Neuronal Cell Fate Specification by the Convergence of Different Spatiotemporal Cues on a Common Terminal Selector Cascade

Abstract

Specification of the myriad of unique neuronal subtypes found in the nervous system depends upon spatiotemporal cues and terminal selector gene cascades, often acting in sequential combinatorial codes to determine final cell fate. However, a specific neuronal cell subtype can often be generated in different parts of the nervous system and at different stages, indicating that different spatiotemporal cues can converge on the same terminal selectors to thereby generate a similar cell fate. However, the regulatory mechanisms underlying such convergence are poorly understood. The Nplp1 neuropeptide neurons in the Drosophila ventral nerve cord can be subdivided into the thoracic-ventral Tv1 neurons and the dorsal-medial dAp neurons. The activation of Nplp1 in Tv1 and dAp neurons depends upon the same terminal selector cascade: col>ap/eya>dimm>Nplp1. However, Tv1 and dAp neurons are generated by different neural progenitors (neuroblasts) with different spatiotemporal appearance. Here, we find that the same terminal selector cascade is triggered by Kr/pdm>grn in dAp neurons, but by Antp/hth/exd/lbe/cas in Tv1 neurons. Hence, two different spatiotemporal combinations can funnel into a common downstream terminal selector cascade to determine a highly related cell fate.

Fig 1. dAp and Tv1 neurons in the Drosophila VNC.

(A) Lateral view of early embryonic Drosophila CNS, showing NB5-6T in the three thoracic segments (red) and the NB that gives rise to the dAp cells (green). Model of NB5-6T lineage, with the Tv1 neuron (red/blue). (B) Model of late embryonic Drosophila VNC (air-filled trachea [AFT] stage), depicting the Ap clusters in the thoracic segments and the dAp cells located in the segments T1-A10.

Fig 2. Dorsal Ap cells are generated by NB4-3.

(A–H) NB marker expression, either direct antibody stain or βgal stain of lacZ constructs, costained with Eya to identify the progenitor of the dAp cell. (A–F) None of the markers overlap with the Eya expression of the dAp cell. (G) Msh staining overlaps with the Eya expression in the dAp neuron. (H) βgal expression from the hkb-lacZ construct shows overlap with the Eya expression in the dAp cell. (I) Model of a hemisegment during mid embryogenesis, showing the NBs with their different markers. Combination of immunostains for specific markers made it possible to rule out certain NBs as progenitors of the dAp cell. The dAp cell is positive for hkb-lacZ and Msh, which indicates that NB4-3 is the progenitor of dAp cells. (J) NB staining for Msh shows overlap with Dpn and the col-dAp-GFP enhancer, which indicates that the NB generating the dAp cell, is NB4-3. (K) Expression of GFP, Pros, Dpn, and pH3 at St11 and St12 shows a pH3/Pros positive and Dpn negative cells (St11 thick yellow dashed circle) overlapping with the GFP expression from the col-dAp-GFP enhancer construct, which suggests that the dAp cell is born in a type I division. (L) GFP, Kr, and Dpn expression at different stages (yellow dashed circles). At StE11, Dpn and GFP are expressed in the NB, but Kr is not detectable. At St11, the NB 4-3 lineage progresses (based on the GFP signal from the enhancer construct), Dpn is still detectable, but Kr is not expressed. By StL11, two strong GFP positive cells are detectable and GFP expression is evident in a cell in the previous NB location, but without a Dpn signal. (M) GFP, Kr, and Eya expression at St12, St13, and St14 shows that one of the cells expressing strong GFP is the dAp cell and turns on Eya expression by stage 13 (thick yellow dashed circle). (N) Kr is not expressed in the dAp cells at stage 16 (yellow dashed circles). (O) Costaining for GFP and βgal of the col-dAp-GFP enhancer together with grn-lacZ, shows that grain, one of the critical factors for the dAp specification is expressed in the NB4-3 lineage (yellow dashed circles). (P) Staining for Eya and βgal shows that grn is expressed in the dAp cells by stage 14 (yellow dashed circles). (Q) GFP (col-dAp-GFP), Col, and (Nub) Pdm expression at different stages in the NB4-3 lineage reveals that GFP is detectable prior to endogenous Col expression at StL11. At this stage, Nub (Pdm) starts being expressed in the two strong GFP positive cells (thick yellow dashed circles). At StM13 the GFP signal remains strongly expressed; furthermore, Col and Nub (Pdm) are expressed robustly. Of note, Col and Nub (Pdm) expression overlaps in one of the strong GFP positive cells (thick yellow dashed circles). At St14, Col expression is still strong, whereas Nub (Pdm) expression is downregulated (thick yellow dashed circles); Col, GFP, Nub (Pdm) expressing cell is the dAp cell. (R) Nub (Pdm) is not expressed in the dAp cells at stage 15 (yellow dashed circles). (S) GFP (col-dAp-GFP), Col, and Eya show an overlap in the dAp cell at St14. (T) Part of the lineage model of the NB 4–3. At StE11, Kr was not expressed in the NB (L). Still, we find Grn and Nub expression in the NB and the daughter cells. Together with the positive pH3 staining, prior to birth of the dAp cell, this model suggests that the dAp cell is born in a type I division mode by St12 and subsequently activates Col, Eya, and by later stages Dimm and Nplp1. Genotypes: (A) mirr-lacZ. (B) unpg-lacZ. (C) ind-lacZ. (D, E) OregonR. (F) en-lacZ. (G) OregonR. (H) hkb-lacZ/+. (J) col-dAp-GFP; col-dAp-GFP. (L, M, Q, and S) col-dAp-GFP; col-dAp-GFP. (N) OregonR. (O and P) col-dAp-GFP/+; col-dAp-GFP/grn-lacZ. (R) ap-Gal4, UAS-mRFP/CyO.

Fig 3. Early temporal genes are critical for dAp specification.

(A–F) Expression of Eya and Nplp1 in control and temporal mutants, at St16 or AFT. (B) In hb mutants (boxed area), we observe two dorsal Ap cells (yellow dotted circles), which both express Eya, Col, and Dimm. Quantification of Nplp1 positive dAp cells in hb mutants fails since hb mutants do not develop into stage AFT at which Nplp1 is expressed. (C and D) Both Kr and pdm mutants show decreased numbers of Eya and Nplp1 expressing dAp cells (long dotted brackets). (E and F) Eya and Nplp1 expression in cas and grh mutants is not affected. (G–I) Dimm and Col expression shows a loss of both factors in the dAp cells in Kr and pdm mutants (long dotted brackets). (J) Cross-rescue of Kr mutants by UAS-pdm from pros-Gal4 does not rescue Eya expression in dAp cells, but can partially rescue Nplp1 expression. (K) Col and Nplp1 expression in dAp cells of control and Kr mutants expressing pdm from pros-Gal4 shows that pdm can restore the Col and Nplp1 expression in Kr mutants. (L) Quantification of Eya and Nplp1 positive dAp neurons in temporal mutants (n > 10; asterisk denotes p < 0.05; Student´s two-tailed t test; see S1 Data). (M) Genetic model of the dAp specification cascade, showing that the early temporal genes Kr and pdm act to specify the dorsal Ap cell fate. (N) Quantification of Eya and Nplp1 positive dAp neurons in (n > 10 VNC; asterisk denotes p < 0.05; Student´s two-tailed t test; see S1 Data). Numbers of Nplp1 positive dAp neurons in Kr mutants expressing pdm are significantly increased compared to Kr mutants. Genotypes: (A) OregonR. (B) hb^P1, hb^FB. (C, H) Kr¹, Kr^CD. (D, I) Df(2L)ED773. (E) cas^Δ1/ cas^Δ1. (F) grh^IM/grh^IM. (G) OregonR. (J, and K) Kr¹, Kr^CD; pros-Gal4/UAS-nub.

Fig 4. grain is critical for dAp specification.

(A, B) Eya and Nplp1 expression in VNCs at stage AFT. In grn mutants, Eya and Nplp1 expression in dAp cells is almost completely lost (long dotted bracket). (C-F) Expression of Dimm, Col and βgal (ap^rK568) in control and grn mutants. In grn mutants all three markers are strongly downregulated, specifically in dAp cells (long dotted brackets). In contrast, expression in Tv1 cells is unperturbed. (G) Quantification of Nplp1 and Eya expressing dAp cells in control and grn mutant VNCs (n > 10 VNCs; asterisks denote significant difference in grn mutants compared to control; p < 0.05, Student´s two-tailed t test; see S1 Data). Genotypes: (A) OregonR. (B) grn^7L12/grn^SPJ9. (C, E) ap^rK568/+. (D, F) ap^rK568/+; grn^SPJ9/grn^7L12.

Fig 5. Cross-rescue reveals that the primary role of grn is to activate col.

(A-D) Eya and Nplp1 expression in grn mutants cross-rescued with UAS-col, from either pros-Gal4 or elav-Gal4. Both early and late misexpression of col rescues the grn mutant phenotype and results in ectopic expression of Eya and Nplp1 positive dAp cells. (E and F) In contrast, cross-rescue of col mutants with UAS-grn, from either pros-Gal4 or elav-Gal4, fails to rescue the col mutant phenotype (long dotted bracket). (G) Quantification of Eya and Nplp1 positive dAp cells shows a significant increase when col is misexpressed in grn mutants from either pros-Gal4 or elav-Gal4 compared to control VNCs (n = 8 VNCs for elav>col and grn; elav>col for all others, n > 10 VNCs; asterisks denote p < 0.05, Student´s two-tailed t test). (H) Quantification of Eya and Nplp1 positive dAp cells in col mutants with grn misexpression shows that grn does not rescue the col mutant phenotype (n = 7 VNCs for pros>grn, Eya cell quantification; for all others, n > 10 VNCs; asterisks denote p < 0.05, Student´s two-tailed t test; see S1 Data). Genotypes: (A) UAS-col/pros-Gal4. (B) UAS-col/pros-Gal4; grn^SPJ9/grn^7L12. (C) elav-Gal4/UAS-col. (D) elav-Gal4/UAS-col; grn^SPJ9/grn^SPJ9. (E) col¹/col¹, UAS-grn; pros-Gal4/+. (F) col¹/col¹, UAS-grn; elav-Gal4/+.

Fig 6. Cross-rescue reveals that Kr and pdm act upstream of grn and col.

(A–D) Eya and Nplp1 expression in cross-rescue of Kr and pdm mutants with either UAS-col or UAS-grn misexpressed from pros-Gal4, at stage AFT. (A) While the number of Eya expressing dAp neurons in Kr mutants is not restored by misexpression of grn, (B) in pdm mutants misexpression of grn results in partial rescue in numbers of dAp neurons expressing both Eya and Nplp1. (C) Cross-rescue of Kr with col can fully rescue the mutant phenotype and results in ectopic numbers of dAp neurons expressing both Eya or Nplp1. (D) Cross-rescue of pdm with col can fully rescue the pdm mutant phenotype with respect to Eya and Nplp1 positive dAp neurons, and results in ectopic Eya and Nplp1 expression. (E, F) Quantification of Eya and Nplp1 positive dAp neurons from the different rescue experiments (n = 9 VNCs for Kr; pros>grn, n = 8 VNCs for pdm; pros>col. For all others n >10 VNCs; asterisk denotes p < 0.05, Student´s two-tailed t test; see S1 Data). Genotypes: (A) Kr¹, Kr^CD; pros-Gal4/ UAS-grn. (B) Df(2L)ED773; pros-Gal4/UAS-grn. (C) Kr¹, Kr^CD; pros-Gal4/UAS-col. (D) Df(2L)ED773; pros-Gal4/UAS-col.

Fig 7. lbe is critical for Ap cluster formation and Tv1 specification.

(B) Eya, Dimm, and Nplp1 expression in control VNCs at stage AFT, showing that Eya is expressed in all four neurons of the Ap clusters and the dAp cells. Dimm is expressed in two out of four Ap cluster cells and the dAp cells, while Nplp1 is expressed in one neuron (Tv1 cell) of each Ap cluster and the dAp cells. (C) In lbe mutants Eya, Dimm and Nplp1 expression is lost in the Ap clusters, whereas their expression in dAp cells is unchanged. (D–E) GFP (lbe(K)-EGFP), Dpn, Col, and Nab expression in NB5-6T at St14 showing that Col expression is lost in lbe mutants while Nab expression is unaffected. (F, G) GFP, Dpn, Lbe, and Nab expression in NB5-6T at St14 in control and col mutants, showing that Lbe expression is not affected in col mutants. (H, I) GFP, Dimm, Nplp1, and Eya expression in the Ap cluster at stage AFT in control, reveals loss of Eya, Dimm, and Nplp1 in lbe mutants. (J, K) GFP, Lbe, Eya, and Nab expression in the Ap cluster at St16 reveals no effect on Lbe expression in col mutants. (L, M) GFP, Lbe, Col, Dpn expression in NB5-6T at St12 and St13 reveals that Lbe is expressed prior to the onset of Col expression. (N, O) Eya, Col, Dimm, and Nplp1 expression at St17 and AFT in the dAp cells of control and lbe mutants, revealing no difference in cell fate specification with respect to Nplp1 expression. Genotypes: (B) OregonR. (C) lbe^12C005/Df(lbl-lbe)B44. (D, L, M) lbe(K)-EGFP. (E) lbe(K)-EGFP/+; lbe^12C005/Df(lbl-lbe)B44. (F) lbe(K)-EGFP/TTG homozygous. (G) col¹/col³; lbe(K)-EGFP/+.

Fig 8. lbe is critical for Ap cluster formation and Tv1 specification.

(A–C) Expression of Eya, Dimm, and Nplp1 in control and in lbe mutants rescued with UAS-lbe, or cross-rescued with UAS-col, driven from elav-Gal4. Rescue of lbe mutants by misexpression of UAS-lbe can rescue the mutant phenotype in the Ap clusters with respect to Nplp1 expression and gives rise to more Eya positive cells. In contrast, the cross-rescue of lbe by misexpression of UAS-col fails to rescue the Ap cluster expression of Eya, Dimm, and Nplp1. (D–G) Expression of Eya, βgal (ap^rK568), Dimm, and Nplp1. Single misexpression of lbe or col results in some ectopic Eya, βgal (ap^rK568), Dimm, and Nplp1 expression. Co-misexpression of lbe and col results in a dramatic increase of ectopic Eya, βgal (ap^rK568), Dimm, and Nplp1 expression. (H) Model of a potential feed-forward cascade for the Nplp1 specification. lbe both activates col and potentially feeds forward on downstream targets such as eya and ap. Genotypes: (A) OregonR. (B) UAS-col/+; lbe^12C005/Df(lbl-lbe)B44, elav-Gal4. (C) UAS-lbe/+; lbe^12C005/Df(lbl-lbe)B44, elav-Gal4. (D) OregonR. (E) ap^rK568/+; elav-Gal4/UAS-lbe. (F) ap^rK568/+; elav-Gal4/UAS-col.(G) UAS-col/ap^rK568; elav-Gal4/UAS-lbe.

Fig 9. Illustration summarizing the findings.

(Left) Lateral view of the early developing Drosophila embryonic CNS depicting the thoracic NB5-6T, which generates the Tv1 cells, and the NB4-3, which generates the dAp cells. (Middle-right) Our results reveal that the critical terminal selector gene col is activated by different spatio-temporal selector genes acting in the two different NB lineages. In the NB5-6T col is activated by the late temporal gene cas, together with Hox input, via Antp/hth/exd, and lbe. In NB4-3 col is activated by the early temporal genes Kr and pdm and the GATA gene grn. Downstream of col, the Nplp1 activation cascade in the NB5-6T and NB 4–3 lineages is near identical and acts to specify the related cell fate of the dAp and Tv1 cells. In the NB5-6T lineage, we identified two new players involved in the Nplp1 cell fate specification: lbe which activates col and feeds forward onto ap and eya, and Kr, which shows a late onset in the Tv1 cell to maintain col expression. Hence, different spatiotemporal selector genes acting in cells of a different developmental history triggers a common terminal selector cascade via the key entry point gene col.