Live Imaging during Arthropod Sequential Segmentation

Among arthropods, most of our small live imaging collection regarding sequential segmentation and axial elongation comes from time-lapse analyses performed on germbands of the isopod crustacean Porcellio scaber (Hejnol et al. 2006) and fluo- rescently labeled embryos of the red flour beetle Tribolium castaneum (El-Sherif et al. 2012; Sarrazin et al. 2012; Benton et al. 2013, 2016; Zhu et al. 2017; Benton 2018) and the spider Parasteatoda tepidariorum (Hemmi et al. 2018). In addition, some studies in the cricket Gryllus bimaculatus, the amphipod crustacean Parhyale hawaiensis and also T. castaneum, have indirectly covered part of these processes (Nakamura et al. 2010; Strobl and Stelzer 2014; Donoughe and Extavour 2015; Wolff et al. 2018).

A conserved feature of malacostracan, the largest crustacean class that includes the wood louse P. scaber and the sand flea P. hawaiensis, is that their posterior or post-naupliar segments are formed by cells that divide in a very stereotyped pattern (see Chapter 6). Using 4D-Nomarski microscopy, Hejnol and colleagues (2006) generated a detailed description of the post-naupliar cell division patterns and tracked the lineages of individual cells at the transitional zone between the naupliar and post- naupliar regions of P. scaber. Within this zone, they detected irregular cell divisions and unexpected cell movements that allowed them to establish phylogenetic relationships between different orders of malacostracan crustaceans (tanaids and isopods).

In 2012, two groups demonstrated, based on different approaches that included live fluorescent imaging, the presence of a segmentation clock operating in the segment addition zone of T castaneum (El-Sherif et al. 2012; Sarrazin et al. 2012). The use of time-lapse imaging and cell tracking was essential to correlate the expression waves of the pair-rule genes Tc-even-skipped (Tc-eve El-Sherif et al. 2012) and Tc-odd-skipped (Tc-odd Sarrazin et al. 2012) suggested by the detailed analysis of multiple fixed embryos with changes in the expression levels within cells rather than cell movements. For this, Sarrazin and colleagues (2012) generated the EFA-nGFP transgenic line that expresses a ubiquitous nuclear-localized GFP under the control of the regulatory sequences of the EFl-a gene. Given that during T. castaneum elongation, the posterior region of the germband, including the SAZ, is surrounded by opaque yolk cells that interfered with fluorescence visualization, they decided to dissect the embryo and mount it flat prior to performing the time-lapse recording, for which they developed a whole embryo culture. In a one-hour time-lapse fluorescence recording of a dissected T. castaneum germband (8 ’-stacks captured every 3 minutes), Sarrazin and coworkers tracked cell nuclei during the dynamic formation of the forth Тс-odd stripe. In the case of El-Sherif and colleagues, they used the same transgenic line as Sarrazin et al. (2012) to track cell movements from blastoderm formation to germband condensation, covering 11 hours (5 z-stacks captured every 5 minutes). Since cell displacements within the SAZ and blastoderm could not explain the oscillatory behavior of Тс-odd and Тс-eve respectively, both groups suggested that a ver- tebrate-like molecular clock was probably underlying the sequential segmentation in arthropods. It is worth noting that very recently Hemmi and colleagues (2018) demonstrated the oscillatory expression of the segment polarity gene hedgehog (Pt-hh) in the opisthosomal region (SAZ) of the spider Parasteatoda tepidariorum using a similar approach. For that they tracked fluorescently labeled cell clones (injected with mRNAs coding for NLS-tdEosFP or NLS-tdTomato) during the dynamic expression of Pt-hh and they found that cells persisted in the area of Pt-hh waves, indicating that cell movements were not responsible for this oscillatory behavior.

Over the following years, three interesting scientific papers were published, which used live imaging and fluorescent labeling approaches in T castaneum (Benton et al. 2013; Strobl and Stelzer 2014, 2016). Strobl and Stelzer performed, for the first time.

long-term live imaging of T. castaneum EFA-nGFP embryos by light sheet-based fluorescence microscopy (LSFM), covering the complete embryogenesis with a high spatiotemporal resolution and reduced photodamage (Strobl and Stelzer 2014). In addition, they presented a new transgenic line (FNL; Strobl and Stelzer 2016) that together with the combined LAN-GFP line (LifeAct-EGFP plus EFAl-nGFP; Sarrazin et al. 2012; Benton et al. 2013) presented by van Drongelen and coworkers (2018), expanded the transgenic and microscopy resources for the study of axial elongation and segmentation in T. castaneum (see Tables 8.2 and 8.3). In the same


Fluorescent Transgenic Lines and Labeling Approaches for Live Imaging Used or Feasible to Use to Study Axial Elongation and Sequential Segmentation in Different Arthropods

In Tribohum (Hexapoda)






Sarrazin et al. 2012* El-Sherif et al. 2012 Strobl and Stelzer 2014

H2B-RFP (for transient expression)


Benton et al. 2013*

LifeAct-EGFP (for transient expression)


Benton et al. 2013*

GAP43-YFP (for transient expression)

Cell membrane

Benton et al. 2013* van der Zee et al. 2015*

LAN-GFP (LifeAct-EGFP plus EFAl-nGFP)

F-actin (LifeAct) Nuclei (EFA1)

van Drongelen et al. 2018**

FNL line (Histone2B-EGFP)


Strobl and Stelzer 2016**

H2B-Venus (for transient expression)


Benton 2018*

NLS-td/:<« (nuclear localization signal-tandem Eos; for transient expression; photoconvertable)

In Cr)’llus (Hexapoda)


Benton 2018*


Nuclei and cytoplasm

Nakamura et al. 2010**

pXLBGact -actin.eGFP


Nakamura et al. 2010**

pXLBGact-Histone2B:eGFP In Parasteatoda (Chelicerata)


Nakamura et al. 2010**

NLS-tdEosFP (for transient expression)


Hemmi et al. 2018*

NLS-td7omnto (for transient expression)


Hemmi et al. 2018*

Hl-tdEosFP (for transient expression) In Parhyale (Crustacea)


Hemmi et al. 2018*



Hannibal et a. 2012

PhHS-H2BmRFPruby (for transient expression)


Wolff et al. 2018*

EGFP-caax (for transient expression)

Cell membrane

Wolff et al. 2018*

^Original article where the transgenic reporter/labeling was first described. 'Live imaging feasible to use.


Time-Lapse Movie Links

Akiyama et al. 2014


Aulehla et al. 2008 (mouse)


Benton 2018 (beetle) 10.137 l/journal.pbio.2005093#sec013

Benton et al. 2013 (beetle)

http://dev.biologists.Org/content/develop/suppl/2013/07/l l/140.15.3210.DCl/DEV096271.pdf

Delaune et al. 2012 (zebrafish)

https://www.sciencedirect.eom/science/article/pii/S 15345807120042007via%3Dihub#app2

Delfini et al. 2005 (chicken)

http://www.pnas.Org/content/102/32/l 1343/tab-figures-data

El-Sherif et al. 2012 (beetle)

Harima et al. 2013 (mouse)

Hejnol et al. 2006 (crustacean)


Horikawa et al. 2006 (zebrafish)


Masamizu et al. 2006 (mouse)

http://www.pnas.Org/content/103/5/l 313/tab-figures-data#M 1

Nakamura et al. 2010 (cricket)


Sarrazin et al. 2012 (beetle) 1218256/DC1

Shimojo et al. 2016 (mouse) /102/suppl/DC 1

Sonnen et al. 2018 (mouse)


Soroldoni et al. 2014 (zebrafish)

Strobl and Stelzer 2016 (beetle)

https://ars.els-cdn.eom/content/image/l -s2.0-S2214574516300955-mmc 1 .mp4

Takashima et al. 2011 (mouse) 10.1073/pnas. 1014418108/-/DCSupplemental/sm01 .avi 10.1073/pnas. 1014418108/-/DCSupplemental/sm02.avi

Wolff et al. 2018 (crustacean) 1 video2

path, Benton and colleagues (Benton et al. 2013) presented the combined use of transient fluorescence labeling and RNAi by the coinjection of the different cell marker constructs H2B-RFP, LifeAct-EGFP, and GAP43-YFP to label chromatin, F-actin, and the cell membrane, respectively, and dsRNA. The combination of these labeling strategies with live imaging allowed the authors to track cell movements during blastoderm formation, germband condensation, and elongation both in WT and RNAi (Tc-caudal knock-down) embryos. Using the same strategy, Benton et al. (2016) observed how difficult it is for the double knock-down Tc-Toll7-Tolll0 embryos transiently labeled with GAP43-YFP to elongate along the anterior-posterior axis and how cell intercalation was affected during embryo condensation. These results were essential to confirm the involvement of a new subfamily of Toll genes, the Long Toll or Loto clade, in germband elongation in T. castaneum, and together with results in six other groups of arthropods, they suggest a conserved role of these genes in axis elongation and that this function was probably present in the last common ancestor of all arthropods (Benton et al. 2016).

Very recently, making use of live cell tracking and a photoconvertible fluorescent construct (the nuclear-localized nls-tdEos), Matthew Benton (2018) challenged the preconceived idea that dorsal epithelium during T. castaneum germband elongation was formed completely by the amnion (an extraembryonic tissue). By photoconverting small groups of dorsal cells he found that some of these cells became part of the embryonic tissue by moving to the ventral epithelium along the entire germband. This finding changed the current fate map from late blastoderm to the elongating germband stages, increasing the number of ectodermal cells involved in the axial elongation and segmentation. In addition, Benton found mediolateral cell intercalations within the SAZ during elongation, a cellular behavior suggested to be absent during this process by previous work based on static images (Nakamoto et al. 2015).

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