Supplementary information from Zebrafish lipid droplets regulate embryonic ATP homeostasis to power early development

In zebrafish embryos, the maternally supplied pool of ATP is insufficient to power even the earliest of developmental events (0–3 hpf) such as oocyte-to-embryo transition (OET). The embryos generate additional pulse (2.5 h long) of ATP (1.25–4 hpf) to achieve the embryonic ATP homeostasis. We demonstrate that the additional pulse of ATP is needed for successful execution of OET. The maternally supplied yolk lipids play a crucial role in maintaining the embryonic ATP homeostasis. In this paper, we identify the source and trafficking routes of free fatty acids (FFAs) that feed the mitochondria for synthesis of ATP. Interestingly, neither the maternally supplied pool of yolk-FFA nor the yolk-FACoA (Fatty Acyl Coenzyme A) is used for ATP homeostasis during 0–5 hpf in zebrafish embryos. With the help of lipidomics, we explore the link between lipid droplets (LDs) mediated lipolysis and ATP homeostasis in zebrafish embryos. Until 5 hpf, the embryonic LDs undergo extensive lipolysis that generates FFAs. We demonstrate that these newly synthesized FFAs from LDs are involved in the maintenance of embryonic ATP homeostasis, rather than the FFAs/FACoA present in the yolk. Thus, the LDs are vital embryonic organelles that maintain the ATP homeostasis during early developmental stages (0–5 hpf) in zebrafish embryos. Our study highlights the important roles carried on by the LDs during the early development of the zebrafish embryos

Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos

<div><p>Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1–8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis.</p></div

Redistribution of blastomeric Myo1C upon MyoI inhibition by PClP.

<p>(A) 3D rendered 100 μm cross section of Myo1C immunostaining profile in control 1 hpf embryo (inset- single confocal slice) (Bi) Normalized immunostaining intensity along the line drawn in Fig 4A inset, n = 5 embryos (Bi, top panel). Actin distribution at the same time, redrawn from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180301#pone.0180301.g003" target="_blank">Fig 3A</a>, left panel, n = 5 (Bi, middle panel). Membrane distribution profile perpendicular to surface of embryo shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180301#pone.0180301.g003" target="_blank">Fig 3D</a>, control)\, (Bi, bottom panel). (Bii) cartoon representation of Myo1C distribution in blastodisc cortex. (C) 3D rendered in 100 μm cross section of Myo1C immunostaining profile in 30 min PClP treated 1 hpf embryo, inset single confocal slice. (D) Comparative normalized intensity calculated long lines drawn in insets of (A&C)-slice views, control-cross, Myo1 inhibited-box, n = 5,. (E) Myo1C profiles for control 2 hpf, (F) Myo1 inhibited 2 hpf and (G) Normalized immunostaining intensity long lines drawn in insets of (E&F)-slice views, control (cross), Myo1 inhibited (box), n = 5, error bar indicates -standard deviation everywhere in Fig 4.</p

Comparison between role of Myo2 and Myo1, in furrow formation and LD dynamics.

<p>(A) Sideview, time-lapse profile of furrow maturation in control (top panel), Blebbistatin treated/Myo2 inhibited (middle panel) and PClP treated/Myo1 inhibited embryos (bottom panel). White dashed arrows in top panel and bottom panel indicate the formation of third furrow (marked as 3), parallel to the first furrow (marked as 1). Filled white arrows in bottom panel indicate the dissolving first-furrow and LD accumulation at that site, in Myo1 inhibited embryo. Filled white arrow in middle panel indicates dis-localization of second and third furrow. Bar 100 μm. (B) Top-view time-lapse profile of furrow maturation in control (top panel), Myo2 inhibited (middle panel) and Myo1 inhibited embryos (bottom panel). Arrows in bottom panel indicate LD accumulation along dissolving first furrow in Myo1 inhibited embryo. Bar 50 μm. (C) Cartoon diagram of embryo with (i) top and (ii) sideview position shown by eye symbol. (D) Scanning electron microscopy showing embryo blastomere surface of 64 cell control (top panel), equivalent time Myo2 inhibited (middle panel) and equivalent time Myo1 inhibited embryo (bottom panel). Bar 100 μM.</p

LDs gradually accumulate at the cleavage furrow of PClP treated embryo.

<p>(A-C) Lateral views of embryo, as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180301#pone.0180301.g002" target="_blank">Fig 2A</a>, Zoomed in on first cleavage furrow, (A) Control embryo from approximately 40 min post fertilization, montage of every 5 min, arrows indicates LDs near the first cleavage furrow. (B) Myo1 inhibited embryo from approximately 40 min post fertilization, montage of every 5 min. Arrows indicate accumulation of LDs at the first cleavage furrow line. (C) Myo2 inhibited embryo from approximately 40 min post fertilization, montage of every 5 min. Arrows indicate LDs near the first cleavage furrow. (D) Nile red staining of first cleavage furrow with lipid droplets shown by arrow in control embryo. (E) Nile red staining of first cleavage furrow with lipid droplet clump shown by arrow in Myo1 inhibited embryo. (F) Control, (G) Myo1 inhibited and (H) Myo2 inhibited embryos, kymograph view of average intensity along 10 μm line on both sides of the first cleavage furrow, ~ for approximately 40 min as shown in panels 5A-5C. Time is indicated along vertical axis and correlates with images in panel (5A-5C), approximate location of start and finish of 40 min time in kymographs are marked by arrows. LDs are indicated by black arrowheads. Scale bars are 50 μm in all images of this figure.</p

Inhibition of Myo1 arrests cell division and affected blastomere shapeof Zebrafish embryos.

<p>Schematic representation of Myo1 domain structures. (A) Semiquantitative RT-PCR profiles from cDNA and RNA templates. For Myo1Ea&b, control (top panel) and with PClP (bottom panel), were compared at 1–4 and 64 cells stages for cDNA and RNA templates, (B) Semiquantitative RT-PCR profiles from cDNA and RNA templates. For Myo1Cb, control (top panel) and with PClP (bottom panel), were compared at 1–4 and 64 cells for cDNA and RNA templates. (C) (i) Western blot for Myo1C relative-levels at 1–4 and 64 cell stages. Control (top panel) and with PClP (bottom panel), GAPDH used as loading control. (ii) Relative change in levels of Myo1C ±PClP, 1–4 and 64 cell stages, control grey, PClP black. Error bars indicate SD, n = 3. (D) (i) Top panel, control embryos at different developmental stages. Bottom panel, PClP treated embryos taken in identical time as in control, dotted line shows boundary between yolk and blastodisc in both panels, (ii) measurement of changes in blastodisk thickness, as indicated by vertical both sided arrows in(Ei) with time, approximately along first cleavage furrow in both, the control (dotted line) and PClP treated embryos (solid line), n = 8, error indicate SD. Black arrow-head in time axis indicates PClP addition. Embryos were observed in lateral or side view position. (E) DAPI stained nucleus profile of 64-cell control (2 hpf) (top panel) and equivalent 2 hpf PClP inhibited 8- cell embryo (middle panel). 1 hpf control 8- cell embryo, showing divided nucleus is also shown (lower panel). bar 120μm in all places.</p