Preprint highlights, selected by the biological community

Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of cell division, differentiation, and loss

Judy Martin, Erin Nicole Sanders, Paola Moreno-Roman, Shruthi Balachandra, XinXin Du, Leslie Ann Jaramillo Koyama, Lucy Erin O'Brien

Preprint posted on February 26, 2018 https://www.biorxiv.org/content/early/2018/02/26/271742

A new method captures the dynamics of tissue homeostasis in the stem-cell based organ of the adult fly midgut

Selected by Natalie Dye

Categories: cancer biology, cell biology, developmental biology, genetics, physiology

Background:

To maintain tissue homeostasis, many adult organs harbour pools of stem cells that divide and differentiate to replenish cells that are lost through ageing and/or injury. How these division and differentiation events are regulated is an important open question in biology.

Due to its genetic tractability, the adult Drosophila midgut (analogous to the mammalian small intestine) has been a powerful model system for identifying the conserved signalling pathways underlying both normal tissue homeostasis and its dysfunction during disease, infection, and ageing. Nonetheless, without methods to support extended live imaging of this tissue (longer than 90min), it has been difficult to use this powerful genetic model to study the cell biology and signalling dynamics underlying stem-cell-based homeostasis.

Summary of the preprint:

The authors of this preprint create a new and vastly improved protocol for imaging the midgut. They first stabilize the abdomen of the animal while providing a feeding tube; then they remove part of the dorsal cuticle, immerse this exposed part in media, and image through this media (Fig 1). The authors develop an image processing pipeline to compensate for global movements of the tissue and use a combination of fluorescent genetic markers and quantification of nuclear size to simultaneously identify and quantify the dynamics of different midgut cell types on a tissue scale for 12-16 hrs.

Fig 1: Method schematic (Fig 1A-C of the preprint): (A) Illustration of the fly digestive track; midgut in white. (B) Mounting the animal for imaging (with an upright microscope). The midgut is accessed through a hole in the cuticle, and the animal is fed through a tube. (C) Steps in sample preparation.

Movie S5 from the preprint showing the a ‘fate sensor’ midgut (esgGal4, UAS-his2b::CFP, GBE-Su(H)-GFP:nls; ubi-his2av::mRFP). With the combination of nuclear size and genetic labels, four midgut cell types can be identified and tracked: stem cells (red), enteroblasts (yellow-green), enterocytes (gray, polyploid – and thus larger), and enteroendocrine cells (gray, diploid – and thus smaller).

To demonstrate the power of their technique, the authors then provide new observations related to three processes occurring during normal tissue homeostasis:

Loss. Novel observations regarding the extrusion of enterocytes (terminally differentiated, nutrient-absorbing cells) include:
1. Basal cross sectional cell area shrinks in a rachet-like pulsatile fashion
2. The nucleus rapidly shoots out of the tissue toward the lumen before recoiling back toward the epithelial surface.
Division. Stem cell divisions occur less frequently during live imaging than would be expected based on staining of fixed tissues. Nonetheless, the authors report observations from 39 mitosis events—far more than ever before. Results not only confirm previous work using fixed tissues but also include the following new observations:
1. Dividing stem cells can rapidly reorient before division.
2. Neighbouring enteroblasts influence stem cell division orientation.
Differentiation. While genetic experiments have clearly implicated Notch signalling in stem cell differentiation, without live imaging data, the dynamics have been elusive, limiting our understanding of the regulatory mechanisms. Here, the authors calibrate a live Notch activity reporter and make three new discoveries:
1. Notch activation and stem cell differentiation can be directly observed live (Fig 2) and occurs over a time-scale of hours (based on observation of 5 events).
2. Proximity between stem cell siblings after division does not correlate with Notch activity.
3. The kinetics of Notch activation and cell differentiation imply a latency period after a cell is born, whereby its differentiation is delayed for several hours.

Fig 2: Direct observation of Notch activation during cell fate transitions (Fig 5D from the preprint). GBE-Su(H)-GFP:nls reports Notch transcriptional activity, while ubi-his2av::mRFP provides a reference signal. Cells additionally express esg>his2ab::CFP to mark progenitor cells. The authors use the GFP:RFP ratio to quantify Notch activation and directly observe transitions between stem and enteroblast states, finding that these transitions require several hours (2.4-6.9hrs).

Why I chose this preprint:

In my humble opinion, methods papers are where the preprint servers are especially valuable. Getting a new method out in the open as soon as possible is a great thing. Included in this paper are videos and detailed diagrams to help the reader use the method. Slight modifications may enable imaging of different parts of the adult fly, making the approach potentially even more widely applicable. Drosophila genetics has proven to be extremely powerful over the decades, but I’m excited to see more and more recent efforts to add live imaging to the toolbox – from the embryo, to the brain, wing disc, and now gut.

While this paper is first and foremost a method paper, there’s also a lot of tantalizing new biology presented. Their new data demonstrate how certain kinds of information can really only be obtained by direct observation over time, something I myself have been aiming for in my own work. The authors provide solid, valuable characterization of the dynamics—but nevertheless their data seem to generate more questions than answers. Thus, I think the best is yet to come. There is great potential for the live imaging approach to synergize with existing genetic tools in Drosophila to make really fundamental mechanistic discoveries. In principle, their techniques may also be combined with laser ablation, to probe the influence of mechanical forces, and pharmacological perturbations to rapidly, locally, and reversibly inhibit various proteins.

Future Directions:

Here are some of the questions I’m looking forward to hearing about in the future:

What in the world is going on with the nucleus in those extruding cells?
Since the duration of stem cell sibling contact (at least at the level of the nucleus) does not seem to be important, what regulates the when and whether Notch is activated?
- Related question: are there filopodia-like protrusions in these cells that may signal long distances through Notch, as in the Drosophila notum?
What is the molecular mechanism underlying the apparent latency phase in stem cell differentiation?
What happens to these dynamics upon injury, infection, ageing, and with varying diets?
- Related comment: another recent preprint demonstrates that dietary lipids affect cell differentiation and Notch signalling in the midgut (Obniski et al 2018: https://www.biorxiv.org/content/early/2018/02/28/273813). This is just one example of the interesting perturbations that can be now studied live using the techniques from O’Brien and colleagues.

Further reading:

Cohen M, Georgiou M, Stevenson NL, Miodownik M, Baum B. “Dynamic filopodia transmit intermittent Delta-Notch signaling to drive pattern refinement during lateral inhibition.” Dev Cell. 2010. doi: 10.1016/j.devcel.2010.06.006.
Gervais L, Bardin AJ. “Tissue homeostasis and aging: new insight from the fly intestine.” Curr Opin Cell Biol. 2017. doi: 10.1016/j.ceb.2017.06.005.
Guo Z, Lucchetta E, Rafel N, Ohlstein B.“Maintenance of the adult Drosophila intestine: all roads lead to homeostasis.” Curr Opin Genet Dev. 2016. doi: 10.1016/j.gde.2016.06.009.
Obniski R, Sieber M, and Spradling A. “Dietary lipids modulate Notch signaling and influence adult intestinal development and metabolism in Drosophila.” BioRxiv 2018. https://www.biorxiv.org/content/early/2018/02/28/273813

Tags: cell extrusion, differentiation, homeostasis, intestine, microscopy, notch, organ renewal, stem cells

Posted on: 20th March 2018

Read preprint

(No Ratings Yet)

Issue: *

Your Name: *

Your Email: *

Details: *

Have your say Cancel reply

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Also in the cancer biology category:

Quantification of microenvironmental metabolites in murine cancer models reveals determinants of tumor nutrient availability

Mark R Sullivan, Laura V Danai, Caroline A Lewis, et al.

Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of cell division, differentiation, and loss

Share this:

Have your say Cancel reply

Sign up to customise the site to your preferences and to receive alerts

Also in the cancer biology category:

Quantification of microenvironmental metabolites in murine cancer models reveals determinants of tumor nutrient availability

Mechanical Stretch Kills Transformed Cancer Cells

Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

A conserved MFS orchestrates a subset of O-glycosylation to facilitate macrophage dissemination and tissue invasion

MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

Applications, Promises, and Pitfalls of Deep Learning for Fluorescence Image Reconstruction

Basal extrusion drives cell invasion and mechanical stripping of E-cadherin

Molecularly distinct models of zebrafish Myc-induced B cell leukemia

The landscape of antigen-specific T cells in human cancers

Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma.

STAT3 promotes melanoma metastasis by CEBP-induced repression of the MITF pigmentation pathway

Memory sequencing reveals heritable single cell gene expression programs associated with distinct cellular behaviors

Anti-angiogenic effects of VEGF stimulation on endothelium deficient in phosphoinositide recycling

Profiling the surface proteome identifies actionable biology for TSC1 mutant cells beyond mTORC1 signaling

Precise tuning of gene expression output levels in mammalian cells

Moving beyond P values: Everyday data analysis with estimation plots

Also in the cell biology category:

Synthetic pluripotent bacterial stem cells

A DNA-based voltmeter for organelles

Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

Minimal membrane interactions conferred by Rheb C-terminal farnesylation are essential for mTORC1 activation

Mechanical Stretch Kills Transformed Cancer Cells

EHD2-mediated restriction of caveolar dynamics regulates cellular lipid uptake

Mechanical Stretch Kills Transformed Cancer Cells

Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

A metabolic switch from OXPHOS to glycolysis is essential for cardiomyocyte proliferation in the regenerating heart

S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate desmosome assembly and cell adhesion.

A complex containing lysine-acetylated actin inhibits the formin INF2

Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

Distinct progenitor populations mediate regeneration in the zebrafish lateral line.

Actomyosin-II facilitates long-range retrograde transport of large cargoes by controlling axonal radial contractility

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

Also in the developmental biology category:

Lineage tracing on transcriptional landscapes links state to fate during differentiation

Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

A direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila

A metabolic switch from OXPHOS to glycolysis is essential for cardiomyocyte proliferation in the regenerating heart

Reconstruction of the global neural crest gene regulatory network in vivo

Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissue

Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

Distinct progenitor populations mediate regeneration in the zebrafish lateral line.

Force inference predicts local and tissue-scale stress patterns in epithelia

Embryo geometry drives formation of robust signaling gradients through receptor localization

Unlimited genetic switches for cell-type specific manipulation

Applications, Promises, and Pitfalls of Deep Learning for Fluorescence Image Reconstruction

Maintenance of spatial gene expression by Polycomb-mediated repression after formation of a vertebrate body plan

The coordination of terminal differentiation and cell cycle exit is mediated through the regulation of chromatin accessibility

Embryo geometry drives formation of robust signaling gradients through receptor localization

Symmetry breaking in the embryonic skin triggers a directional and sequential front of competence during plumage patterning

A SOSEKI-based coordinate system interprets global polarity cues in Arabidopsis

Also in the genetics category:

Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

A direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila

MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

Distinct progenitor populations mediate regeneration in the zebrafish lateral line.

The coordination of terminal differentiation and cell cycle exit is mediated through the regulation of chromatin accessibility

Ribosomal DNA and the rDNA-binding protein Indra mediate non-random sister chromatid segregation in Drosophila male germline stem cells

A non-canonical arm of UPRER mediates longevity through ER remodeling and lipophagy.

Psychiatric risk gene NT5C2 regulates protein translation in human neural progenitor cells

The Toll pathway inhibits tissue growth and regulates cell fitness in an infection-dependent manner

Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 in Drosophila

Evidence for an Integrated Gene Repression Mechanism based on mRNA Isoform Toggling in Human Cells

Signaling dynamics control cell fate in the early Drosophila embryo

PUMILIO hyperactivity drives premature aging of Norad-deficient mice

Arterio-Venous Remodeling in the Zebrafish Trunk Is Controlled by Genetic Programming and Flow-Mediated Fine-Tuning

CRISPR/Cas9-mediated gene deletion of the ompA gene in an Enterobacter gut symbiont impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes

Also in the physiology category:

A DNA-based voltmeter for organelles

Quantification of microenvironmental metabolites in murine cancer models reveals determinants of tumor nutrient availability

Regulation of modulatory cell activity across olfactory structures in Drosophila melanogaster

A non-canonical arm of UPR^ER mediates longevity through ER remodeling and lipophagy.