Lin A/15 as model to study how a stereotyped number of neurons is produced by a NB

(A) Drawing of an adult fly showing the position of the CNS (white (cortex), blue (neuropiles)) and Lin A/15 leg MNs (green cell bodies and dendrites in the VNC and axons in the legs). Black box indicates the VNC imaged in B. VNC: ventral nerve cord.

(B1) Maximum projection of confocal sections of an adult VNC where the six Lin A/15s are genetically labeled with mCD8::GFP (green). (B2) Confocal section of the VNC in B1 immunostained with anti-Elav (neuronal marker, blue) and anti-Repo (glia marker, yellow). T1, T2 and T3 indicate the Prothoracic, Mesothoracic and Metathoracic neuromere respectively. (B3-B4) Confocal section of first left prothoracic neuromere (T1L) (the boxed region in B2), arrowheads and arrows indicate Lin A/15 MN and glia cell bodies, respectively. NG: neuropile glia; NP: neuropile; MN: motor neuron.

(C) Drawing of the anterior region of a third instar larva showing the position of the CNS (white (cortex) blue (neuropiles)) and immature LinA/15 leg MNs (green).

(D1) Maximum projection of confocal sections of the second left thoracic hemisegment (T2L) where Lin A/15 is genetically labeled with mCD8::GFP (green). (D2-D4) Confocal section of the second left thoracic hemisegment (T2L) in (D1) immunostained with anti-Elav (neuronal marker, blue) anti-Dpn (NB marker, cyan) and anti-Repo (glia marker, yellow). Arrowheads, doted arrows and arrows indicate immature Lin A/15 MNs (iMNs), Lin NB and Lin A/15 proliferative glia (PG) respectively. iNP: immature neuropile.

(E1-E2) Plots of the relative position of each Lin A/15 cell from two perspectives: E1 ventral view, E2 lateral view. Axes: Anterior (A), Lateral (L), Ventral (V). Lin A proliferative Glia (PG) are in yellow, Lin A/15 immature MNs (iMNs) are in blue, Lin A/15 GMCs are in white and Lin A/15 NB is in Cyan. Arrows indicate the positions of the confocal sections in (D2-D4).

(F) Graph of the number of Elav+ MNs in a late third instar larva (LL3) vs that in an adult fly.

(G) Schematic of the Lin A/15 type Ib division. NGB: neuroglioblast, NB: neuroblast, IMC: intermediate mother cell, GMC: ganglion mother cell, GB: glioblast, MN: motoneuron. Note 1: the destiny of the MN sister cell during the second phase of division is unknown. Note 2: Lin A/15 development has not been studied during pupal stages.

The MN sibling cells die through PCD during the second phase of Lin A/15 NB division

(A1-F3) Graphs and confocal images showing the development of Lin A/15 during larval stages, the developmental time points are indicated on top. (A1, B1, C1, D1, E1, F1): Graphs of the relative position of each Lin A/15 cells in (A3, B3, C3, D3, E3, F3) from a lateral perspective. Axes: Anterior (A), Ventral (V). Lin A/15 proliferative Glia are in yellow, Lin A/15 immature MNs are in blue, Lin A/15 GMCs are in white, Lin A/15 NB is in Cyan, Lin A/15 cDcp1+ cells are in red. The black lines indicate the positions of the confocal section in (A3-F3). (A2, B2, C2, D2, E2, F2): Maximum projection of confocal sections of the second left thoracic hemisegment (T2L) where Lin A/15 is genetically labeled with mCD8::GFP (green). The boxes in (C2-F2) are confocal sections showing Lin A/15 NB immunostained with anti-Dpn (cyan). Note: in A2-B2 the NB is easily recognizable by its size (arrowheads). (A3, B3, C3, D3, E3, F3): are magnified confocal sections of samples in (A2, B2, C2, D2, E2, F2) immunostained with anti-cDcp1(red), anti-Elav (neuronal marker, blue) and anti-Repo (glia marker, yellow) (A2, B2) or with anti-Dpn (NB marker, cyan) (C2, D2, E2, F2) . Asterisk in (B3, C3, D3, E3, F3) indicate of the cDcp1+ Elav apoptotic cell.

(G1) Maximum projection of confocal sections of a second right thoracic hemisegment (T2R) with a Lin A/15 MARCM clone genetically labeled with mCD8::GFP (green) under the control of tub-Gal4 and mCherry (red) under the control of VGlut-LexA::GAD. (G3) Confocal section of the second right thoracic hemisegment (T2R) (boxed region in G1) immunostained with anti-Elav (blue) and anti-cDcp1 (cyan). The arrowheads indicate the cDcp1+ Elav-VGlut-apoptotic cell. (G2) Graph of the relative position of each Lin A/15 cell (excluding the proliferative glia) in (G1) from a lateral perspective. Axes: Anterior (A), Lateral (L), Ventral (V). Lin A/15 immature MNs are in blue (Elav+), the blue cells surrounded in red are the GFP+ Elav+ Vglut+ immature MNs, the blue cells surrounded in green are the GFP+ Elav+ VGlut-immature MNs (last born MNs) and the cyan cells surrounded in green are the cDcp1+ GFP+ VGlut-Elav-apoptotic cell. Note: the NB (white cell surrounded in green) has been identified by its size. The black line indicates the position of the confocal section in (G2). (G3) Confocal section of the second right thoracic hemisegment (T2R) (boxed region in G1) immunostained with anti-Elav (blue) and anti-cDcp1 (cyan). The arrowheads indicate the cDcp1+ Elav-VGlut-apoptotic cell.

(H) Schematic of the Lin A/15 type Ib division. NGB: neuroglioblast, NB: neuroblast, IMC: intermediate mother cell, GMC: ganglion mother cell, GB: glioblast, MN: motoneuron. The markers used to label each type of Lin A cells are indicated.

Lin A/15 NB decommissions at 24h APF through PCD

(A1-C3) Confocal images showing the development of Lin A/15 during pupal stages, the developmental time points are indicated on top. APF: after pupa formation.

(A1, B1, C1) Maximum projection of confocal sections of the first left thoracic hemisegment (T1L) where Lin A/15 is genetically labeled with mCD8::GFP (green). (A2, B2, C2) Confocal sections showing Lin A/15 immunostained with anti-Elav (blue), anti-Dpn (cyan) and anti-pH3 (red, phospho-Histone3, mitosis-specific marker (A3, B3, C3) magnifications of the boxed region in (A2, B2, C2). Arrowheads indicate the proliferative Lin A/15 NBs (Dpn+ pH3+).

(D1) Maximum projection of confocal sections of three thoracic ganglions (T1, T2, T3) at 24h APF where all 6 Lin A/15s are genetically labeled with mCD8::GFP (green). (D2) Confocal section of thoracic ganglions in (D1) immunostained with anti-Elav (blue), anti-Dpn (cyan) and anti-cDcp1 (red). (D2) magnifications of the boxed region in (D2) (left Mesothoracic neuromere, T2L). Arrowheads indicates the apoptotic Lin A/15 NB (Dpn+ cDcp1+).

(E1-F1) Confocal images showing the absence vs presence of Lin A/15 NB at 28h APF in Control vs P35 OE conditions. 6 Lin A/15s are genetically labeled with mCD8::GFP (green), Lin A/15 NB and MNs are visualized with anti-Dpn (red) and anti-Elav (blue) respectively. (E2-F2) Magnifications of the boxed region in (E1-F1) (first right thoracic hemisegment, T1R) indicate the presence of NB (Dpn+, arrowhead) in P35 OE condition.

(G) Graph of the frequency of NB presence (Number of Lin A/15 samples analyzed is indicated on each bar) at different developmental time points under different genetic conditions: absence of NB (white), NB presence in Control (green), NB presence in P35 OE (purple).

(H) Schematic of the Lin A/15 type Ib division during larval and pupal stages. NGB: neuroglioblast, NB: neuroblast, IMC: intermediate mother cell, GMC: ganglion mother cell, GB: glioblast, MN: motoneuron. The markers used to label each type of Lin A/15 cells are indicated.

Spatio-temporal expression of Imp/Syp in Lin A/15

(A1-J2) Confocal images showing the development of Lin A/15 during larval and pupal stages, the developmental time points are indicated on top. ALH: after larva hatching; APF: after pupa formation. (A1, B1, C1, D1, E1, F1, G1, H1, I1, J1) Maximum projection of confocal sections of the second left thoracic hemisegment (T2L) where Lin A/15 is genetically labeled with mCD8::GFP (white). (A2, B2, C2, D2, E2, F2 G2, H2, I2, J2) Magnified confocal sections of boxed regions in (A1, B1, C1, D1, E1, F1, G1, H1, I1, J1) showing Lin A/15 NB in immunostained with anti-Elav (neuronal marker, blue), anti-Dpn (NB marker, cyan) and anti-Imp (red) (A2, B2, C2, D2, E2) or anti-Syp (yellow) (F2 G2, H2, I2, J2). The dashed lines indicates GFP labeled cells including NB and new-born MNs. White arrowheads indicate Lin A/15 NB.

(K) Schematic of the Lin A/15 type Ib division correlating Imp/Syp protein level and spatio-temporal PCD pattern in Lin A/15 cells. NGB: neuroglioblast, NB: neuroblast.

Opposite temporal expressions of Imp/Syp control the timing of Lin A/15 NB decommissioning

(A1-O2) Confocal images showing the development of WT (A1-C2), Imp RNAi (D1-F2), from pupal stages until the adult stage.

(A1, B1, C1, D1, E1, F1) Maximum projection of confocal sections of the three right thoracic hemisegments (T1R, T2R, T3R) during pupal stages where Lin A is genetically labeled with mCD8::GFP (green). (A2, B2, C2, D2, E2, F2) Confocal sections of boxed regions in (A1, B1, C1, D1, E1, F1) immunostained with anti-Elav (neuronal marker, blue) and anti-Dpn (NB marker, cyan). The developmental time points are indicated on the top of each panel. Arrowheads indicate the presence of NB (Dpn+).

(G) Graph of the frequency of NB presence (Number of Lin A/15 samples analysed is indicated on each bar) at different developmental time points under different genetic conditions: absence of NB (white), NB presence in Control (green)and NB presence in Imp RNAi (orange°.

Opposite temporal expressions of Imp/Syp control the timing of Lin A/15 NB decommissioning

(A1-I2) Confocal images showing the development of WT (A1-C2), Imp OE (D1-F2) and Syp RNAi Lin A/15 (G1-I2) from pupal stages until the adult stage.

(A1, B1, C1, D1, E1, F1, G1, H1, I1) Maximum projection of confocal sections of the three right thoracic hemisegments (T1R, T2R, T3R) during pupal stages or in adult VNCs where Lin A is genetically labeled with mCD8::GFP (green). (A2, B2, C2, D2, E2, F2, G2, H2, I2) Confocal sections of boxed regions in (A1, B1, C1, D1, E1, F1, G1, H1, I1) immunostained with anti-Elav (neuronal marker, blue) and anti-Dpn (NB marker, cyan). The developmental time points are indicated on the top of each panel. Arrowheads indicate the presence of NB (Dpn+).

(J) Graph of the frequency of NB presence (Number of Lin A/15 samples analysed is indicated on each bar) at different developmental time points under different genetic conditions: absence of NB (white), NB presence in Control (green), NB presence in Imp RNAi (orange), NB presence in Imp OE (blue) and NB presence in Syp RNAi (purple).

The last-born Lin A/15 MNs are eliminated by PCD during early pupal stages

(A1-C3) Graphs and confocal images showing the PCD pattern in Lin A/15 MNs during pupal stages, the developmental time points are indicated on top.

(A1, B1, C1) Graphs of the relative position of each Lin A/15 cell of the boxed regions in (A2, B2, C2) from a lateral perspective. Axes: Anterior (A), Ventral (V). Lin A/15 immature MNs are in blue, Lin A/15 GMCs are in white, Lin A/15 NB is in Cyan and cDcp1+ MNs are in red and blue. The black lines indicate the positions of the confocal sections in (A3, A4, B3, B4, C3, C4).

(A2-C3) Confocal sections of Lin A/15 genetically labeled with mCD8::GFP (green). The confocal sections showing the Elav+, cDcp1+ cells immunostained with anti-Dpn (cyan), anti-Elav (blue) and anti-cDcp1 (red) are indicated in (A1, B1, C1). Arrowheads indicate the apoptotic MNs (Elav+, cDcp1+).

(D1) Maximum projection of confocal sections of three left thoracic hemisegments at 5 hour APF where Lin A/15 is genetically labeled with mCD8::GFP (green). (D2, D3, D4) Confocal sections of the three left thoracic hemisegments (boxed regions in (E1)) immunostained with anti-cDcp1(red), anti-Elav (neuronal marker, blue) and Edu (yellow). Arrowheads indicate the Elav+, cDcp1+ Edu+ cells. Note: the larvae were fed with Edu from 74 to 96 h ALH to label only the last born motoneurons with Edu (close to the NB). The cDcp1+ Elav+ cells are always Edu+. (D5, D6, D7) Graphs of the relative position of each Lin A/15 cell of the boxed regions in (D1) from a lateral perspective. Axes: Anterior (A), Ventral (V). Lin A/15 immature MNs are in blue, Lin A/15 GMCs are in white, Lin A/15 NB is in Cyan and Elav+, cDcp1+ cells are in red and blue, Edu+ cells are circled in yellow. Note: the last born MNs and the GMCs as well as NB are all Edu+ (circled yellow) and the apoptotic Elav+ cDcp1+ cells is always part of this population of Edu + cells demonstrating that the last born MNs are dying.

(E) Graph of the number of Edu+ Lin A/15 MNs at 5h APF and 17h APF, of which larvae are fed with Edu from 74 to 96 hours ALH. Note: the number of Edu+ cells neurons decrease significantly between 5h and 17h APF demonstrating that the last born MNs are eliminated.

(F) Graph of the number of Elav+ VGlut+ Lin A/15 cells in third instar larvae (LL3) and adults (Adult) of WT vs VGlut>P35 Lin A/15 MARCM clones.

(G-J) Maximum projection of confocal sections of the left T1 segment (T1L) of third instar larva (G-H) and the left prothoracic neuromere (T1L) in adult fly (I-J) containing a WT (G, I) or a VGlut>P35 (H,J) Lin A/15 MARCM clone. The boxed regions inserted in each picture in (G-J) indicate the Elav+ GFP+ cells.

(L) Graph of the number of Elav+ Lin A/15 neurons at different developmental time points.

(M) Schematic of the Lin A/15 type Ib division during larval and pupal stages. NGB: neuroglioblast, NB: neuroblast, IMC: intermediate mother cell, GMC: ganglion mother cell, GB: Glioblast, MN: motoneuron.

The opposite expression pattern in immature neurons of Imp/Syp instructs the number of surviving MN

(A1) Maximum projection of confocal sections of the T1R hemisegment where Lin A/15 is genetically labeled with mCD8::GFP (green) at 5h APF. (A2, A3, A4, A5) are confocal sections of the Lin A/15 in (A1) showing the apoptotic MN immunostained with anti-Imp (red), anti-Elav (blue) and anti-cDcp1 (cyan) of (A1). Arrowheads indicate the apoptotic MN (Elav+, cDcp1+) is absent of Imp (Imp-).

(B1-B2) Graph of the number of apoptotic MNs (Elav+, cDcp1+) observed in Lin A/15 at different developmental time points under different genetic conditions: Control (green), Imp RNAi (orange), Imp OE (blue) and Syp RNAi (purple).

(C1, D1, E1, F1, G1, H1, I1, J1) Maximum projection of confocal sections of the second right thoracic hemi-segments (T2R) where Lin A/15 is genetically labeled with mCD8::GFP (green) under different genetic conditions control (C1, E1, H1), Imp RNAi (D1), Imp OE (F1, I1,) and Syp RNAi (G1, J1). (C2, D2, E2, F2, G2, H2, I2, J2) Confocal sections of the second right thoracic hemisegment (T2R) in (C1, D1, E1, F1, G1, H1, I1, J1) immunostained with anti-cDcp1(red), anti-Elav (neuronal marker, blue and anti-Dpn (NB marker, cyan). The boxed region in (D2, E2, H2, J2) indicated the presence of Elav+ cDcp1+ cells. (C3, D3, E3, F3, G3, H3, I3, J3) Graphs of the relative position of each Lin A/15 cell in (C1, D1, E1, F1, G1, H1, I1, J1) from a lateral perspective. Axes: Anterior (A), Ventral (V). Lin A/15 immature MNs are in blue, Lin A/15 GMCs are in white, Lin A/15 NB is in Cyan and cDcp1+ Elav+ neurons are in red and blue.

(K) Graph of the number of Elav+ Lin A/15 neurons observed in adult flies under different genetic conditions: control (green), Imp RNAi (orange), Imp OE (blue) and Syp RNAi (purple).

(L1-M1) Maximum projection of confocal sections of the left prothoracic neuromere (T1L) containing a control (L1) or a VGlut>Imp (M1) Lin A/15 MARCM clone. The boxed regions in (L1-M1) are confocal sections showing the Lin A/15 Elav+ (anti-Elav, Blue) GFP+ cells. (L2-M2) Graphs of the relative position of each Lin A/15 cell in (L1, M1) from a lateral perspective. Axes: Anterior (A), Ventral (V). Lin A/15 immature MNs are in blue.

(N) Graph of the number of Elav+ VGlut+ Lin A/15 cells of control and VGlut>Imp Lin A/15 MARCM clones in third instar larvae (LL3) and adult flies.

The last-born MNs eliminated by PCD are primed with a specific combination of TFs under control of Imp and Syp

(A1, B1, C1, E1, F1, G1, I1, J1, K1) Plots of the relative position of each Lin A/15 cell from a lateral perspectives showing the expression of Jim (A1, B1, C1,), RunXA (E1, F1, G1), and Nvy (I1, J1, K1) in purple, orange and green respectively, the Elav+ MNs (blue), the NB (cyan) and the GMC in control (A1, E1, I1,), Imp OE (B1, F1, J1) and Syp RNAi (C1, G1, K1).

(A2-A3, B2-B3, C2-C3, E2-E3, F2-F3, G2-G3, I2-I3, J2-J3, K2-K3) confocal sections showing the expression of Jim (A2-A3, B2-B3, C2-C3,), RunXA (E2-E3, F2-F3, G2-G3,), and Nvy (I2-I3, J2-J3, K2-K3) in RED, the Elav+ MNs (blue), the Dpn+ NB (cyan). The position of the sections are shown in (A1, B1, C1, E1, F1, G1, I1, J1, K1). The asterisk indicate the NBs. Note 1: The arrowhead in B2 indicates a jim+ neurons close to NB, this is never seen in the control LinA/15. Note 2: The arrowheads in E2 indicate RunXA+ neurons close to NB, this is never seen in ImpOE and Syp RNAi LinA/15. Note3: the arrowheads in I2, J2 and K2 indicate NVy + neurons close to the NB, the expression of Nvy is barely detectable in ImpOE and Syp RNAi LinA/15.

(A4, B4, C4, E4, F4, G4, I4, J4, K4) Graphs of the frequency of Jim (A4, B4, C4), RunXA (E4, F4, (G4,) and Nvy (I4, J4, K4) expression as a function of x’ (x’: MN ordering axis according to their relative distance from the NB among >15 specimens before (gray bare) and after (colored lines) applying a Savitzky-Golay filter (see material and methods) in control (A4, E4, I4), Imp OE (B4, F4, J4) and Syp RNAi (C4, G1, K4). The horizontal bar indicates the Jim+ cell cluster detected with the PCCD method.

(D1-D2, H1-H2, L1-L2) Graphs of the number of Elav+ Lin A/15 MNs (D1, H1 and L1) and graphs of the number of Elav+ Lin A/15 MNs expressing Jim (D2), RunXA (H2) and Nvy (L2) in control, Imp OE and Syp RNAi LinA/15.

(M) Schematic of the TF codes expressed in each iMN predicted by the PCCD method in an L3 larva in control, Imp OE and Syp RNAi LinA/15). Bottom: schematic of the cell body of Lin A/15 iMNs. The numbers inside indicate their relative distances from NB. Top: the horizontal bars indicate the TF+ cell clusters detected with the PCCD method. The dotted lines indicate the coverage index at the border (see material and methods).

Note 1: The arrowheads in B2 indicate a Jim+ neurons

Changing the combination of TF in last-born MNs leads to MN survival

(A1-D3) Maximum projection of confocal sections of a right prothoracic hemisegment (T2R) with a Lin A/15 MARCM clone genetically labeled with mCD8::GFP (green) under the control of VGlut-Gal4 (A1, B1, C1, D1) and confocal sections trough LinA/15 (green) and labeled with Elav (red) (A2-A3, B2-B3, C2-C3, D2-D3) in different genetic conditions: control (A2-A3), nvy-/- (B2-B3), UAS-jim (C2-C3), nvy-/-; UAS-jim (D2-D3).

(E) Graphs of the number of Elav+ VGlut+ MNs in control, nvy-/- (B2-B3), UAS-Jim (C2-C3), nvy-/-; UAS-jim (D2-D3) MARCM clones.

The MN sibling cells die through PCD during the second phase of Lin A/15 NB division

(A1-C2) Lin A/15 during pupal stages: 0 hour APF (A1-A2), 8 hours APF (B1-B2), 24 hours APF (C1-C2). (A2, B2, C2): Maximum projection of confocal sections of the third left (T3L) (A1), first right (T1R) (B1) and second right (T2R) (C1) thoracic hemisegments where Lin A/15 is genetically labeled with mCD8::GFP (green). (A2, B2, C2): Confocal sections of the corresponding Lin A/15 in (A1, B1, C1) immunostained with anti-cDcp1 (red), anti-Elav (neuronal marker, blue) and anti-Dpn (NB marker, cyan), boxed region showing the dying sibling cells (Elav-, cDcp1+), indicated with arrowheads.

The volume of the Lin A/15 NB continuously decrease during development

(A) Graph of the volume of the Lin A/15 NB at different time point. X axis: different time points during larval and pupal stages, Y axis: volume of the NB in nanom3.

Lin A/15 NB does not enter into autophagy

(A1-B3) Lin A/15 during pupal stages: 17 hours APF (A1-A3), 20 hours APF (B1-B3). (A1, B1) Maximum projection of confocal sections of the first two thoracic hemisegments where Lin A/15 is genetically labeled with mCherry (red). (A2-A3, B2-B3): Confocal sections of the boxed regions in (A1, B1) corresponding to the second right thoracic hemisegment (T2R). Lin A/15 is inmunostained with anti-Elav (neuronal marker, blue) and anti-Dpn (NB marker, cyan) and genetically labeled with Atg8::GFP . Arrowheads indicate the NBs.

(C1-C3) Lin A/15 at 23 hours APF. (C1) Maximum projection of confocal sections of the first two thoracic hemisegments where Lin A/15 is genetically labeled with GFP (green). (C1-C3): Confocal sections of the boxed regions in (C1) corresponding to the second right thoracic hemisegment (T2R). Lin A/15 is immunostained with anti-Elav (neuronal marker, blue) and anti-Dpn (NB marker, cyan) and labeled with Lysotracker. Arrowheads indicate the NBs.

Syp OE d not change the number of Mns produced by Lin A

(A-B) Maximum projection of confocal sections of the prothoracic segments with one (A) wt or two syp OE (B) Lin A/15 MARCM clones (mcherry, red) labeled with Elav (green). Graph of the number of Elav+ VGlut+ Lin A MNs in control and Syp OE MARCM clones.

No mutual inhibition between Imp and Syp in Lin A/15

(A1-D3) Graphs and confocal images showing Syp level at 94-97 AEL in Imp RNAi (A1-B3) and Imp level at 120 AEL in Syp RNAi (C1-D3), AEL: after egg laying. (A1, B1, C1, D1) Graphs of the relative position of each Lin A/15 cells of samples in (A2-D2) from a lateral perspective. Axes: Anterior (A), Ventral (V). The black lines indicate the positions of the confocal section in (A3-D3). (A2, B2, C2, D2) Confocal sections comparing Syp expression in Lin A/15 NB between WT (A2) and Imp RNAi (B2); Imp expression in Lin A/15 NB between control (C2) and Syp RNAi (D2). Stainings: anti-Dpn (NB marker, cyan) and anti-Imp (red) and anti-Syp (yellow). Boxed regions highlight Imp expression in Nbarrowheads indicated Lin A/15 NB, dashed lines indicate GFP labeled NB. (A3, B3, C3, D3) Confocal sections comparing Syp expression in Lin A/15 progenies between control (A3) and Imp RNAi (B3); Imp expression in Lin A/15 progenies between control (C3) and Syp RNAi (D3). Boxed regions highlight Syp expression in MNarrowheads indicated Lin A/15 progenies, dashed lines indicates GFP labeled cells. (E) Graph showing the number of Lin A/15 cells (left panels in each graph) and number of Syp/Imp-expressing Lin A/15 progenies (right panel in each graph) in indicated genetic backgrounds.