Ap1 and Ap4 neurons can be identified by the expression of the two neuropeptides Nplp1 (Ap1) and FMRFa (Ap4). Boxes to the right depict a T3 Ap cluster (dashed box in left panel), with the expression of Eya, Nplp1, and proFMRFa in separate panels. (A–D) are composed from multiple images. Genotypes: (A) lbe(K)-lacZ. (B) lbe(K)-Gal4, UAS-nmEGFP/+; lbe(K)-Gal4, UAS-GFP/+. (C) ap^lacZ/+; lbe(K)-Gal4/UAS-nmEGFP/+. (D) w¹¹¹⁸.

Figure 2. Expression of Hox, Pbx/Meis, and temporal factors within neuroblast lineage 5–6.

(A) Cartoon depicting the NB 5–6 lineage in the 18 embryonic Drosophila CNS segments, with the anteroposterior limits of expression of Hox factors. Although the NB 5–6 lineage is present in all segments, the Ap clusters only appear in thoracic segments. (B) Cartoon depicting the NB 5-6A and NB 5-6T lineage, with expression of Hox, Pbx/Meis, and temporal factors outlined. During stage 9 and 10, only expression of early temporal genes is evident. At stage 11, expression of Hth and Exd commences, followed by Antp and Ubx at stage 12 (for simplicity, expression of Abd-A and Abd-B is not depicted). However, Ubx is not expressed in NB 5-6T at any point. Both NB 5-6A and 5-6T display the typical temporal gene progression of Hb-Kr-Pdm, but only NB 5-6T continues dividing and progressing into the Cas/Grh window. Previous studies reveal a striking drop in Cas expression in the neuroblast at stage 13, after which it commences again. After exiting the cell cycle at stage 12 (NB 5-6A) and 15 (NB 5-6T), both neuroblasts undergo apoptosis.

Figure 3. Homothorax levels rise sharply at stage 13.

(A–L) T2 hemisegments of stage (st) 11 (A–C), 12 (D–F), 13 (G–I), and 14 (J–L) embryos, showing the expression of Hth within the NB 5-6T lineage. Small boxes show the expression of Hth and lbe(K)-Gal4 in separate panels. All images were from embryos processed on the same slide, and scanned using identical confocal settings. There is a sharp increase in Hth levels between stage 12 (D–F) and 13 (G–I). Genotype: lbe(K)-Gal4, UAS-nmEGFP.

Figure 4. Genetically blocking cell death in the abdominal NB 5–6 lineage does not result in appearance of Ap cluster neurons.

(A and B) Expression of lbe(K)-Gal4 at stage 16, in control (A) and H99 (B), reveals a clear difference in both the NB 5–6 thoracic and abdominal lineage size (side view, T1–A1). (C and D) Expression of Eya and Nplp1 at stage 18 h AEL, in control (C) and H99 mutants (D) (side view, T1–A1). There is no evidence of ectopic Ap clusters in abdominal segments. (E) Quantifying NB 5–6 lineage cells in the thoracic versus the abdominal area, in control and H99, reveals that additional cells appear in both lineages when apoptosis is blocked. (F and G) Stage 13, ventral and dorsal confocal stacks of NB 5-6A visualized using lbe(K)-Gal4. Small boxes show the expression of pH3, Dpn, and lbe(K)-Gal4 in separate panels. In contrast to control (F1 and F2), H99 (G1 and G2) mutants reveal expression of Dpn in the NB. In contrast, no phospohistone-3 (pH3) staining is observed in either genotype beyond stage 13. (H) Quantifying NB 5-6A lineage cells at stage 13 in control and H99 mutants reveals no significant increase in cell number in H99 past stage 13. Data for stage 16 were copied from bar graph (E) for easy comparison. (E and H) Asterisks denote significant difference (p<0.01; Student two-tailed test). Genotypes: (A and F) lbe(K)-Gal4,UAS-nmEGFP. (B and G) lbe(K)-Gal4,UAS-nmEGFP;H99. (C) w¹¹¹⁸. (D) H99.

Figure 5. Homeotic transformations of the abdominal NB 5–6 lineage.

(A–F) Analysis of Ap clusters, defined by expression of Eya, FMRFa, and Nplp1 in Bx-C single, double, and triple mutants, reveals the appearance of Ap clusters in abdominal segments. Due to genetic redundancy, in Ubx mutants (B), only A1 and partly A2 are transformed; in abd-A (C) and Abd-B (D), no segment is transformed; in Ubx, abd-A double mutants (E), A1–A8 are transformed, whereas in the triple mutant, all abdominal segments (A1–A9) are transformed (F). Stage 18 h AEL embryos. Genotypes: (A) w¹¹¹⁸. (B) Ubx¹/Ubx^9.22. (C) abd-A^MX1. (D) Abd-B^M1/Abd-B^M2. (E) Ubx¹⁰⁹. (F) Dp(3;1)P68; ss¹ Ubx¹ abd-A^D24 Abd-B^D18.

Figure 6. Suppression of thoracic-like NB 5–6 lineage progression by Ubx and Pbx/Meis.

(A) At stage 15, lbe(K)-Gal4 reveals a larger NB 5–6 lineage in thoracic segments, when compared to abdominal ones. Expression of Cas and Col is only evident in the NB 5-6T lineage. (B–D) At stage 15, in Ubx, hth, and exd mutants, NB 5-6A lineage is larger and expression of Cas and Col is evident. (E and F) Quantification of GFP, Cas, and Col positive cells/NB 5-6A lineage, in control, Ubx, hth, and exd mutants, at stage 14 (n>8 lineages). Asterisks denote significant difference compared to thoracic control (p<0.01, Student two-tailed test). Genotypes: (A) lbe(K)-Gal4, UAS-nmEGFP/+; lbe(K)-Gal4, UAS-nmEGFP/+. (B) lbe(K)-Gal4,UAS-nmEGFP;Ubx. (C) lbe(K)-Gal4,UAS-nmEGFP;hth^5E04/hth^Df3R. (D) exd^B108, FRT^18D/y; lbe(K)-Gal4, UAS-nmEGFP/+; lbe(K)-Gal4, UAS-nmEGFP/+.

Figure 7. collier is able to rescue homothorax, but not Antp.

(A–G) Expression of Eya, FMRFa, and Nplp1 in thoracic segments, at 18 h AEL. Small boxes show the expression of Eya, Nplp1, and proFMRFa in separate panels. (A–C) In control (A), Ap clusters are present, whereas in Antp (B) and hth (C) mutants, expression of Eya, FMRFa, and Nplp1 is lost. (D) Rescue of hth by hth and (F) of Antp by Antp. Both rescues restore Ap-clusters, as evident from the expression of Eya, FMRFa, and Nplp1. In contrast, whereas cross-rescue of hth by col is successful (E), cross-rescue of Antp by col does not restore Ap clusters (G). The extra Ap cluster neurons observed in cross-rescue of hth by col (E) is an effect of ectopic col in the early NB 5–6 lineage when using this Gal4 driver . (H) Quantification of Eya, Nplp1, and FMRFa positive cells/hemisegment (n>20 hemisegments). Asterisk denote significant difference compared to hth rescue, (p<0.01, Student two-tailed test). Genotypes: (A) w¹¹¹⁸. (B) Antp²⁵/Antp^Ns-rvC12. (C) hth^5E04/hth^Df3R. (D) UAS-hth/+; elav-Gal4,hth^5E04/hth^Df3R. (E) UAS-col/+; elav-Gal4,hth^5E04/hth^Df3R. (F) UAS-Antp/+; elav-Gal4,Antp¹⁴/Antp^Ns-rvC12. (G) UAS-col/+; elav-Gal4,Antp¹⁴/Antp^Ns-rvC12.

Figure 8. Overexpression of homothorax triggers the Ap window by activating collier.

(A and B) Images showing expression of Col and Eya within the dorsal (A1 and B1), intermediate (A2 and B2), and ventral (A3 and B3) part of the NB 5-6T lineage; shown are top views, with anterior up. Cartoons represent the NB 5-6T lineage, based upon lineage mapping data . Small boxes show the expression of Eya and Col in separate panels. In control, Ap cluster neurons coexpress Col and Eya at stage 15 and occupy intermediate (A2) and ventral (A3) layers, close to the neuroblast. In hth overexpression embryos, ectopic Col and Eya expressing cells appear in the intermediate (B2) and dorsal (B1) layers of the NB 5-6T lineage. (C and D) Expression of Eya, Dimm, Nplp1, and FMRFa within single T2 Ap clusters at stage 18 h AEL; shown are side views, with anterior to the left. Small boxes show the expression of Eya, Dimm, Nplp1, and proFMRFa in separate panels. (C) In control, Eya is specifically expressed within the four Ap cluster neurons, and Nplp1/Dimm and FMRFa/Dimm are expressed within Ap1/Nplp1 and Ap4/FMRFa neurons, respectively. (D) When hth is overexpressed within the NB 5-6T lineage, extra Eya, Dimm, and Nplp1 expressing cells appear. (E) Quantification (n>8 VNCs) of Col and Eya cell numbers, at stage 15, and NB 5-6 T lineage cell numbers, at stage 14. (F) Quantification (n>8 VNCs) of Eya, Nplp1, Dimm, FMRFa, and NB 5-6 T lineage cell numbers at stage 18 h AEL. Values as mean number of expressing cells within single NB 5-6T lineages, error bars show SD. Asterisks (*) denote significant difference compared to control (p<0.01, Student two-tailed t-test). Genotypes: (A, C, and E) lbe(K)-Gal4/+. (B, D, and F) UAS-hth/+; lbe(K)-Gal4/+.

Figure 9. Rescue of homothorax at different time points reveals its dual role.

(A and B) Control and hth VNCs reveal the loss of differentiated Ap cluster neurons in hth mutants. However, as shown above, in hth mutants, Ap cluster cells are still present in NB 5-6T. In the abdomen, hth mutants fail to truncate the NB 5-6A lineage, resulting in the appearance of Ap cluster cells. (C and D) Rescue of hth using a late (C) and an early (D) Gal4 driver reveals the dual role of hth. Small boxes show the expression of Eya, Nplp1, and proFMRFa in separate panels. (C) Late expression allows for hth to play its late role in Ap cluster neuron specification, and Ap clusters appear in the majority of abdominal segments, evident by the expression of Eya, Nplp1, and FMRFa. However, using this Gal4 driver, hth is reintroduced too late to truncate the NB 5-6A lineage. (D) In contrast, early expression of hth reintroduces hth early enough to truncate the NB 5-6A lineage, evident by fewer Ap clusters in the abdomen. In addition, in the thoracic segments, hth still can play its late role—specifying Ap cluster neurons—evident by the reappearance of Ap clusters in thoracic segments. (E) Quantification of Ap clusters, defined as the presence of Eya, Dimm, and at least one of the two neuropeptides FMRFa and Nplp1 in abdominal lateral segments (abdominal Ap clusters/VNC; n>7). Asterisk denotes significant difference compared to control, † denotes significant difference compared to elav>hth; hth^−/− (p<0.01; Student two-tailed test). Genotypes: (A) w¹¹¹⁸. (B) hth^5E04/hth^Df3R. (C) UAS-hth/+; elav-Gal4,hth^5E04/hth^Df3R. (D) lbe(K)-Gal4/UAS-hth;hth^5E04/hth^Df3R.

Figure 10. Misexpression of Antp triggers Ap cluster formation in anterior NB 5–6 lineages.

(A and B) Compared to control (A), pan-neuronal misexpression of Antp (B) triggers ectopic formation of Ap-clusters into brain segment B2, as evident by the coexpression of Eya, ap^lacZ, and Nplp1. Small boxes show the expression of Eya, Nplp1, and ap^lacZ expression in separate panels. (C) Misexpression of col triggers ectopic Eya and ap^lacZ expression throughout the anterior CNS. However, despite extensive Eya/ap^lacZ coexpression, we find no evidence of Nplp1 expression, confirming the notion that Antp plays additional roles beyond activating col. (D) Misexpressing Antp in a col mutant background, reveals a complete loss of Ap clusters in thoracic segments (the col mutant phenotype), and a failure to trigger ectopic anterior Ap cluster, evident by the absence of Eya/ap^lacZ/Nplp1 expressing clusters, demonstrating that Antp requires col to trigger Ap cluster formation. Genotypes: (A) ap^lacZ;elav-Gal4/+. (B) ap^lacZ/UAS-Antp;elav-Gal4/+. (C) ap^lacZ/UAS-col;elav-Gal4/+. (D) col³, ap^lacZ/col¹; elav-Gal4/UAS-Antp.

Figure 11. Comisexpression of Antp with grainyhead triggers complete Ap cluster specification in anterior NB 5–6 lineages.

(A and B) Compared to control (A), misexpression of Antp (B) triggers partial Ap clusters, evident by anterior Eya/Nplp1 clusters. However, there is no ectopic expression of FMRFa. Small boxes show the expression of Eya, Nplp1, and proFMRFa expression in separate panels. (C) Misexpression of grh does not trigger ectopic Ap clusters, evident by absence of Eya/Nplp1 cluster. (D) However, co-misexpression of Antp and grh, leads to the appearance of ectopic anterior Ap clusters, evident by the coexpression of Eya/Nplp1 with FMRFa. (E) Quantification of the number of Ap clusters/hemisegment, combined for B2–S3, as defined by the presence of Eya/Nplp1 and Eya/FMRFa (clusters/hemisegment; n>10 CNSs). (F) Individual quantification of the number of cells expressing Eya, Nplp1, and FMRFa, in each of the six brain segments (B2–S3), and the thoracic cluster T2 (cells/lineage, n>10). (G) Control and (H) Antp, grh co-misexpression in Ubx, abd-A, Abd-B triple mutant. In wild type (G), Ap clusters are confined to the three thoracic segments. Small boxes show the expression of Eya, Dimm, Nplp1, and proFMRFa in separate panels. (H) In triple Bx-C mutants that co-misexpress Antp and grh, Ap clusters form throughout the neuroaxis. (G and H) are composed from multiple images. Genotypes: (A) ap^lacZ;elav-Gal4/+. (B) ap^lacZ/UAS-Antp;elav-Gal4/+. (C) ap^lacZ/UAS-grh;elav-Gal4/+. (D) ap^lacZ/UAS-Antp,UAS-grh;elav-Gal4/+. (E and F) Genotypes as in (A–D). (G) C155-Gal4/+. (H) C155-Gal4/y; UAS-Antp, UAS-grh/+; Ubx, abd-A, Abd-B.

Figure 12. Summary of Hox/Pbx/Meis and temporal control of NB 5–6 development.

The NB 5–6 lineage is generated in all CNS segments, but the genetic pathway leading to Ap cluster formation is only triggered in the NB 5-6T lineages. Three separate mechanisms act to ensure this segment-specific event. In abdominal segments, the Pbx/Meis genes hth and exd act with Bx-C Hox genes to truncate the NB 5-6A lineage by triggering neuroblast cell cycle exit within an early temporal (Pdm) window. This occurs prior to generation of Ap cluster cells, and prior to progression into the Cas/Grh late temporal window. In thoracic segments, the absence of Bx-C expression in the NB 5-6T neuroblast allows it to progress further and generate a larger lineage, thereby generating the Ap cluster cells. Importantly, this also allows for the lineage to progress into the Cas/Grh late temporal window. Combined with the expression of the thoracic Hox gene Antp, and increasing levels of Hth, this allows for integration of anteroposterior and temporal cues and the specification of Ap cluster cells into Ap cluster neurons, primarily by the activation of the critical Ap cluster determinant col. Grh plays a postmitotic role in specifying the Ap4/FMRFa cell fate. In anterior segments, the NB 5–6 lineage, although varying in size when compared to thoracic segments, does contain a Cas window. However, the absence of Antp expression, coupled with weak or absent expression of the late temporal gene grh, prevents specification of Ap cluster neurons.