Autosomal contributions to furrow regulation.

<p>(A) The major <i>Drosophila</i> chromosomes, with the metacentric autosomes indicated in red. (B) Schematic depicting segregation of compound chromosomes generating 2L, 2R, 3L or 3R aneuploidy. (C) Aneuploid 2L furrow dynamics (n = 3). (D) Aneuploid 2R furrow dynamics (n = 4). (E) Aneuploid 3L furrow dynamics (n = 3). (F) Aneuploid 3R furrow dynamics (n≥3). (G) Maximal furrow length of WT, aneuploid 2L, 2R, 3L and 3R during cycles 11–13 (n≥3). *:p<0.05; **: p<0.005; ***: p<0.0005; <i>ns</i>: not significant. (H) Average and maximal Ingression II rates of WT, aneuploid 2L and 2R embryos during cycles 11–13 (n≥3). *:p<0.05; ***: p<0.0005; <i>ns</i>: not significant. (I) Average and maximal Ingression II rates of WT, aneuploid 3L and 3R embryos during cycles 11–13 (n≥3). *:p<0.05; **: p<0.005; ***: p<0.0005; <i>ns</i>: not significant.</p

Differentially-dimensioned furrow formation by zygotic gene expression and the MBT

<div><p>Despite extensive work on the mechanisms that generate plasma membrane furrows, understanding how cells are able to dynamically regulate furrow dimensions is an unresolved question. Here, we present an in-depth characterization of furrow behaviors and their regulation in vivo during early <i>Drosophila</i> morphogenesis. We show that the deepening in furrow dimensions with successive nuclear cycles is largely due to the introduction of a new, rapid ingression phase (Ingression II). Blocking the midblastula transition (MBT) by suppressing zygotic transcription through pharmacological or genetic means causes the absence of Ingression II, and consequently reduces furrow dimensions. The analysis of compound chromosomes that produce chromosomal aneuploidies suggests that multiple loci on the X, II, and III chromosomes contribute to the production of differentially-dimensioned furrows, and we track the X-chromosomal contribution to furrow lengthening to the <i>nullo</i> gene product. We further show that checkpoint proteins are required for furrow lengthening; however, mitotic phases of the cell cycle are not strictly deterministic for furrow dimensions, as a decoupling of mitotic phases with periods of active ingression occurs as syncytial furrow cycles progress. Finally, we examined the turnover of maternal gene products and find that this is a minor contributor to the developmental regulation of furrow morphologies. Our results suggest that cellularization dynamics during cycle 14 are a continuation of dynamics established during the syncytial cycles and provide a more nuanced view of developmental- and MBT-driven morphogenesis.</p></div

Quantitation of furrow dynamics and ingression rates in WT embryos.

<p>(A) Phases of furrow dynamics from cycle 10–13 in WT. The duration of each phase as well as total cycle times are indicated. The pie chart shows the percentage of duration of each phase for the identified cycle. (B) Maximal furrow length in WT embryos for cycles 10–13 (n≥4). (C) Duration of ingression phase in WT cycles (n≥4). (D) Maximal WT furrow ingression rate (n≥4). *:p<0.05; **: p<0.005; <i>ns</i>: not significant. Maximal rates are calculated from a 2 minute rolling window. (E) Average WT furrow ingression rate during Ingression I or Ingression II (n≥4). *:p<0.05; **: p<0.005; <i>ns</i>: not significant. (F) Duration of stabilization phase in cycle 10–13 (n≥4). *:p<0.05; <i>ns</i>: not significant.</p

Regulation of furrow dynamics by X chromosome zygotic loci.

<p>(A) The major <i>Drosophila</i> chromosomes, with the telocentric X chromosome indicated in red. (B) Schematic depicting segregation of compound chromosomes generating X aneuploidy. (C) Aneuploid X chromosome embryo measurements of intact furrow depths (black curve) or deepest extent of fragmented furrows (blue curve) (n = 3). (D) <i>nullo</i> Df embryo measurements of intact furrow depths (black curve) or deepest extent of fragmented furrows (blue curve) (n = 3). (E) Disrupted furrow phenotype in aneuploid X and <i>nullo</i> Df embryos during cycle 13 and slow phase of cycle 14. A region just adjacent to the furrow tips is shown. Scale bar = 5 μm (F) Maximal furrow length in WT, aneuploid X and <i>nullo</i> Df during cycles 11–13 (n≥3). *:p<0.05; **: p<0.005; ***: p<0.0005; <i>ns</i>: not significant. (G) Maximal furrow ingression rate of WT, aneuploid X and <i>nullo</i> Df during cycles 11–13 (n≥3). *:p<0.05; ***: p<0.0005; <i>ns</i>: not significant.</p

Developmental regulation of furrow dimensions and morphologies in the early <i>Drosophila</i> embryo.

<p>(A) Model of syncytial furrows indicating the apical region, furrow, and furrow canal (i). A planar view of the furrow canal regions is also shown (ii), as well as live-imaging data (iii). Scale bar = 5 μm (B) Still images of furrow dynamics from live-imaged cycle 13 embryos (Gap43:mCh) at t = 0min, 2min, 4min, 6min, 8min, 10min, 12min, 14min, 16min, and 18min, and z-planes at 0, -2, -4, -6, and -8μm. z = 0μm is most apical plane, z = -8μm is most basal. Scale bar = 5 μm (C) Wild type furrow dynamics from cycle 10–13 (cycle 10: n = 4; cycle 11: n = 7; cycle 12 and 13: n = 8). (D) Wild type apical actin displacement (GFP:moeABD, black curve), basal actin displacement (grey curve), and furrow dynamics (Gap43:mCh, blue curve) from cycle 10–13 (n = 4). Dashed blue curves are supplemented from independent data for the out-of-view furrow dynamics. Basal actin displacement curves (grey) end due to actin disbandment at anaphase (cycle 10–12) or to actin moving beyond the field of view (cycle 13).</p

Maternal gene decay is a minor contributor to furrow dynamics.

<p>(A) <i>smg</i> mutant furrow dynamics (cycle 11: n = 3; cycle 12 and 13: n = 5). (B) Furrow morphology in WT and <i>smg</i> mutant embryos at 2 min and 10 min. A region just adjacent to the furrow tips is shown. Scale bar = 5 μm (C) Mitotic defects in α-amanitin injected and <i>zld</i> mutant embryos. Chromosomes are labeled with Histone:GFP. Red, dotted lines highlight individual mitotic figures that fail to properly segregate chromosomes resulting in polyploid nuclei, and asterisks indicate missegregating chromosomal complements. (D) Percent of nuclei that experience mitotic defects in WT, α-amanitin injected, and <i>zld</i> mutant embryos. (E) Percent of adjacent nuclear fusion (black bar) and mitotic nuclear fusion (grey bar) in α-amanitin injected and <i>zld</i> embryos (n = 3) in cycle 13. (F) Furrow length and mitotic defects are inversely correlated. The percentage of mitotic defects at cycle 13 in α-amanitin injected, <i>zld</i>, Aneuploid 2L, Aneuploid X, <i>nullo</i>, Aneuploid 2R, and WT embryos is presented, as well as deepest furrow lengths during metaphase in cycle 13. Intact furrow length and corresponding mitotic defects are measured in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007174#pgen.1007174.s003" target="_blank">S3J Fig</a> (G) Model for developmental regulation of furrow dimensions in the early <i>Drosophila</i> embryo.</p

Zygotic gene activation is required for changes in furrow dimensions.

<p>(A) Furrow dynamics for α-amanitin injected embryos during cycle 11–13 (cycle 11 and 12: n = 5; cycle 13: n = 4). (B) <i>zld</i> mutant furrow dynamics from cycle 11–13 (n = 5). (C) Furrow morphology in WT, α-amanitin injected, and <i>zld</i> mutant embryos at 2 min and 10 min in cycle 13. A region just adjacent to the furrow tips is shown. Scale bar = 5 μm (D) Broken furrow phenotype in α-amanitin injected and <i>zld</i> mutant embryos at 6 min and 12 min in cycle 13. A region just adjacent to the furrow tips is shown. Scale bar = 5 μm (E) Maximal furrow ingression rates of α-amanitin injected and <i>zld</i> mutant embryos from cycle 11–13 (n≥4). *:p<0.05; **: p<0.005; ***: p<0.0005; <i>ns</i>: not significant. (F) Total cycle time of α-amanitin injected and <i>zld</i> mutant embryos from cycle 11–13 (n≥4). *:p<0.05; **: p<0.005; ***: p<0.0005; <i>ns</i>: not significant.</p

AGO1, deadenylation complex components, and decapping factors are absent from GW-bodies.

<p>(A-C) Gw foci do not colocalize with AGO1. (D-I) Gw foci also do not colocalize with deadenylation complex components Not1 and Twin. (J-O) Gw does not colocalize with decapping activators Me31B and Hpat. (P) Quantifications of colocalization with Gw foci. A total of 1608, 1801, 1858, 1440, and 1450 Gw particles were counted respectively for AGO1, Not1, Twin, Me31B, and Hpat double labelings with Gw. The Pearson's correlation coefficient (PCC) value (mean ± SD) for AGO1, Not1, Twin, Me31B, and Hpat double labelings with Gw are .011 ± .007, .017 ± .002, .010 ± .004, .013± .003, and .011 ± .006 respectively. All images are from apical sections of cellularizing (stage 5) embryos. Scale bar = 1 μm.</p

Smg foci colocalize with AGO1.

<p>(A-C) Smg foci colocalize with AGO1. (D-I) Smg foci do not colocalize with deadenylation complex components Not1 and Twin. (J-O) Smg does not colocalize with decapping activators Me31B and Hpat. (P) Quantifications of colocalization with Smg foci. A total of 454, 642, 510, 467, and 642 Smg particles were counted respectively for AGO1, Not1, Twin, Me31B, and Hpat double labelings with Smg. The Pearson's correlation coefficient (PCC) value (mean ± SD) for AGO1, Not1, Twin, Me31B, and Hpat double labelings with Smg are .383 ± .160, .039 ± .034, -.005 ± .007, -.005± .005, and .032 ± .022 respectively. All images are from apical sections of cellularizing (stage 5) embryos. Scale bar = 1 μm.</p

GW-bodies are composed of non-translating RNAs.

<p>Live imaging of Gw:EGFP (A,B), Me31B:GFP (C,D), and Twin:GFP (E,F) injected with water, puromycin, or cycloheximide (CHX). (A) Presyncytial blastoderm embryo expressing Gw:EGFP. Puromycin injection does not induce GW-bodies. (B) Cellularizing embryo expressing Gw:EGFP. Puromycin injection induces GW-body formation while cycloheximide injection causes GW-body disintegration. (C) Presyncytial blastoderm embryo expressing ME31B:GFP. Puromycin injection increases P-body size. P-bodies are stable in the presence of cycloheximide. (D) Cellularizing embryo expressing Me31B:GFP. Puromycin injection induces P-body formation while cycloheximide injection causes P-body disintegration. (E) Presyncytial blastoderm embryo expressing Twin:GFP. (F) Cellularizing embryo expressing Twin:GFP. Puromycin injection does not induce the incorporation of Twin into P-bodies (E,F). Scale bar = 5 μm.</p