Combinatorial action of Grainyhead, Extradenticle and Notch in regulating Hox mediated apoptosis in <i>Drosophila</i> larval CNS

<div><p>Hox mediated neuroblast apoptosis is a prevalent way to pattern larval central nervous system (CNS) by different Hox genes, but the mechanism of this apoptosis is not understood. Our studies with Abdominal-A (Abd-A) mediated larval neuroblast (pNB) apoptosis suggests that AbdA, its cofactor Extradenticle (Exd), a helix-loop-helix transcription factor Grainyhead (Grh), and Notch signaling transcriptionally contribute to expression of RHG family of apoptotic genes. We find that Grh, AbdA, and Exd function together at multiple motifs on the apoptotic enhancer. <i>In vivo</i> mutagenesis of these motifs suggest that they are important for the maintenance of the activity of the enhancer rather than its initiation. We also find that Exd function is independent of its known partner homothorax in this apoptosis. We extend some of our findings to Deformed expressing region of sub-esophageal ganglia where pNBs undergo a similar Hox dependent apoptosis. We propose a mechanism where common players like Exd-Grh-Notch work with different Hox genes through region specific enhancers to pattern respective segments of larval central nervous system.</p></div

Grh, Abd-A and Notch transcriptionally regulate apoptotic enhancer.

<p>(A-D) Shows <i>F3B3-lacZ</i> levels in abdominal pNBs in response to <i>grh</i> (B), <i>abd-A</i> (C) and <i>Notch</i> (D) knockdown compared with pNB blocked for apoptosis by p35 expression (A). (E) Shows the plot of LacZ intensities quantitated and compared from VNCs where abdominal pNBs are blocked from undergoing death by expression of p35 versus VNCs with <i>grh</i>, <i>abd-A</i> and <i>Notch</i> knockdown. (F-G) Comparison of control and Abd-A over expressed larval VNCs with <i>F3-lacZ</i>. (F) Shows basal level of <i>F3-lacZ</i> expression in thoracic NBs. (G) Shows induction of <i>F3-lacZ</i> (white channel) in additional cells in response to ectopic expression of AbdA in thoracic segments of CNS. The dotted lines in these panels enclose abdominal segments of larval VNC which normally express Abd-A. (H) Shows enlarged thoracic region of VNC shown in panel-G. Induction of <i>F3-lacZ</i> and Abd-A in Dpn marked pNB in thoracic segments is shown. The dotted lines in these panels enclose thoracic segments of larval VNC. Yellow arrowheads indicate pNBs. Abdominal and Thoracic segments are indicated as “A” and “T”. Average values are shown as central lines in error bars. Error bars indicate standard deviation.</p

Hox-Grh expression code in pNBs and progeny is same for Dfd-SEG and abdominal segments.

<p>(A) Schematic of L2 VNC showing the extent of Dfd-SEG region as seen by Dfd staining <i>in vivo</i>. Ten pNBs in L2 stage is shown as green filled circles. Approximate location of six pNBs surviving in L3 stage are shown as four filled red circles (showing SA1 and SA2 lineages indicated as 1 and 2) and two hollow red circles (representing SA3 lineage indicated as 3). Approximate location of four ectopic lineages when death is blocked by p35 expression from L1 are shown by hollow blue circles (<i>ect1</i>^<i>Dfd</i> and <i>ect2</i>^<i>Dfd</i> are indicated as e1 and e2) (B-C) Show that pNB in Dfd-SEG region of early L2 and late L3 larval VNCs express Grh. (D-E) Shows that Hox-Grh code of the pNB (Hox^-/Grh⁺) and progeny (Hox⁺/Grh^-) is same in abdominal and Dfd-SEG region. GFP marked lineage is enclosed by a dotted line. Biggest cell in the entire lineage is pNB which is Grh⁺, smaller cells are progeny neurons which are Hox⁺ and Grh^-. pNB death in abdominal segments is blocked by <i>gal80</i>^<i>ts</i><i>; inscGAL4</i> driven UAS-p35 expression specifically in larval stages. (F) Plot shows that all the pNB marked by Dpn in EL2 (10) and LL3 (6) are Grh⁺. Small “n” indicates number of larval VNCs counted. All larval VNCs expressed <i>inscGAL4</i> driven <i>UAS-mCD8-GFP</i> from embryonic stages. Yellow arrowheads indicate pNBs. Average values are shown in middle of bars. Error bars indicate standard deviation.</p

<i>Enhancer-lacZ</i> expression sustains till late L3 stage in abdominal NBs.

<p>(A-B) Show expression of <i>NBRR F3</i> and <i>F4 enhancer-lacZ</i> lines in abdominal NBs resulting from block of apoptosis in <i>M22/MM3</i> transheterzygotic deletion combination. (C) Show that pNBs resulting from blocking of death by expression of p35 in L1 stage (t-shift as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007043#pgen.1007043.s008" target="_blank">S8A Fig</a>) express <i>F3B3-lacZ</i>. Yellow arrowheads indicate pNBs.</p

Exd but not Hth plays an important role in abdominal pNB apoptosis.

<p>(A-B) Show abdominal MARCM clone for Exd (<i>exd</i>^<i>1</i>) and Hth (<i>hth</i>^<i>P2</i>) mutations. pNB is seen in <i>exd</i>^<i>1</i> mutant clone marked by Dpn (A’) but <i>hth</i>^<i>P2</i> clone doesn’t show any surviving pNB (B). (C) Knockdown of Exd using RNA interference in Dfd-SEG region results in ectopic pNBs. Yellow and white arrowheads indicate ectopic NBs.</p

AbdA-Exd-Grh and Su(H) binding sites are required for maintenance of the enhancer.

<p>(A) Shows a comparative schematic of 1Kb <i>F3B3-lacZ</i>, <i>717-lacZ</i> and its mutant versions <i>717-Grh</i>^{<i>mutant</i>}<i>-lacZ</i>, <i>717-HEG</i>^{<i>mutant</i>}<i>-lacZ and 717-Su(H)</i>^{<i>mutant</i>}<i>-lacZ</i>. (B) Vl pNBs resulting from blocking of death by expression of p35 in L1 stage (t-shift as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007043#pgen.1007043.s008" target="_blank">S8A Fig</a>) shows expression of <i>717-lacZ</i> in late L3 stage. (C-E) Mutant versions <i>717-Grh</i>^{<i>mutant</i>}<i>-lacZ</i> (C), <i>717-HEG</i>^{<i>mutant</i>}<i>-lacZ</i> (D) <i>and 717-Su(H)</i>^{<i>mutant</i>}<i>-lacZ</i> (E) show no lacZ expression at the same stage. Yellow arrow arrowheads indicate Vl-pNBs. “G” written within green motifs indicate that only Grh binding sites found in the motif are mutated. “X” sign on green motifs indicate that all Hox-Exd and Grh binding sites found within motif are mutated. “X” sign within blue [Su(H)] motifs indicate that Su(H) binding site is mutated. Details of specific mutation are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007043#pgen.1007043.s011" target="_blank">S1 Text</a>.</p

Model for pNB apoptosis.

<p>(A) Regulation of apoptotic genes <i>grim</i> and <i>reaper</i> in abdominal and Dfd-SEG happen through two distinct enhancers. Enhancer for Dfd-SEG is yet to be identified but lies outside <i>MM3</i> and is arbitrarily shown 5’ to <i>MM3</i>. (B) Model for regulation of RHG genes in abdominal pNBs. Grh, Hox-Exd and Notch signaling play a direct role in regulation of RHG genes through 1Kb <i>F3B3</i> enhancer. (C) An approximate schematic suggesting possible assembly of AbdA-Exd-Grh-DNA tetracomplex on motif-30. The Hox, Exd and Grh binding sites are indicated as H1, H2, G1, G2 and Exd. The table below the schematic is for various mutant oligos and has (X) indicating that binding is abrogated, (√) indicating the binding is present and (X/√) indicating that binding is reduced.</p

Enhancer for abdominal NB apoptosis lies in <i>NBRR F3</i> and <i>F4</i> overlap.

<p>(A) Schematic showing 22kb <i>NBRR</i> region with approximate extents of <i>M22</i> deletion and overlapping genomic fragments used for making <i>enhancer-lacZ</i> lines. MiMIC transposon used to generate the <i>M22</i> deletion is inserted at approximate 9kb from 5’ end of <i>NBRR</i>, indicated by blue arrowhead. (B-E) Show mid L3 larval VNCs for <i>NBRR F3</i>, <i>F4</i>, <i>F3B3</i> and <i>717 enhancer-lacZ</i> lines. Yellow arrowheads indicate pNBs.</p

AbdA-Exd are critical for tetracomplex assembly on motif-30.

<p>(A) DNA Sequence for motif-30 is shown in capital letters and various mutation in this sequence are shown in small case. (B) EMSA with Exd binding site mutant oligo (45–52) show disruption of Hox-Exd complex (lane 50) and tetra-complex (lane 52), while Grh binding (lane 46) and Hox-Grh binding are still present (lane 51). (C) EMSA with oligo mutant for Hox1 binding site (lane 58–65) show that Hox-Exd (lane 63) and Hox-Grh binding is slightly reduced (lane 56 vs 64) but tetra-complex is dramatically reduced (lane 57 vs 65). (D) EMSA with oligo mutant for Hox2 binding sites (lane 72–77) show that Hox binding is completely disrupted on DNA (lane 74) while Grh binding on DNA is normal (lane 73) and tetracomplex binding is largely abolished (lane 77).</p

Role of Notch in pNB apoptosis is independent of Grh.

<p>(A-B) Show abdominal pNB MARCM clone for Notch mutation (<i>N</i>^<i>55e11</i>). Grh expression is found to be normal (panel-A) and Abd-A is found to be expressed (panel-B) in pNBs. (C) Shows that Grh expression is unaffected in ectopic pNBs obtained in Dfd-SEG region in response to Notch knockdown by RNA interference. (D) Simultaneous knockdown of Notch (by RNA interference) and over expression of Abd-A in abdominal pNBs block their apoptosis. (E) Plot showing a comparison of the total number of pNBs in Dfd-SEG and abdominal region of larval VNCs in wild type and Notch knockdown by RNA interference. “n” indicate the number of VNCs counted for each genotype. Average values are shown in bars. Error bars are standard deviations.</p